IE65218B1 - Agents for use in the prophylaxis and therapy of fungal infections - Google Patents

Description

AGENTS FOR USE IN THE PROPHYLAXIS AND THERAPY OF FUNGAL INFECTIONS This invention relates to agents for use in the prophylaxis and therapy of Candida albicans infections, especially recurring C. albicans infections.

The yeast C. albicans is a normal constituent of the microflora in healthy individuals. However, C. albicans is also an opportunistic pathogen. It is the causative organism of yeast infections, including polysystemic chronic candidiasis, genital candidiasis, yeast vaginitis, intertrigo, onychia and paronychia and thrush.

Factors which reduce the effectiveness of the immune system, including disease, antibiotics and immunosuppressive agents used in other medical therapies may pre-dispose the individual to yeast infection. Typically, one will observe recurring episodes of infection in individuals susceptible to such infection either as a result of an ongoing iatrogenic condition, by reinfection resulting from environmental situations or by being reinfected by contact with an asymptomatic partner in the case of yeast vaginitis.

Conventional medical therapy for yeast infections includes the use of antibiotics with anti-eucaryotic activity, such as Nystatin. The repetitive use of any antimicrobial agent is undesirable and for this reason, the use of a prophylactic agent to prevent reinfection is a desirable objective.

The natural body response to any infection is the production of antibodies of the class IgG in systemic infection and secretory IgA in the case of infections of the mucus membrane, including the oral cavity, the digestive tract and genitourinary tract. As C. albicans is a constituent of the normal microflora , it tends to be tolerated and does not stimulate antibody formation as effectively as other pathogens. The ability to use an extraneous source of anti-Candida antibodies to aw* * supplement the natural defence mechanism would be a desirable objective in the case of the prophylaxis of C. albicans infections.

Conventional sources of bulk polyclonal antibody include immune equine and bovine serum. Apart from aesthetic objections, these polyclonal antibodies are antigenic in themselves and are, therefore, unsuitable for long term application in the human. Most humans are tolerant to egg protein as it is consumed regularly as a food and it is an ingredient in many cosmetics and topical lotions. Likewise, most humans are tolerant to milk of various ruminant species.

EP-A 0 152 270 discloses a method of passively immunising a mammal against a condition caused by an antigen which involves administering to the mammal immunising amounts of an antibody obtained from a domesticated fowl or bovid which has been immunised against the antigen; the mammal being tolerant to the antibody by virtue of having a history of consumption of antibody-containing material derived from the egg of a fowl or milk of a bovid.

Thalley, B.S. and Carroll, S.B. (Bio/Technology (1990); Vol. 8, page 934) demonstrate that antivenoms purified from the egg yolks of laying hens immunised with Crotalus atrox rattlesnake venom and Leiurus quinquestriatus hebraeus scorpion venom neutralise the lethal effects of these venoms in vivo. It is concluded that antivenoms purified from chicken eggs may be pharmaceutically safer and more economical to produce than current horse antivenoms.

It is an object of the present invention to provide an agent effective in the prophylaxis and therapy of C. albicans infections.

Conventional therapy of C. albicans infections can result in a reaction known as the Herxheimer reaction. The latter reaction is somewhat similar to anaphylactic shock and is attributable to an immune reaction to debris resulting from the lysis of C. albicans cells during treatment with antifungal agents.

It is a further object of the present invention to provide an agent for use in the prophylaxis and therapy of C. albicans infections in association with an antimicrobial agent, while minimising the risk of, or obviating the occurrence of, the Herxheimer reaction.

The invention provides use of immunoglobulin or a fraction thereof specific for Candida albicans and obtained from a hyperimmunised animal in the manufacture of a medicament for use in the prophylaxis or therapy of C. albicans infections.

By Candida albicans herein is meant C. albicans and varieties thereof capable of becoming pathogenic. Pathogenicity in the case of C. albicans is normally associated with morphosis of the yeast from a single cell to a hyphal form.

By fraction of immunoglobulin herein is meant an antibody, antibody fragment or antibody precursor capable of binding to C. albicans or a fragment thereof so as to render it non-pathogenic.

