WO2012033427A1 - Procédé de traitement des infections mycosiques et produit pour sa mise en oeuvre - Google Patents

Procédé de traitement des infections mycosiques et produit pour sa mise en oeuvre Download PDF

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Publication number
WO2012033427A1
WO2012033427A1 PCT/RU2010/000492 RU2010000492W WO2012033427A1 WO 2012033427 A1 WO2012033427 A1 WO 2012033427A1 RU 2010000492 W RU2010000492 W RU 2010000492W WO 2012033427 A1 WO2012033427 A1 WO 2012033427A1
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WIPO (PCT)
Prior art keywords
lytic
exoenzyme
drug
strain
per
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Application number
PCT/RU2010/000492
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English (en)
Russian (ru)
Inventor
Михаил Викторович ДАЛИН
Эдуард Георгиевич КРАВЦОВ
Елена Александровна ВАСИЛЬЕВА
Ирина Викторовна АНОХИНА
Надежда Павловна САЧИВКИНА
Наталия Вячеславовна ЯШИНА
Original Assignee
Dalin Mikhail Viktorovich
Kravtsov Eduard Georgievich
Vasilieva Elena Aleksandrovna
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
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Application filed by Dalin Mikhail Viktorovich, Kravtsov Eduard Georgievich, Vasilieva Elena Aleksandrovna filed Critical Dalin Mikhail Viktorovich
Priority to PCT/RU2010/000492 priority Critical patent/WO2012033427A1/fr
Publication of WO2012033427A1 publication Critical patent/WO2012033427A1/fr

