EP1124560A1

EP1124560A1 - Use of antifungal agents for treating scleroses

Info

Publication number: EP1124560A1
Application number: EP99950850A
Authority: EP
Inventors: Roumen Antonov
Original assignee: Candida Medical BV
Current assignee: Candida Medical BV
Priority date: 1998-10-27
Filing date: 1999-10-27
Publication date: 2001-08-22
Also published as: JP2002528064A; AU1036999A; BR9914792A; IL142641A0; CN1328466A; AU6347199A; WO2000024403A1; WO2000024402A1; CA2348160A1

Abstract

The invention concerns the use of active antifungal agents on fungi of the Candida genus, and as the case may be of the Rodhotorula genus, for preparing medicines for preventing or treating tissue scleroses, and other derived pathologies, in humans and animals, related to the presence in the human or animal organism of fungi derived from those mentioned above.

Description

USE OF ANTIFUNGALS IN THE TREATMENT OF SCLEROSES

The present invention relates to the use of antifungals active on fungi of the genus Candida, and if necessary on those of the genus Rodhotorula, for the preparation of medicaments intended for the prevention or treatment of tissue sclerosis, and other derived pathologies , in humans or animals, linked to the presence in the human or animal body of fungi derived from those mentioned above. This invention stems from the discovery by the inventor of the fact that an antifungal active on Candida, known under the international common name (INN) Nystatin, makes it possible to absorb sclerosis of the tissues of the organism, when it is used, on the one hand at a dosage significantly lower than its usual dosage in the context of the treatment of candidiasis, and, on the other hand, for a duration of treatment much longer than the usual duration of treatment of candidiasis.

This demonstration is the result of many years of observations of the effects of Nystatin in vivo during long periods of treatment carried out by the Inventor on himself.

In his autobiographical book published in Bulgaria on April 27, 1998 ("Chronicle of a Dance with the Devil", by Roumen Antonov, Pygmalion Press ISBN954-8336-39-1), the Inventor reports on the tests carried out on himself, and some other volunteers, with a drug supposedly active against Candida albicans, without designating this drug.

Having found that this drug, which in reality was Nystatin, had a resorption effect on tissue necrosis or sclerosis originating from old scars, the inventor hypothesized that these tissue sclerosis could result from the development of Candida albicans in the regions from the body where dead cells and tissue debris accumulate.

This is how the inventor sought the presence of Candida albicans in atherosclerotic plaques in vitro. The experiment consists of incising an atheroma plaque and treating it with a local liquid antifungal active against Candida albicans. We then see reproducibly the in vitro decomposition of the atheroma plaque after addition of the antifungal.

Furthermore, the cultivation of these atheroma plaques makes it possible to observe a Y-shaped structure which, although relatively atypical, could suggest the presence of Candida albicans inside this atheroma plaque.

However, this simple observation alone did not allow us to conclude with certainty on the presence or not of Candida albicans in atheroma plaques. Such confirmation required the implementation of additional experiments.

Recent experiments of cultures on Sabouraud medium and of specific colorings, in particular by fluorescence, of the microorganisms present in the atheroma plaques, have enabled the inventor to conclude without ambiguity on the presence of a fungus related to the fungi of the Candida genus in atherosclerotic plaques, and other sclerotic tissues, although having filaments having dimensions never described until now for Candida fungi. In addition, the Inventor was thus able to demonstrate that the above-mentioned fungus was sometimes accompanied by a fungus of the genus Rodhotorula, the latter itself being described for the first time by the Inventor in filamentous form.

Furthermore, the in vivo studies carried out for a long time by the inventor have enabled him to determine the dosages of antifungal agents necessary for the prevention or the treatment of pathologies, in particular of the various scleroses, linked to the development of these fungi in the organism.

The subject of the invention is a fungus derived, in particular by mutation, from a fungus of the genus Candida, such as Candida lipolitica, or Jarowia lipolitica, or derived from other germs or fungi, in particular other species of lipolytic fungi, said being able to develop in perfect sexual phase in filamentous form in the human or animal organism, in particular in the vascular system, the various organs, as well as in the skin.

