RU2802388C2 - Method for treatment of fungal ulcer of cornea - Google Patents

Abstract

FIELD: medicine; ophthalmology.

SUBSTANCE: eye drops are instilled, while instillation of a terbinafine solution diluted to 100 mcg/ml (0.1%) is performed, 1 drop into the conjunctival cavity of the affected eye 4 times a day with monitoring of microflora on days 1, 2 and 3 from the start of treatment to clinical recovery and laboratory sanitation.

EFFECT: method makes it possible to achieve relief of the infection, which is confirmed by the cultural and cytological methods of testing, accelerate epithelialization, low risk of stromal opacification due to rapid sanitation and epithelization, and clinical recovery.

1 cl, 1 ex, 3 dwg

Description

The invention relates to medicine, namely ophthalmology, and is intended for the treatment of fungal ulcers under experimental conditions.

There are known methods of conservative treatment of herpetic keratitis: Method of treatment of herpetic keratitis (Patent RU No. 2328252 C1, IPC A61F 9/00, A61K 31/522, A61K 36/00, A61P 27/02 - 07/10/2008, Bulletin No. 19) combined with secondary immunodeficiency, by local administration of acyclovir and intravenous drip administration of an acyclovir solution of 250 mg 3 times a day, characterized in that the acyclovir solution is administered under the conjunctiva of the lower fornix in a dose of 1.0 ml 1 time per day for 10 days, intravenous administration is carried out also for 10 days, Immunomax 1.0 ml is administered intramuscularly 1 time per day on days 1, 2, 3, 8, 9, 10 of treatment.

A method for the treatment of herpetic keratitis with ulceration (Patent RU No. 2192898 C1, IPC A61N 1/30, 2/00 - 20.11.2002, Bulletin No. 19), including local administration of Oftalmoferon eye drops at a concentration of 20,000 IU/ml in the form of magnetophoresis using low-frequency alternating magnetic field 1 time per day until complete epithelization of the cornea.

A method of treating herpetic keratitis (Patent UA 46642 A 05.15.2002) involves instilling antibacterial and aviral substances into the eyes along with subalin as an inducer of endogenous interferon. Subalin is diluted in physiological solution before use.

The common disadvantage of the above methods in this context is that they are not intended for fungal keratitis.

There is a known method of treating deep stromal keratitis (Patent RU No. 2440801 C1, IPC A61F 9/00, A61K 35/14, A61P 27/00 - 01/27/2012, Bulletin No. 3) by administering autologous blood mononuclear cells (AM) against the background of traditional pharmacotherapy , including specific antiviral and/or antibacterial drugs, non-steroidal anti-inflammatory and antiallergic drugs, as well as keratoprotectors, characterized in that after local anesthesia, a cell suspension of AM in a volume of 0.5 ml is injected into the thickness of the corneal stroma of the diseased eye around the pathological focus immediately after it preparation, for which AM is isolated from the patient’s heparinized blood by fractionation in a density gradient using a ficoll-verografin separating solution with a ratio of the volumes of the gradient and the separated suspension of 1:3-1:4, at 2000 rpm, at room temperature for 15 minutes, add 1.0 ml of isotonic sodium chloride solution and the resulting suspension is centrifuged again for 7 minutes at 1500 rpm, then the resulting cell suspension is placed in a sterile vial with the addition of 3.0 ml of isotonic sodium chloride solution, and during the treatment the procedure is repeated 1 -2 times with an interval of 4-6 days, depending on the severity of the disease and the dynamics of the clinical picture.

The disadvantages of this method of treatment are the technical complexity of use in the form of intrastromal injections, the dosage form is not stable, and there is no information about the effectiveness of fungal keratitis.

There are currently no standardized local treatment methods for fungal keratitis in the Russian Federation due to the lack of licensed commercial antifungal ophthalmic solutions. Today, the treatment of fungal eye infections is carried out using non-standard off-label methods, which includes installation of a solution of fluconazole for intravenous administration, diluted lyophilisates of voriconazole, amphotericin, instillation of dissolved crushed nystatin tablets, oral and parenteral administration of antifungal drugs into the affected eye.

In countries such as the United States of America, developed countries of Western Europe, and India, the commercial drug Natacyn (in the form of eye drops, 5% natamycin suspension) and its analogues (Fungal Keratitis - Europe. October, 2016. Accessed 08/10/21) are available. https://www.aao.org/topic-detail/fungal-keratitis-europe#disqus_thread), (Guidelines for the management of Fungal keratitis. Accessed 08/01/21. https://kipdf.com/guidelines-for-the -manage-ment-of-fungal-keratitis_5acebde17f8b9a6b478b460c.html). However, this drug is not licensed or certified for use in the Russian Federation.

