Terpinolene as a potentiator of the action of conventional antifungal drugs.

Abstract

Terpinolene as a potentiator of the action of conventional antifungal drugs. The present invention describes a process for obtaining antifungal products for the control of dermatophytosis in humans and animals, composed of the natural product terpinolene and various combinations of terpinolene and terbinafine. Dermatophytosis is one of the most prevalent mycoses worldwide. Currently, a high rate of fungal resistance is reported, justifying the search for natural products that can act as new modulating agents of the action of conventional antifungals. Thus, the association of terpinolene with terbinafine can lead to a reduction in their effective concentrations for inhibiting fungal growth and treatment. Therefore, the present invention offers an innovative alternative of a natural product that exhibits antifungal activity in its isolated form and synergistic activity when associated with terbinafine at subinhibitory concentrations.

Description

Terpinolene as a potentiator of the action of conventional antifungal drugs. Field of invention

[001] The present invention relates to a monoterpene (terpinolene) with antifungal activity and potentiating the action of conventional antifungals. More specifically, the present invention relates to a natural product with innovative action, which has antifungal properties, aiming at its use for the control of dermatophytoses in humans and animals, caused especially by Microsporum and Trichophyton. Background of the invention

[002] Dermatophytoses are infections that affect keratinized tissues in humans and animals. Although the diversity found in the frequency of dermatophyte species in Brazil is evidenced in several studies, epidemiological records indicate the predominance of Microsporum canis and Microsporum gypseum species among the main etiological agents of tinea capitis and tinea corporis in children, mainly (Havlickova and Czaika. Mycoses, 2008).

[003] Microsporum canis is one of the main pathogens causing tinea capitis worldwide, generally associated with scalp lesions in children under ten years of age, but an increase in tinea capitis is also observed in adults and the elderly. Onychomycosis caused by dermatophytes is generally caused by Trichophyton spp; however, Petition 870190026589, dated 03 / 20 / 2019, page 10 / 21 Two out of ten cases have been reported as secondary to M. canis, primarily in patients with HIV / AIDS (Yin et al., Mycopathologia, 2013).

[004] Some agents stand out in clinical use for the treatment of dermatophytoses, among which are imidazole drugs, triazoles, griseofulvin, and terbinafine. Terbinafine is a synthetic antifungal drug from the allylamine group that has the ability to specifically interfere with the initial step in the biosynthesis of fungal sterols, decreasing growth and resulting in fungal death (Martins et al. Journal of Medical Microbiology, 2016). The antifungal drugs available on the market are grouped into a few chemical classes and often have a restricted spectrum of action. In addition, the success of treatment with antifungals can be compromised because dermatophytoses are generally associated with high recurrence rates and drug resistance.

[005] The treatment of dermatophytoses has been a major concern worldwide. This is justified by the increased prevalence of these diseases globally and, especially, by the emergence of strains resistant to the main antifungals used in clinical therapy, such as terbinafine (Ughachukwu and Unekwe. Annals of Medical and Health Sciences Research, 2012). The reduction of drug accumulation in the intracellular environment through the expression of efflux pumps is the most described molecular mechanism of dermatophyte resistance in the literature. Increased drug efflux due to pump activation results in a small amount of drug at the site of action (Singh et al. Mycoses, 2018). Previous studies state that dermatophyte fungi exposed to antifungal drugs showed increased levels of mRNA transcription for the mdr2 gene, which also encodes an ABC transporter - ATP-binding cassette. Petition 870190026589, dated 03 / 20 / 2019, page 11 / 21 3 / 10 conferring resistance to antifungals (Fachin et al. Journal of Medical Microbiology, 2006).

[006] Given this, natural products have proven to be an important tool in the search for new antifungal drugs or as a potentiator of known antifungals (Danielli et al. Industrial Crops and Products, 2017). And, in this context, the use of monoterpenes – components of essential oils from aromatic plants – as potential antifungal agents presents itself as a promising strategy.

[007] Terpinolene (C10H16) is a monocyclic monoterpene found in essential oils of Manilla elemi, Nectranda elaiophora, and Dacrydium colensoi (Turkez et al. Cytotechnology, 2015). Its repellent activity is reported (Laznik and Trdan, Journal of Pest Science, 2016), in addition to being a chemical constituent of essential oils that possess analgesic and anti-inflammatory properties (Macedo et al. Brazilian Journal of Medical and Biological Research, 2016).

[008] In the patent literature, there is no record of a process / product similar to the one presented here. The records found that are close to the subject matter of this invention deal with the use of terpinolene as an antibacterial for everyday products (CN104706540-A), as an antibacterial in products for use in the oral cavity (JP2007099782A), or as an antibacterial against microorganisms of importance in food (KR20140075650-A). Furthermore, there are records of inventions for products containing terpinolene in their formulation with applications as a food preservative and insecticide (PE07912003-A1), as a repellent (US2004213822-A1), and as an insecticide (CN107711896-A).