The above-mentioned fungus is also characterized in that it is capable, after having been taken from a host, in particular in plaques of human atheromas, to develop in vitro in a nutritive medium, such as Sabouraud medium, in the form of filamentous colonies of whitish color, and of a substrate mycelium having clearly visible filaments in the nutritive medium. It can also be in the form of whitish yeast colonies with a fatty appearance compared to the nutrient medium, and corresponding to the imperfect fungus in the asexual phase.

The fungus derived from the genus Candida mentioned above according to the invention, is further characterized in that it comprises filaments whose diameter varies between approximately 4 to approximately 8 μ, and can reach up to more than 30 μ in nutritive medium m vitro , or in the body.

The above-mentioned fungus of the invention is also characterized in that it develops in an anaerobic medium, in vivo or in vitro. The invention also relates to a fungus as defined above, deposited at the

National Collection of Culture of Microorganisms (CNCM) of the Institut Pasteur in Paris under the number 1-2336 on October 15, 1999.

The above-mentioned fungi, and more particularly those derived from the genus Candida as defined above according to the invention, are more characterized in that they can develop in the human or animal organism, in association or not with a fungus of the genus Rodhotorula, or a derivative of the latter, in particular in the presence of Rodhotorula rubra or Rodhotorula glutinis or other subspecies of Rodhotorula, this fungus of the genus Rodhotorula being optionally in filamentous form corresponding to a sexual form of these fungi in perfect phase , and, as already mentioned above, more particularly in blood vessels (such as veins, capillaries, arteries, arterioles, coronaries, etc.), or other human tissues, the aforementioned fungi being extractable from of this organization.

A subject of the invention is also the use of antifungal agents active on fungi of the genus Candida, in particular on Candida albicans, Candida lipolitica, or Jarowia lipolitica, and, if appropriate on fungi of the genus Rodhotorula, such as

Rodhotorula rubra or Rodhotorula glutinis, for the preparation of a medicament intended for the prevention or treatment of pathologies linked to the presence in the human or animal body of a mushroom mentioned above, and more particularly of a mushroom derived from the genus Candida as defined above, in particular of a fungus derived from Candida lipolitica, or other germ, having the above-mentioned atypical filamentous structure, where appropriate in association with a above-mentioned fungus of the genus Rodhotorula, and / or in association with yeasts of the Candida lipolitica type. A more particular subject of the invention is the use of antifungals as defined above and below, for the preparation of a medicament intended for the prevention or treatment of sclerosis or tissue necrosis, or of pathologies resulting from these scleroses or necroses, and being linked to the presence in the human or animal organism of fungi as described above, and more particularly of a fungus derived from the genus Candida as defined above, where appropriate in combination with an aforementioned fungus of the genus Rodhotorula.

A more particular subject of the invention is the above-mentioned use of antifungal agents as described above, for the preparation of a medicament intended for the prevention or treatment of pathologies linked to the development of sclerosis or necrosis in the body, namely a medicament intended to prevent or reduce sclerosis or necrosis resulting from the deposition and accumulation of cellular debris in the tissues, as well as from their maintenance in the form of clumps by the fungi as described above , and more particularly by fungi related to those of the genus Candida as defined above, if necessary in association with a aforementioned fungus of the genus Rodhotorula, developing in these sclerotic or necrotic structures.

The invention also relates to the use of antifungals as defined above and below, for the preparation of a medicament intended for the prevention or treatment of atherosclerosis linked to the presence in the blood vessels of the human or animal organism, fungi as described above, and more particularly a fungus derived from the genus Candida as defined above, where appropriate in combination with a aforementioned fungus of the genus Rodhotorula, as well as different pathologies associated with the aforementioned atherosclerosis, such as myocardial infarction, phlebitis, and cerebral vascular thrombosis.