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/10—Antimycotics

Definitions

  • the group of inventions relates to medicine, namely to the treatment of mycotic infections and can be used, in particular, in gynecological practice.
  • VK vaginal candidiasis
  • Candida albicans the dominant pathogen of which is Candida albicans.
  • VC vaginal candidiasis
  • Relapses of candidiasis of the mucous membranes in certain categories of patients are a significant problem (see, for example, the “Practical Guide to Anti-Infectious Chemotherapy,” p / p L.S. Strachunsky, Yu. S. Belousov, S. N. Kozlova. -M ., 2002).
  • a known method of correcting the microflora of the vagina (RU2131258, Smirnov and others, 06/10/1999).
  • the method consists in the intravaginal administration of Biosporin (in suppositories or tampons).
  • the invention allows to correct the microflora of the vagina in case of its disorders caused by pathogenic and conditionally pathogenic microorganisms of various taxonomic groups.
  • the effect of this drug is indirect and, in case of any violation of the microbiota, VC recurrence is possible.
  • a well-known pharmaceutical composition for the treatment of candidal vulvovaginitis, including as an active component prebiotic lactulose in the amount of 200 mg in the form of vaginal suppositories, and as an active component antimycotics (azoles: intraconazole, terconazole, econazole; nifuratel and natamycin) in an amount of 100 mg or 500 mg.
  • an active component antimycotics azoles: intraconazole, terconazole, econazole; nifuratel and natamycin
  • VK RU2254857, Kolomoytseva et al., June 27, 2005
  • Multitabs, Rekitsen-RD drugs which can significantly improve the condition of patients in the acute period, but are not specific antimycotic therapy, and do not guarantee the absence of relapse within six months after treatment.
  • vaginal suppositories containing, in particular, benzalkonium chloride, benzoic acid, and vitamin tex with a certain content of ingredients.
  • Benzalkonium chloride has a bactericidal effect on a whole gamut of bacteria, however, it is less active against pathogenic LPG, in particular Candida albicans, and benzoic acid is a pH regulator, it inhibits the growth of pathogenic LPG, but, not related to the means of direct effect on DPG does not lead to the complete elimination of the latter from the vagina.
  • the complex of lytic exoenzymes was extracted from strains of Corpinus comatus, Licoperdon depressum or Licoperdon pyriforme. Enzymes were identified by laminarinase (chitinase, chitobiasis). However, these enzymes destroy the component of the cell wall of DPG - chitin, but do not affect the mannan layer, which determines the main wall structure of the VK pathogen.
  • the invention is directed to the treatment of recurrent mycotic infections, which make it possible to exert a specific effect directly on Candida albicans without inhibiting normal microflora. This ensures the irreversibility of Candida losing its virulent properties and viability, which leads to the absence of relapse.
  • the lytic exoenzyme obtained from the strain Cellulomonas cellulans VKPM AC-870 has important differences in the mechanism of action, which makes it possible to obtain a drug with an activity more than 2 times higher than that of a commercial drug lyticase. Antimycotic properties are ensured by the destruction of two surface layers - manan and fibrillar, while maintaining the glucan and chitin layers. It is this circumstance that makes it possible to use the lytic exoenzyme - lyticase as an antimycotic etiotropic agent that eliminates the very cause of the disease.
  • the drug does not destroy the cells of the macroorganism and normal microflora, since they do not contain those components that They are targets for the lytic action of the enzyme, which additionally contributes to the selectivity of its action.
  • a patented method for treating mycotic infections using a lytic exoenzyme in the form of a topical preparation as an active substance is characterized in that they use a lytic exoenzyme of the strain Cellulomonas cellulans VKPM AC-870, having a molecular weight in the range from 100 to 30 kD and specific activity of at least 48.3 U / mg protein.
  • the method can be characterized by the fact that said exoenzyme is a lyticase, as well as by the fact that with intravaginal and rectal administration, said exoenzyme is used at a dose of 10-50 units once a day for 5-7 days.
  • the method can also be characterized by the fact that, when applied topically, the mentioned exoenzyme is used in an amount of 2-10 U per 1 g of a biologically acceptable carrier, mainly in the form of an ointment or gel, 1 time per day for 5-7 days.
  • the method can also be characterized by the fact that a contrical proteinase inhibitor is additionally prescribed, which is administered together with the said exoenzyme in an amount of 500-1000 ATPE per dose of the latter.
  • a preparation for treating mycotic infections using a lytic exoenzyme as an active substance in the form of a topical application is characterized in that they use a lytic exoenzyme of the strain Cellulomonas cellulans VKPM AC-870, having a molecular weight in the range from 100 to 30 kD and specific activity of not less than 48.3 U / mg protein, as well as the fact that said exoenzyme is a lyticase.
  • a preparation for local, intravaginal and rectal administration contains 10-50 units of the mentioned exoenzyme per unit pharmaceutical form, mainly a suppository, tampon or sanitary pad.
  • the preparation may be characterized in that it contains the said exoenzyme in an amount of 2.0-10.0 UNITS per 1.0 g of a biologically acceptable carrier, and is in the form of an ointment or gel, and also contains a proteinase inhibitor in an amount of 500-1000 ATPE per dose of said lytic exo-enzyme.