The immunoglobulin or fraction thereof is preferably obtained from a body fluid of an animal which has been hyperimmunised with C. albicans antigen.

Polyclonal anti-Candida immunoglobulin has the advantage that it is an aesthetically acceptable and non-allergenic source of extraneous antibody for prevention of recurring yeast infection.

The animal is suitably an avine.

For example, the avine can be a domestic fowl such as an egglaying hen, duck, guinea fowl, goose or turkey, more especially a domestic hen. Thus, the body fluid is suitably egg yolk.

The immunoglobulin or fraction thereof is suitably avian IgY or a fragment thereof.

Egg immunoglobulin is classified as avian IgY which is similar to mammalian secretory IgA and, therefore, naturally suited to the mucus epithelial environment.

In an alternative embodiment, the body fluid is milk from a ruminant.

An especially suitable ruminant is a caprine.

The invention also provides a pharmaceutical composition for use in the prophylaxis or therapy of C. albicans infections comprising immunoglobulin or a fraction thereof specific for C. albicans and obtained from a hyperimmunised animal optionally in association with a pharmaceutically acceptable carrier or diluent therefor.

The immunoglobulin can be administered without any conventional diluent or excipient as hereinafter described.

The pharmaceutical composition according to the invention is preferably administered in a form suitable for oral or buccal administration, topical application or in the form of an enema, pessary or suppository according to the principal sites of C. albicans infection. Indeed, in the case of multiple site infection by the organism one or more different forms of the anti-C. albicans active agent according to the invention can be used as a combined therapy.

Furthermore, the pharmaceutical composition according to the invention can also be used in conjunction with, or include, an antimicrobial agent of the kind used in conventional therapy or C. albicans infections, especially an antifungal agent.

The invention also provides a product containing immunoglobulin or a fraction thereof specific for C. albicans and obtained from a hyperimmunised animal and an antimicrobial agent as a combined preparation for simultaneous, separate or sequential use in the prophylaxis or therapy of C. albicans infections.

One egg yolk can contain up to 200 mg of antibody. The human population, in general, can tolerate up to 200 mg of egg yolk antibody per day without stimulating an allergic response. Thus, it will be appreciated that the C. albicans specific antibody in accordance with the invention can be administered in a daily dose of up to 200 mg or higher expressed as milligrams of purified polyclonal IgY antibody in the therapy of C. albicans infections. However, as a prophylactic agent an amount of the order of 2 mg would probably suffice to achieve the desired effects.

The mode of action of the C. albicans specific antibody is related to the number of C. albicans organisms present at a given site. It will be appreciated that there is a direct molecular correlation between antibody entities attaching to each yeast cell and the numbers of cells present. The dose level will also be dictated by the total surface area of affected tissue and biological parameters which affect wash out rates.

The appropriate formulation(s), dose and duration of treatment will be determined by the attending physician.

It is possible for the C. albicans specific antibody in accordance with the invention to be administered in the form of a powder, especially for oral administration and topical application. For example, the antibody when isolated from egg yolk as hereinafter described can be obtained as a freeze-dried or lyophilised powder.

When the powder is to be administered by the oral route, the powder will preferably contain a buffering agent which can optionally be administered in the form of a suspension.

In the case of formulations according to the invention for use against oral thrush, yeast vaginitis or similar localised infection of the mucus epithelium, such formulations will preferably be in the form of a lyophilised tablet, pessary or suppository containing a mucoadhesive polymer, as appropriate.

Suitable mucoadhesive polymers are known and include various cross-linked polyacrylic acids. For example, the mucoadhesive polymer could include varying amounts of the polymers sold under the Trade Marks CARBOPOL 934P and POLYCARBOPHIL NOVEON AA-1 to achieve the desired rheological, adhesive and other characteristics required of the formulation. CARBOPOL and CARBOPHIL NOVEON are Trade Marks of B.F. Goodrich Company, Cleveland, Ohio, U.S.A.

Enemas, pessaries and suppositories for use in accordance with the invention will contain conventional auxiliaries in addition to the C. albicans specific antibody and antimicrobial agent, if present.