Another subject of the invention is the use of antifungals as defined above and below, for the preparation of a medicament intended for the prevention or treatment of sclerosis or necrosis of the skin, such as eczema, psoriasis, erythemas, ichthyoses, linked to the presence in the human or animal organism, of fungi as described above, and more particularly of a fungus derived from the genus Candida as defined above, the if necessary in combination with a aforementioned fungus of the genus Rodhotorula, or a drug intended for the detersion of necrotic scars and wounds and the elimination of sphaceles, especially in cases of ulcers (for example of the legs) of venous origin, bedsores, traumatic, chronic, ulcerated or dull wounds, superficial and deep burns, necrosis skin of traumatic origin, in which the aforementioned mushroom (s) is located.

The invention also relates to the use of antifungals as defined above and below, for the preparation of a medicament intended for the prevention or treatment of sclerosis or necrosis of the joints, in particular in the case of osteoarthritis , linked to the presence in the human or animal organism of fungi as described above, and more particularly of a fungus derived from the genus Candida as defined above, where appropriate in association with a aforementioned fungus of the genus Rodhotorula.

The subject of the invention is also the use of antifungals as defined above and below, for the preparation of a medicament intended for the prevention or treatment of pulmonary pathologies of sclerotic origin, and more particularly of asthma or spasmodic cough, linked to the presence in the human or animal organism of fungi as described above, and more particularly of a fungus derived from the genus Candida as defined above, where appropriate in association with an aforementioned fungus of the genus Rodhotorula.

The term “antifungals active on fungi of the genus Candida, and on those of the genus Rodhotorula, is understood in particular to mean any compound active on any species of the genus Candida and Rodhotorula, in particular any compound active on Candida Albicans, Candida lipolitica, or Jarowia lipolitica, and any compound active on Rodhotorula rubra, or on any species or subspecies derived, in particular by mutation, from the above-mentioned fungi.

Among the antifungal agents capable of being used in the context of the present invention, there may be mentioned:

- Flucytosine (DCI),

- antifungal contact antibiotics of the polyene family,

- antifungals from the imidazole group, such as:

. Miconazole (DCI),. Ketoconazole (DCI),

. Fluconazole (INN) (bistriazole antifungal). A more particular subject of the invention is the above-mentioned use of antifungal contact antibiotics of the polyene family, and more particularly those extracted from cultures of bacteria of the genus Streptomyces, such as:

. Amphotericin B (INN) extracted from culture of Streptomyces nodosus,

. Nystatin (INN) extracted from culture of Streptomyces noursei, or any derivative of these antifungals, such as those capable of being obtained by chemical synthesis.

By way of illustration, the polyenes capable of being used in the context of the present invention are the following:

- the following tetraenes of formula 1:. nystatin of formula la

. pimarycin of formula lb in which R = CH ₃ and arenomycin in which R = CH ₂ -CH ₂ -CH ₂ -CH ₃

. the formula rimocide the

tetrine A of formula ld in which R = H, and tetrine B in which

R = OH

- Pentaenes of the following formulas 2:

. the philipine of formula 2a in which R = H, and the pentamycin in which

. mycotitzin A of formula 2b in which R = H, and mycotitzin B of formula 2b in which R = CH ₃

. eirotitzine A of formula 2c in which R = CH ₃ , and eirotitzine B of formula 2c in which R = H

the compound of formula 2d below

- hexaenes of the following formulas 3:

. dermostatin A of formula 3 in which R = CH ₃ , and dermostatin B of formula 3 in which R = CH ₂ -CH ₃

the following formula 4 heptaenes. amphotericin B of formula 4a

the candidine of formula 4b

Advantageously, the antifungal used in the context of the present invention is Nystatin.

Advantageously still, the doses of antifungals used within the framework of the present invention are approximately 2 to approximately 100 times, in particular approximately 5 to approximately 50 times, lower than the usual doses of these same antifungal agents (namely at the usual doses of approximately 10 500,000 IU tablets per day for 10 days) as part of the treatment of candidiasis, especially buccopharyngeal, intestinal or vaginal.