  • EFFECT reduced recurrence rate in the treatment of mycotic infections, increased antimycotic activity without inhibition of normal microflora. This is ensured, as indicated above, due to the action of the mentioned exoenzyme both on the mannan layer of the cell wall of Candida albicans and on the fibrillar layer with its subsequent cleavage.
  • An additional technical result is a synergistic effect when using the aforementioned lytic exoenzyme and contrikal together: a developed surface is formed in the Candida genetically modified form, however, it does not exhibit pathogenic properties.
  • FIG. 1 presents the growth dynamics of the producer culture and changes in the proteolytic activity of the culture fluid (QL); in FIG. 2 - change in the activity of lyticase in the dynamics of cultivation of Cellulomonas cellulans; in FIG. 3, 4 - vaginal clearance in relation to DPG under the action of lithicase at a dose of 2 U / ml and 10 U / ml, respectively.
  • a lytic exoenzyme is used - lithase of the strain Cellulomonas cellulans VKPM AC-870.
  • C. cellulans culture is stabilized by creating a cold shock (inoculum exposure for 20 minutes at a temperature of + 4 ° ⁇ ).
  • the strain is cultivated with constant juggling for 2.5-3 days at a temperature of 37 ° C on a modified cardiac cerebrum broth (SMB) with the addition of killed baking yeast in the amount of 9.5-10.5 g per 1 liter of medium as an inducer of lithicase. Growth dynamics is controlled by optical density (OD).
  • QOL in a volume of 1.5 liters is centrifuged at 3000 rpm for 15 minutes. and passed through a sterilizing filter with a pore diameter of 0.02 ⁇ m. Further, QOL is subjected to diafiltration on AMICON cells using XM-300, XM-100 and RM-30 membranes. Each of fractions are concentrated to a volume of 20 ml, after which the activity of lithium is determined. It was found that the enzyme activity was absent in fractions containing biopolymers with a molecular mass of more than 100 kD and 300 kD, but was fully manifested in the range from 100 to 30 kD. The obtained parameters are consistent with the characteristics described in the literature. After washing and concentrating this fraction, the specific activity of the final product is not less than 48.3 U / mg protein, which is more than 2 times the specific activity of the commercial drug Lyticase from Sigma (20 U / mg protein), which we adopted as a standard.
  • Acute toxicity of the preparation was evaluated in animals, for which 0.4 mice of a solution containing 117 mg of protein were injected into 10 mice in the dorsal tail vein. The animals remained healthy for 10 days, from which it was concluded that the drug is not toxic at doses that are hundreds of times higher than the therapeutic ones.
  • the effect of the obtained exoenzyme - lyticase is manifested in the form of lysis of DPG cells, is expressed in a decrease in the number of viable cells and depends on the dose of the enzyme used in the experiment.
  • a decrease in the optical density of the suspension to 53% relative to the control within 1 hour is observed.
  • a dose-dependent effect is observed when evaluating the results of seeding of the remaining viable cells. Both processes are described by two regression equations that reflect the dependence of the lytic effect on the dose of the enzyme:
  • y is the magnitude of the decrease in the optical density of DPG suspension under the influence of lithase, compared with the control;
  • x is the dose of lithicase;
  • the antimycotic effect of lithicase can be supplemented by the introduction of the drug "Contrical”.
  • the drug “Contrical” (Aprotinin / APROTININ), an inhibitor of proteolytic enzymes and plasma proteases, is usually used to treat pancreatitis (manufactured by AWD pharma GmbH & Co., Germany).
  • the applicant for the first time established the previously unknown property of contracal to inhibit the production of aspartyl proteinase aggression enzymes in DPG. Inhibition of aspartyl proteinases by contrical prevents the realization of the invasive properties of the pathogen, blocks its transition to a pathogenic mycelial form and promotes the elimination of the pathogen.
  • the counter-counter is administered in an amount of 500-1000 ATPE together with lithase, per dose of the latter.
  • the patented preparation contains lithicase and counter-cal in a single composition.
  • the synergistic antimycotic effect of the two drugs is proved by the mechanism found in an in vitro experiment. Litikase provides a lysing effect, and contracal blocks the aggression enzymes of aspartyl proteinase in DPG, which have remained viable.
  • albicans N ° 40 is clinical, isolated from a pregnant woman with VK. Therefore, experiments with lithase and C. albicans strain N ° 40 in vitro and in vivo on the VK model can be considered as experimentally substantiated evidence of the drug's applicability for the treatment of mycotic infections.
  • Candida genus DPG their adhesion on the surface of target cells (vaginal epithelium) decreases, but the adhesive activity against representatives of the normal microflora of the vagina (lactobacilli) does not change.
  • the recommended dose of lithicase is much lower than necessary for cell lysis. This allows to reduce the drug load while maintaining the therapeutic effect.
  • the proposed preparation uses lithicase, the initial activity of which is at least 48.3 U / mg protein.
  • the specified lytic exoenzyme can be introduced in various amounts into the composition of different pharmaceutical forms of the drug to achieve an activity of 10-50 units per dose.
  • the drug lithicase can be used for local, vaginal or rectal use in the form of an aqueous solution obtained by stationary or industrial technology.
  • a solvent buffered saline, pH 7.2 can be used.