As indicated above, the C. albicans specific antibody according to the invention can be used in combination therapy with conventional agents for the treatment of C. albicans infections.

The formulations according to the invention can be controlled or sustained release formulations, if required, especially when used in a body cavity.

The formulations according to the invention are useful for combating the aberrant growth of C. albicans known to be the cause of chronic yeast infections known commonly as candidiasis and also variously referred to as poly systemic chronic candidiasis, genital candidiasis, yeast vaginitis, intertrigo, onychia and paronychia and thrush.

As indicated above, aberrant growth of the yeast can be attributed to a multiplicity of factors, including long-term exposure to antibiotics which disrupt the normal balance of the microflora. By microflora herein is meant the microflora of the gastrointestinal tract, the genitourinary tract, skin and hair.

Current medical treatment for candidiasis is repetitive use of Nystatin or other eucaryotic antibiotics and/or a dietary regimen Ί designed to reinstate the normal balance of microflora. The formulations in accordance with the invention can be used as an alternative to, or supplement to, such treatments.

Methods of hyperimmunisation of any vertebrate species are well known and well illustrated in the literature. A generic description has been provided by Makoto, et al. (J. Food Science (1988); Vol. 53, No. 5) and also Thalley, B.S. and Carroll, S.B. ((1990) supra).

The invention will be further illustrated by the following Examples.

Example 1 Preparation of anti-Candida egg immunoglobulin.

A suspension of C. albicans was heat shocked by flash pasteurisation; total kill being demonstrated by failure to grow in liquid culture. Killed cells were washed in saline and freeze-dried in ampoules; 2.0 χ 108 yeast cells per ampoule. Twice weekly inoculations were carried out intramuscularly in domestic hens, using 1.0 ml purified water to resuspend each ampoule. Yolks of eggs collected from hyper-immunised hens were assayed to determine peak antibody titre using an ELISA (enzyme linked immunosorbent assay) specific for C. albicans IgY.

At 3 to 4 weeks, when peak titre had been achieved, egg yolks were harvested by separation from the egg white and removing the contents of the yolk sac using a hypodermic syringe. The immunoglobulin fraction was purified using industry standard supercritical CO2 equipment to dissolve the lipid, leaving the proteinaceous polyclonal immunoglobulin in a purified state. The immunoglobulin fraction was diluted using purified glass distilled water to a concentration of 10 mg/ml and lyophilised in trays. The resulting cake was used to evaluate the prophylactic potential of the anti-Candida IgY.

Example 2 Preparation of yeast cells C. albicans strain CBS 2710 and other strains of C. albicans isolated from mucus epithelium from infected individuals were used in an in vitro experiment to demonstrate the prophylactic potential of egg immunoglobulin isolated from domestic hens hyper-immunised with C. albicans antigen.

The yeast cells were grown in 500 ml Erlenmeyer flasks containing 100 ml of 2% glucose, 0.15% yeast nitrogen base, 0.5% ammonium sulphate supplemented, where necessary, with amino acids. The flasks were shaken at 200 r.p.m. in a rotary incubator at 37°C for 24 hours.

Example 3 Cell adhesion assay Adhesion of C. albicans to mucus epithelium cells is considered to be the primary stage in infection. In this Example buccal epithelial cells (BEC) were used to evaluate adhesion.

BECs were harvested from volunteers using a cell scraper (Costar, Cambridge, Ma, USA). The exfoliated cells were washed and re-suspended in phosphate buffered saline (PBS) at a density of 5 x lOW Fresh 24 hour cultured yeast cells were washed by centrifugation in PBS, re-suspended and mixed with BEC at a ratio of 100:1 in PBS and incubated at 37°C for 2 hours. After incubation, the culture was filtered through a 45pm filter to remove any unadhered cells, washed in PBS and re-suspended.

The adherence of yeast cells to BEC was evaluated by microscopic examination at 400 magnifications using a stage micrometer grid to facilitate accurate counting. Adherence was expressed as a percentage of BEC with visibly adhering yeast cells.

A washed suspension of yeast cells was divided in two. One half was treated with immunoglobulin from immunised hens, the other with immunoglobulin from normal eggs. The two suspensions were cocultured with BEC as described and following filtration and washing, examined microscopically.