In addition, the duration of treatment is much longer, in particular at least about 5 times longer, in the case of the present invention, than in the case of the treatment of the abovementioned candidiasis, and is more particularly at least about

2 months, especially at least around 6 months.

By way of illustration, the dosages used in the case of the use of antifungals, and more particularly of Nystatin, in the context of the present invention, are the following: - less than approximately 100,000 units per day in the adult, especially in people between the ages of about 20 and 30 to 40, or

- between approximately 25,000 and approximately 50,000 units per day in people under the age of about 20, and in children,

- between approximately 25,000 and approximately 125,000 units per week, advantageously approximately 25,000 units per week, in people over 40 years of age, for an unlimited period of treatment.

The dosages indicated above are given for one or more intakes daily during the duration of the treatment, or, preferably, for one or more intakes per day spaced apart by intervals of approximately 3 to approximately 7 days during the duration of the treatment .

At the dosages indicated above, the duration of treatment is approximately at least 6 months for children and people under the age of about 20, up to one or more years in people over the age of 20.

It can be noted that after such periods of treatment, the immune system becomes able to take over against the development of these scleroses or necroses due to the development of fungi as described above, and more particularly of the aforementioned fungi derived from the genus Candida, through a process immunization, for a period of between approximately 3 years and approximately 5 years. Treatment with the above-mentioned antifungals may then stop.

In the event that this immunization disappears, treatment with the abovementioned antifungal agents can then resume, preferably with dosages approximately two times lower than those indicated above, and for a period approximately twice as short.

The dosages and durations of treatment indicated above make it possible to significantly reduce the amounts of fungi as described above, and more particularly of the aforementioned fungi derived from the genus Candida, as well as, if appropriate, those of the genus Rodhotorula, in the body.

The invention also relates to the aforementioned use of antifungals as described above, in dosages which make it possible to stop the growth of said fungi in the body. In this case, the dosage used in the context of the use of antifungals, and more particularly of Nystatin, is advantageously around 25,000 units per week (in one or more doses), in people over 40 years old, for an unlimited period of processing.

The invention also relates to the methods of treatment of the abovementioned pathologies, by administration to the patients likely to be treated, of the abovementioned antifungal agents, in particular at the doses indicated above, and during the abovementioned durations.

Advantageously, the abovementioned antifungals are used, in the context of the present invention, for the preparation of medicaments capable of being administered orally, in particular in the form of tablets or oral suspensions, or by injectable route. The invention will be further illustrated with the aid of the following detailed description of the demonstration of fungi as defined above in biposias of human origin.

1 Patients For this research, biopsies of vessels carried out during cardiovascular operations performed on 69 patients were used, coming from two different hospitals. The patients are distributed as follows: 50 men and 19 women, aged between 32 and 81 years.

The materials collected during the biopsies are ^" 137 in number, distributed according to their gender between: aorta 35; saphenous vein 30; carotid artery 17; mammary artery 39; pulmonary artery, radial artery, etc. 6.

4 patients underwent 4 biopsies, 19 patients 3 biopsies, 13 patients 2 biopsies and 33 patients only one biopsy.

2. Methodology The biological materials were removed under the best possible sterility conditions guaranteed by hospitals during surgical operations. During the operation, the biopsies were placed in sterile containers and transported immediately to the laboratory. In the laboratory, while the strictest sterility conditions were respected and the air controlled, the biological materials were cut into smaller pieces for the rest of the work to be carried out and in particular the preparation of slides for direct observation, l inoculation in nutritive medium and conservation in freezing for further research.

For the various biological materials, a maximum number of slices was sought in order to increase the number of slices sown. An air control was carried out by the method of sedimentation on a solid nutritive medium in the field of work and during the whole period of work.