  • the drug lyticase may also optionally contain a filler, usually used in the manufacture of the corresponding prescription forms.
  • a filler usually used in the manufacture of suppositories or suppositories, for example, confectionery fat, lanolin, cocoa butter, aluminum hydroxide gel, paraffin, and the like are used as filler.
  • confectionery fat, lanolin, cocoa butter, aluminum hydroxide gel, paraffin, and the like are used as filler.
  • the drug can also be lyophilized.
  • Example 1 Obtaining a liquid preparation of lithicase.
  • the cultivation of the Cellulomonas cellulans strain VKPM AC-870 is carried out in a reactor / fermenter with culture medium at a temperature of 35-37 ° C for 10-24 hours until the culture reaches the stationary growth phase.
  • QOLs are separated and, by fractionation on cartridges, an extracellular biopolymer with a molecular weight in the range from 100 to 30 kD is isolated and concentrated.
  • the activity of lyticase is determined by the lysis of yeasts of the genus Sacharomyces, and the resulting preparation with a specific activity of not less than 48.3 U / mg protein is considered suitable for subsequent use and poured into vials with the introduction of known stabilizing additives.
  • a liquid preparation in an amount containing a single dose for example, 5 ml of buffered saline (PBS), pH 7.2, containing 50 IU of lyticase
  • PBS buffered saline
  • pH 7.2 pH 7.2
  • 50 IU of lyticase a single dose
  • Example 2 Obtaining the drug in the form of a suppository.
  • the liquid preparation obtained in Example 1 is dehydrated on a spray or vacuum-drying unit, combined with fillers (confectionery fat, lanolin, paraffin, vitepsol, etc.) in an amount providing at least 10-50 UNITS of activity lithicases per suppository. So, for the preparation of 100 suppositories weighing 5 g. each, it is necessary to introduce 103.5 mg of lyticase per 500 g of excipient (with initial activity of 48.3 U / mg of protein). If suppositories contain a concical, then it is administered simultaneously with lithase according to the calculation: for one suppository - 500-1000 ATre. Further, suppositories are formed on a special installation.
  • Example 3 Obtaining the drug in the form of an ointment or gel.
  • the liquid preparation obtained in Example 1 is dehydrated on a spray or vacuum dryer, combined with an ointment (petrolatum, lanolin, etc.) or gel (methyl cellulose, sodium alginate, etc.) base in an amount that provides not less than 2-10 PIECES of lithicase activity per gram of base. So, for the preparation of an ointment (gel), it is necessary to introduce 20.7 mg of lyticase per 100 g of base (with an initial activity of at least 48.3 U / mg of protein).
  • the ointment (gel) contains contrycal, then it is administered simultaneously with lithase in accordance with the calculation: for one gram of ointment (gel) 100 - 200 ATPE. Next, the ointment (gel) is Packed in tubes or bottles.
  • mice are administered intravaginally.
  • the drug is in a volume of 20 ⁇ l.
  • mice are administered intravaginally.
  • the drug is in a volume of 20 ⁇ l.
  • mice are administered intravaginally.
  • the drug is in a volume of 20 ⁇ l.
  • mice are weighing 15-16 g are used.
  • the drug is considered harmless if all the mice remain alive for 5 days of observation and no disease is detected.
  • the drug lithicase is characterized by fungicidal activity against the test strain C. albicans N ° 40, which is an internal standard for determining the antimycotic effect of the claimed drug.
  • the fungicidal effect of lithicase was determined by the method of serial dilutions in a liquid nutrient medium (Labinskaya).
  • a suspension containing 10 5 microbial bodies in 1 ml of broth was prepared from a culture of C. albicans using a bacterial optical standard.
  • a series of dilutions of lithicase (containing from 10 to 5120 PIECES) in 2 ml meat-peptone broth was carried out, 0.2 ml of C. albicans culture was added to all tubes.
  • Two controls were carried out: control of the viability of C. albicans culture without lithicase administration and control of the microbiological purity of the enzyme containing 5120 units of lyticase without culture.
  • the fungistatic and fungicidal effect of the enzyme in relation to C. albicans was also determined in an experiment on a dense AGV medium. 10 samples of 1 ml of a lithicase solution in PBS with an initial specific activity of 10, 20, 40, 80, 160, 320, 640, 1280, 2560, 5120 IU were layered on AGV medium and kept for 5 hours at a temperature of 37 ° ⁇ to absorb liquid. PBS was added to the control sample. Thereafter, 10 ⁇ l of a suspension of C. albicans was seeded, providing growth of about 50 CFU in the control. The results were taken into account after 24 hours of incubation in a thermostat according to the percentage of viable C. albicans after treatment with the enzyme.
  • the conducted studies allow us to conclude that the patented method of treating mycotic infections using lithicase, a lytic exoenzyme of the strain Cellulomonas cellulans VKPM AC-870, with a specific activity of at least 48.3 U / mg of protein contained in extracellular fractions of a biopolymer with a molecular mass in the range from 100 to 30 kDa and selectively destroying the main structure of the cell wall of DPG, contribute to the complete rehabilitation of the infectious focus. Moreover, the elimination of the pathogen from the vagina is so effective that provoking a relapse of the infection becomes impossible.
  • the drug is based on the strain Cellulomonas cellulans VKPM AC-870, deposited in the All-Russian collection of industrial microorganisms FSUE State Research Institute of Genetics, can be obtained through traditional technologies for microbiological and pharmaceutical industry, equipment and equipment.