According to the official instructions for the substance natamycin, most pathogenic yeast fungi are sensitive to it, especially Candida albicans, some protozoa (T. vaginalis) (Natamycin description. Access date 10.10.21. https://www.vidal.ru/drugs /molecule/1466). However, the spectrum of antifungal action of Natacyn 5% eye drops, judging by the instructions, is much wider and includes fungi of the genus Candida, Aspergillus, Cephalosporium, Fusarium and Penicillium (NATACYN® (natamycin ophthalmic suspension) 5% Sterile. Accessed 10.10.21. https:// www.novartis.us/sites/www.no-vartis.us/files/natacyn.pdf). The most relevant pathogens of fungal keratitis are Fusarium spp., Candida [Samoilov A.N., Davletshina N.I. Analysis of the etiology and antimicrobial sensitivity of fungal keratitis pathogens in a series of clinical cases. Ophthalmosurgery. 2020; (1):71-76.], and changes in the virulence of fungal agents leads to the need to select antifungal therapy taking into account possible resistance.

Methods for treating fungal keratitis based on the substance natamycin are being studied: “Gel therapeutic film with natamycin based on alginic acid and its preparation method” (priority CN202110386922.0A - 2021-06-04, Affiliated Hospital of Qingdao University), “Natamycin eye drops modified with oxidized sodium alginate, and a method for their preparation" (priority CN202110386927.3A - 2021-07-09, Affiliated Hospital of Qingdao University), "Natamycin eye drops modified with chondroitin sulfate oxide, and a method for their preparation" (priority CN202110239339.7A - 2021-06- 15, Affiliated Hospital of Qingdao University), “Contact lenses with natamycin nanoparticles and the method for their production” (Priority CN202110093319.3 A - 2021-06-04, Weifang Medical University).

Applications for patent inventions for the treatment of fungal keratitis using alternative methods have been published: “Dimethyl fumarate eye drops, a method for their preparation and the use of dimethyl fumarate eye drops for the treatment of fungal keratitis” (priority CN202110695312.9A - 07/23/2021), “Cladinostat fibrous membrane from oxidized alginic acid and method obtaining it" (priority CN202110386905.7A - 2021-07-06, affiliated hospital of Qingdao University), "Corneal contact lenses with carrageenan containing voriconazole, and the method for their preparation" (priority CN202110249461.2A - 2021 -06-22, affiliated hospital Qingdao University), “Method for producing polyquercetin through enzymatic catalysis and the use of polyquercetin” (priority CN202110232800.6A - 2021-06-22 Affiliated Hospital of Qingdao University), “Antifungal eye drops and method of their preparation” (priority CN202110168153.7A - 2021- 05 -04 Henan Provincial People's Hospital), "The use of thymol in the preparation of drugs for the treatment of fungal keratitis" (priority CN202011167133.HA - 2021-01-26 Affiliated Hospital of Qingdao University), "The use of mouse lacrimal gland secretory protein in the preparation of drugs for the treatment of fungal keratitis "(priority CN202010929166.7A - 2020-12-11 Affiliated Hospital of Qingdao University).

The main advantage of the above methods lies in the microbial sanitation and clinical effectiveness declared by the authors. The main unifying drawback of the proposed methods is the lack of stable ophthalmic dosage forms and scant information on the antifungal activity of these substances.

The objective of the proposed method is to create an effective and safe antifungal drug for topical use in ophthalmology.

The technical result of the claimed method is the sanitation of the infectious focus, confirmed by cultural and cytoscopic research methods, clinical recovery from fungal keratouveitis caused by Fusarium spp., which is the main endemic etiological factor of keratomycosis.

The technical result of the claimed method is achieved due to the fact that as a result of instillation of a terbinafine solution onto the cornea affected by sensitive fungal agents, the infection is cleared, confirmed by cultural and cytoscopic studies, accelerated epithelization with a minimal risk of cloud formation and clinical recovery.

The advantage provided by the above set of symptoms is clinical recovery, potential availability of the drug, and broad antifungal activity.

Details, features, and advantages of the present invention can be seen from the following description of the implementation of the claimed technical solution using the figures that show:

Fig. 1 - Keratouveitis with corneal ulceration in a rabbit as a result of experimental infection of the rabbit cornea with a clinical suspension of fungal isolates of Fusarium spp.

Fig. 2 - View of the cornea of an experimental animal on the 3rd day of treatment.

Fig. 3 - The dynamics of the decrease in colonies of microorganisms at the peak of infection (top left), after the first day of treatment (top right) and by the end of the third day of treatment (bottom).

The method is carried out as follows.

First, fungal keratitis is reproduced.