The results showed that the adhesion of C. albicans to BEC was reduced by more than 50% in the case of yeast treated with immunoglobulin from immunised hens, when compared with both untreated yeast and yeast treated with extract of normal egg.

Example 4 Formation of germ tube (hyphal) assay The morphosis of C. albicans from cellular growth to hyphal growth in the presence of standard foetal calf serum is a diagnostic procedure for C. albicans infection.

The organisms, growth medium and cell culture were as described in the case of Example 2.

A washed yeast suspension was divided in two, one half treated with immunoglobulin from immunised hens, the other with immunoglobulin from normal eggs. Both fractions were suspended in foetal calf serum and observed during a six hour period. None of the cells treated with immunoglobulin from immunised eggs became hyphal; 80-90% of the cells treated with normal egg immunoglobulin became hyphal.

Example 5 Colony formation Standard YEPD (Yeast Extract Peptone Dextrose) agar plates were spread with a solution containing 50 mg egg immunoglobulin and dried at 37°C. The plates were used to study the effect of the immunoglobulin on colony formation.

Petri dishes containing agar treated with immunised egg extract were inoculated with C. albicans. After 24 hours, the colony formation on these plates was compared with colonies on plates treated with normal egg immunoglobulin.

Colonies on immunised egg immunoglobulin plates showed distinctly smaller colonies with evidence of inhibited growth and distorted colony shape. φ φ φ $ φ The above in vitro experiments demonstrate that purified immunoglobulin fractions extracted from the yolk sac of eggs laid by hens previously hyperimmunised with C. albicans antigen inhibit growth, epithelial cell adhesion and the formation of germ tubes.

These experiments enable one to conclude that C. albicans specific egg immunoglobulin can be used in the prophylaxis and therapy of recurring yeast infection.

Claims (22)

CLAIMS:

1. Use of immunoglobulin or a fraction thereof specific for Candida albicans and obtained from a hyperimmunised animal in the manufacture of a medicament for use in the prophylaxis or therapy of 5 C. albicans infections.

2. Use according to Claim 1, wherein the immunoglobulin or fraction thereof is obtained from a body fluid of an animal which has been hyperimmunised with C. albicans antigen.

3. Use according to Claim 2, wherein the animal is an avine. 10

4. Use according to Claim 3, wherein the animal is a domestic fowl.

5. Use according to Claim 4, wherein the animal is a domestic hen.

6. Use according to any one of Claims 3-5, wherein the body 15 fluid is egg yolk.

7. Use according to any one of Claims 2-5, wherein the immunoglobulin or fraction thereof is avian IgY or a fragment thereof.

8. Use according to Claim 2, wherein the body fluid is milk from a ruminant. 20

9. Use according to Claim 8, wherein the ruminant is a caprine.

10. A pharmaceutical composition for use in the prophylaxis or therapy of C. albicans infections comprising immunoglobulin or a fraction thereof specific for C. albicans and obtained from a 25 hyperimmunised animal optionally in association with a pharmaceutically acceptable carrier or diluent therefor.

11. A composition according to Claim 10, for oral administration.

12. A composition according to Claim 10, for use in the buccal cavity.

13. A composition according to Claim 10, for topical application.

14. A composition according to Claim 10, in the form of a pessary.

15. A composition according to Claim 10, in the form of a suppository.

16. A composition according to any one of Claims 10-15, wherein the immunoglobulin or fraction thereof is in a controlled release form.

17. A composition according to any one of Claims 10-16, 15 which additionally includes an antimicrobial agent.

18. A composition according to Claim 17, wherein the antimicrobial agent is an antifungal agent.

19. A product containing immunoglobulin or a fraction thereof specific for C. albicans and obtained from a hyperimmunised 20. Animal and an antimicrobial agent as a combined preparation for simultaneous, separate or sequential use in the prophylaxis or therapy of C. albicans infections.

20. Use according to Claim 1, substantially as hereinbefore described. 25

21. A pharmaceutical composition according to Claim 10, substantially as hereinbefore described.

22. A product according to Claim 19, substantially as hereinbefore described.