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Veterinary Medicine (AREA)
  • Chemical & Material Sciences (AREA)
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  • General Health & Medical Sciences (AREA)
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  • Bioinformatics & Cheminformatics (AREA)
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  • Gastroenterology & Hepatology (AREA)
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  • Proteomics, Peptides & Aminoacids (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
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Abstract

Pour traiter des infections mycosiques à récidives on propose d'utiliser un exoferment lytique de la souche Cellulomas cellulans classification VKPM AS-870 possédant une masse moléculaire entre 100 et 30 kD et une activité spécifique d'au moins 48,3 U/mg de protéine, qui se présente comme une lyticase ainsi qu'un moyen de son application locale sur sa base. L'exoferment lytique a une action spécifique directement sur Candida albicans sans supprimer la microflore normale grâce à son action tant sur la couche mannane de la paroi cellulaire de Candida albicans que sur la couche fibrillaire, laquelle action est suivie par le détachement de cette dernière.
PCT/RU2010/000492 2010-09-08 2010-09-08 Procédé de traitement des infections mycosiques et produit pour sa mise en oeuvre WO2012033427A1 (fr)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4062941A (en) * 1975-06-11 1977-12-13 G. D. Searle & Co. Ltd. Method for treating fungal infections using cell lytic enzymes
RU2152398C1 (ru) * 1994-11-08 2000-07-10 Глаксо Вэллкам С.А. Производные сордарина и обладающий противогрибковой активностью фармацевтический состав на их основе
RU2262948C1 (ru) * 2004-04-12 2005-10-27 Иркутский институт химии им. А.Е. Фаворского Сибирского отделения Российской академии наук (ИрИХ СО РАН) Способ лечения кандидозного вульвовагинита у беременных

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4062941A (en) * 1975-06-11 1977-12-13 G. D. Searle & Co. Ltd. Method for treating fungal infections using cell lytic enzymes
RU2152398C1 (ru) * 1994-11-08 2000-07-10 Глаксо Вэллкам С.А. Производные сордарина и обладающий противогрибковой активностью фармацевтический состав на их основе
RU2262948C1 (ru) * 2004-04-12 2005-10-27 Иркутский институт химии им. А.Е. Фаворского Сибирского отделения Российской академии наук (ИрИХ СО РАН) Способ лечения кандидозного вульвовагинита у беременных

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
KRAVTSOV E.G. ET AL.: "Izuchenie antimikoticheskoy aktivnosti litikazy", BYULLETEN EKSPERIMENTALNOY BIOLOGII I MEDITSINY, no. 9, 2009, pages 180 - 183, Retrieved from the Internet <URL:http://www.fesmu.ru/elib/Article.aspx?id=207230> [retrieved on 20110511] *

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