2. a. Biological materials prepared for direct observation under the microscope

(smear)

The smears were prepared as follows: a small piece of biological material was placed between two slides. It is allowed to dry and then the slides are fixed using heat. From each biopsy, a Gram-colored smear is prepared - Modification of Bôzuc: staining with violet crystal and carbon nitrate, lugol; discoloration with acetone alcohol and obtaining the contrast with safranine. The smear thus colored is observed under the immersion microscope under objective x 100. Another smear is prepared for observation under fluorescence. 2.b. Seeding

The microbiological inoculation is done on a solid nutritive medium of Sabouraud, while seeking to obtain a maximum contact of the biological material with the nutritive medium. For this, the solid medium is cut and the biological material is seeded in the cut obtained.

The nutrient medium contains:

10 g Peptone Difco 20 g Glucose Difco 15 g Agar Difco distilled water up to 1000 ml.

The medium is sterilized for 10 minutes at 121 ° C.

Once the seeding has been carried out, the Petri dishes are rolled up with a plaster to reduce the risk of contamination during the cultivation time. The culture of the material is done at 28 ° C for 30 days by carrying out daily checks without removing the sticking plaster from the Petri dishes.

2.c. Transplanting of the pure strains obtained

When one locates a growth of the plate towards the medium, one proceeds, respecting the conditions of sterility mentioned above, to subculturing the strain obtained and again preparing a smear with Gram staining to observe the strain under the microscope.

2.d. Identification

To identify the fungal strains obtained, API 32C from Bio-Mérieux is used, which is used for yeasts, as well as macroscopic and microscopic observation methods used for the morphological identification of other types of mycelium.

From 137 biopsies, 535 materials were cut and seeded primarily on the solid nutrient medium. 3. Results

3. a. Microscope results

Out of 137 biopsies, 115 smears were taken for direct observation during the primary cutting of the biological pieces for sowing. Yeasts and / or filaments are then observed under the microscope on 87% of the smears.

3.b. Isolated fungal strains

In a large part of the biological materials sown and in variable periods of time, we observe shoots of fungal strains, the major part of which is a filamentous fungal strain of whitish color and with a substrate mycelium with clearly visible filaments in the nutritive medium. These filaments emerge directly from the biological material while very visibly making concentric shapes.

The time necessary for the clear appearance of a fungal colony is between the 4 ^th and the 20 ^th day after the primary seeding, with two climaxes: the 6 ^th and the 13 ^th day. From September 21 to October 6, out of 50 patients, 21 had biopsies with positive results (development of the strain), ie 42%. It is interesting to note that the percentage of strain development is 75% for patients who have had 4 biopsies, 70% for those who have had 3 biopsies and that this percentage drops to 29-26% for those who had 2 and 1 biopsies. By biopsy, the percentage of positive results is 28%.

The second type of fungal strain found according to its rate of presence in biological materials is Rhodotorula (Rubra, Glutinis). It grows directly from biological material while entering the environment. The identification of this strain was carried out by API 32C and it turned out that it is Rhodotorula Rubra and Rhodotorula Glutinis. Rhodotorula cases are distributed equally between patients in both hospitals. Rhodotorula grows faster, between 3 and 7 days from sowing. In the majority of cases, it grows from a patient's biopsy, where later the whitish filamentous strain will also grow, and represents 12% of patients. The third fungal strain based on its presence rate in biopsies was found to be Candida Lipolitica (on 2 patients). In all cases, it was isolated next to the whitish filamentous fungal strain. During the time of research, on 3 of the 535 seedings carried out, we saw growing strains of a different kind which, depending on where they had grown in the petri dishes, were judged as contaminants because they had no direct relationship with the biological material.

In none of the Petri dishes with nutrient medium and air control, yeasts or contaminants could not be found.

Claims

1. Mushroom derived, in particular by mutation, from a fungus of the genus Candida, such as Candida lipolitica, or Jarowia lipolitica, said fungus being capable of developing in perfect sexual phase in filamentous form in the human or animal organism, in particular in the vascular system, the various organs, as well as in the skin.

2. Mushroom according to claim 1, characterized in that it comprises filaments whose diameter varies between approximately 4 to approximately 8 μ, and can reach up to more than 30 μ in a nutritive medium in vitro or in the organism.