Next, fungal keratouveitis is treated, which includes instillation of a 0.1% solution of terbinafine, 1 drop, into the conjunctival cavity of the affected eye 4 times a day under the control of microflora from the beginning of treatment to clinical recovery and laboratory sanitation.

The presence of microflora was monitored by cultural and cytological methods. A smear for cultural examination was taken from the conjunctiva of the eye under study immediately before infection, then when the corneal ulcer was reached (in different rabbits 14-16 days from the start of infection), then on the 1st, 2nd, 3rd day from the start of treatment. Under local installation anesthesia with a 0.1% solution of oxybuprocaine, the material collected with a sterile swab was placed in a neutral transport medium and on the same day inoculation was carried out on Sabouraud's nutrient medium in the mycological laboratory. Corneal scrapings for cytological examination were also taken when the corneal ulcer was reached, then on the 1st, 2nd, 3rd day from the start of treatment. Immediately before infection, an imprint smear was taken from the cornea to avoid deepithelialization, corneal trauma, and deliberate creation of an entry gate for future infection. The dynamics of cultivation on a nutrient medium is shown in Fig. 3. (from left to right): before the start of treatment, exponential growth of airy flocculate-felt colonies of grayish-white color; on the third day there was a sharp decrease in the number of colonies; on the third day the nutrient medium turned out to be sterile, which correlated with the clinical picture of the ulcer.

Terbinafine was selected based on the specific sensitivity of fungi in cases of fungal keratitis in patients [Samoilov A.N., Davletshina N.I. Analysis of the etiology and antimicrobial sensitivity of fungal keratitis pathogens in a series of clinical cases. Ophthalmosurgery. 2020; (1):71-76]. Terbinafine is effective against fungi that cause diseases of the skin, hair and nails, incl. dermatophytes such as Trichophyton (for example, T. rubrum, T. mentagrophytes, T. verrucosum, T. tonsurans, T. violaceum), Microsporum (for example, M. canis), Epidermophyton floccosum, as well as yeast fungi of the genus Candida (for example, C. albicans) and Pityrosporum. In low concentrations, terbinafine has a fungicidal effect against dermatophytes, molds and some dimorphic fungi. Activity against yeast fungi, depending on their type, can be fungicidal or fungistatic. [Terbinafine (Terbinafinum). Date of appeal - 10.10.2021. https://www.rlsnet.ru/mnn_index_id_1475.htm].

During the experiment, it turned out that the maximum possible soluble concentration of the substance is 100 μg/ml (0.1%), which was taken as the basis for the study. In ophthalmic practice, terbinafine is not widely used due to the lack of an ophthalmic form of the drug, and possibly a solution for intravenous administration. However, a number of researchers note the high sensitivity of pathogenic fungi that cause keratitis and corneal ulcers to terbinafine.

The invention is aimed at eliminating the following disadvantages: the lack of licensed commercial antifungal ophthalmic solutions, reliably effective and safe treatment of fungal keratitis, ulcers and/or keratouveitis of fungal etiology, a wide antifungal spectrum of activity.

In total, the experiment was carried out on 6 rabbits, 12 eyes.

The method is illustrated by the following experimental example.

Example 1.

Cornea of a rabbit (Fig. 1), in which fungal hypopyon-keratouveitis was modeled.

Bacteriological and mycological culture of a smear of conjunctival cavity discharge taken on the day of completion of infection revealed the growth of Fusarium spp. According to cystoscopy of corneal scraping, active elements of Fusarium spp were detected.

On the 3rd day from the start of instillation of a 0.1% terbinafine solution, the hypopyon was stopped, the infiltrate was resorbed, the ulcer was epithelialized, and a slight clouding of the corneal stroma was formed (Fig. 2). Bacteriological and mycological culture of a smear of discharge from the conjunctival cavity taken on this day did not reveal any growth. Cytoscopy of corneal scrapings also did not reveal any living fungal elements.

A cultural study was carried out immediately before the start of instillations, on the first and third days of treatment (Fig. 3).

The use of terbinafine makes it possible to completely sanitize the conjunctival cavity of experimental animals with fungal keratitis, which is confirmed by cultural data.

Thus, the claimed method allows you to sanitize the fungus from the corneal infiltrate, stop inflammation of the layers of the cornea, prevent deep ulceration and perforation of the cornea, and achieve clinical recovery.

Claims (1)

A method of treating a fungal ulcer of the cornea, including instillation of eye drops, characterized in that instillation of a terbinafine solution diluted to 100 mcg/ml (0.1%), 1 drop into the conjunctival cavity of the affected eye 4 times a day under the control of microflora for 1 1st, 2nd and 3rd days from the start of treatment to clinical recovery and laboratory sanitation.