3. Mushroom according to claim 1 or 2, characterized in that it develops in an anaerobic environment.

4. Mushroom according to one of claims 1 to 3, deposited at the National Collection of Culture of Microorganisms (CNCM) of the Institut Pasteur in Paris under the number 1-2336 on October 15, 1999.

5. Mushroom according to one of claims 1 to 4, characterized in that it can develop in the human or animal organism, in association with a fungus of the genus Rodhotorula, or with a derivative of the latter, in particular in the presence of Rodhotorula rubra or Rodhotorula glutinis.

6. Use of antifungal agents active on fungi of the genus Candida, and, where appropriate on fungi of the genus Rodhotorula, for the preparation of a medicament intended for the prevention or treatment of pathologies linked to the presence in the body human or animal of a fungus defined in one of claims 1 to 5, where appropriate in association with a fungus of the genus Rodhotorula.

7. Use of antifungal agents according to claim 6, for the preparation of a medicament intended for the prevention or treatment of sclerosis or tissue necrosis, or from pathologies resulting from these scleroses or necroses, and being linked to the presence in the human or animal body of a fungus defined in one of claims 1 to 5, if necessary in association with a fungus of the genus Rodhotorula.

8. Use of antifungal agents according to claim 6 or 7, for the preparation of a medicament intended for the prevention or the treatment:

- atherosclerosis linked to the presence in the blood vessels of the human or animal organism, of a fungus defined in one of claims 1 to 5, if necessary in association with a fungus of the genus Rodhotorula, as well as different pathologies associated with the aforementioned atherosclerosis, such as myocardial infarction, phlebitis, and cerebrovascular thrombosis,

- sclerosis or necrosis of the skin, such as eczema, psoriasis, erythemas, ichthyoses, linked to the presence in the human or animal body of a fungus defined in one of claims 1 to 5, the if necessary in combination with a fungus of the genus Rodhotorula, or a drug intended for the cleansing of scars and necrotic wounds and the elimination of sphaceles in particular in the case of ulcers of venous origin, bedsores, wounds traumatic, chronic, ulcerated or dull, superficial and deep burns, skin necrosis of traumatic origin, within which the fungus defined in one of claims 1 to 5 is situated, if necessary in association with a fungus of the genus Rodhotorula,

- sclerosis or necrosis of the joints, in particular in the case of osteoarthritis, linked to the presence in the human or animal body of a fungus defined in one of claims 1 to 5, if appropriate in association with a fungus of the genus Rodhotorula, - pulmonary pathologies of sclerotic origin, and more particularly of asthma or spasmodic cough, linked to the presence in the human or animal body of a fungus defined in one of claims 1 to 5 , if necessary in association with a fungus of the genus Rodhotorula.

9. Use according to one of claims 6 to 8, of antifungals chosen from the following:

- Flucytosine (DCI), - antifungal contact antibiotics of the polyene family,

- antifungals from the imidazole group, such as:

. Miconazole (DCI),. Ketoconazole (DCI),. Fluconazole (INN) (bistriazole antifungal).

10. Use according to one of claims 6 to 8, anti-fungal contact antibiotics of the polyene family, and more particularly those extracted from cultures of bacteria of the genus Streptomyces, such as:. Amphotericin B (INN) extracted from culture of Streptomyces nodosus,

. Nystatin (INN) extracted from culture of Streptomyces noursei.

11. Use according to one of claims 6 to 8, Nystatin.

12. Use according to one of claims 6 to 11, antifungals, and more particularly Nystatin, in the following dosages:

- less than around 100,000 units per day in adults, especially in people between the ages of around 20 and around 30 to 40, or

- between approximately 25,000 and approximately 50,000 units per day in people under the age of about 20, and in children, or

13. Use according to claim 12, at the rate of one or more daily intakes during the duration of the treatment, or, preferably, at the rate of one or more intakes per day spaced apart by intervals of approximately 3 to approximately 7 days. for the duration of treatment, the duration of treatment being approximately at least 6 months for children and persons under approximately 20 years, up to one or more years in persons over 20 years.