[009] To date, no reports have been found regarding the application of terpinolene as an antifungal product against dermatophytes, nor Petition 870190026589, dated 03 / 20 / 2019, page 12 / 21 4 / 10 as an enhancer of antifungal action. Therefore, the object proposed in the present invention is characterized as novel. Objective of the invention

[010] The objective of the invention is to present terpinolene as a natural product with antifungal activity and potentiating the action of conventional antifungals. Summary of the invention

[011] One of the objects of the present invention is to provide an antifungal product (terpinolene) for the control of mycoses in humans and animals, especially those caused by Microsporum and Trichophyton. Preferably, terpinolene inhibits the in vitro growth of fungal strains of Microsporum canis and Trichophyton interdigitale and potentiates the action of terbinafine against these fungi. Terpinolene acts synergistically when combined with terbinafine.

[012] A further object of the present invention is to provide a process for obtaining terpinolene dilutions for antifungal activity assays, comprising the steps of: • Weighing the drugs on an analytical balance to achieve the concentrations used in the work (2048 pg / mL down to 1 pg / mL). • Solubilization of terpinolene in distilled water with the aid of the emulsifying agent dimethyl sulfoxide (DMSO) at 100%.

[013] It is an additional object of the present invention to provide a process for proving the antifungal activity and enhancing the action of Petition 870190026589, dated 03 / 20 / 2019, page 13 / 21 5 / 10 terbinafine terpinolene against strains of Microsporum canis and Trichophyton interdigitale, comprising the following steps: • Determination of the minimum inhibitory concentration (MIC) of terpinolene, terbinafine, and chlorpromazine (a pharmacological tool as an efflux pump inhibitor). • Analysis of the modulating activity of terpinolene on the action of terbinafine. • Analysis of the effects of the association between terpinolene and terbinafine.

[014] Figure 1 represents the results of the K+ ion release rate of Microsporum canis LM 216 in the absence (control) and presence of sodium dodecyl sulfate (SDS), terbinafine and terpinolene. Detailed description of the invention

[015] The present invention relates to terpinolene as a natural product with antifungal activity and potentiating the antifungal action of terbinafine.

[016] For the purpose of this invention, the following strains were used: Microsporum canis LM 216, Trichophyton interdigitale H6 (ATCC MYA-3108) and its derivative Δ mdr2. The fungal strains were initially cultured on potato dextrose agar at 28°C for 7 days. The newly formed fungal colonies were covered with sterile saline solution (0.9% NaCl). The densities of the suspensions of each strain were adjusted in a spectrophotometer at 520 nm to a transmittance value of 70-72%, which corresponds to an inoculum of approximately 0.5 - 5 x 10⁶ colony-forming units in 1 mL (CFU / mL).

[017] Solutions of terpinolene, terbinafine, and chlorpromazine should be prepared by first dissolving them in 100 µL of 100% dimethyl sulfoxide and then using sufficient sterile distilled water. Petition 870190026589, dated 03 / 20 / 2019, pp. 14 / 21 6 / 10 to obtain an initial concentration of 2048 pg / mL. From this concentration, serial dilutions should be made in a ratio of two until lower concentrations are reached using RPMI 1640 culture medium.

[018] To verify the antifungal activity of terpinolene, terbinafine, and chlorpromazine against dermatophyte strains, tests were performed to determine the minimum inhibitory concentration (MIC) and evaluate the effects on plasma membrane functionality (K+ release). To verify the activity of terpinolene as a potentiator of terbinafine action, the MIC of terbinafine was determined in the presence of subinhibitory concentrations of terpinolene. The effects of this combination of terpinolene and terbinafine were analyzed using the checkerboard technique. All tests were performed in triplicate. Example 1: Determination of the minimum inhibitory concentration (MIC)

[019] The MIC of terpinolene, terbinafine, and chlorpromazine was determined by the microdilution technique (Clinical and Laboratory Standards Institute, document M38-A, 2008). The MIC was defined as the lowest concentration of the drugs capable of visually inhibiting 100% of fungal growth. MIC values ​​are described in Table 1. Terpinolene inhibited fungal growth from a concentration of 128 pg / mL. The fungi were also sensitive to terbinafine and chlorpromazine (see Table 1).

[020] Table 1: Minimum inhibitory concentration (MIC) values ​​of terpinolene, terbinafine and chlorpromazine against dermatophytes. CIM Strains (pg / mL)* Petition 870190026589, dated 03 / 20 / 2019, pp. 15-21 7 / 10 Terpinolene Terbinafine Chlorpromazine Microsporum canis LM 216 1024 1 2 Trichophyton interdigitale H6 1024 0.5 0.5 Trichophyton interdigitale Δmdr2 128 0.5 1 *Modal values ​​from three experiments. Example 2: Study to evaluate the effects of terpinolene on the fungal membrane.

[021] The concentration of free potassium ions (K+) in the fungal inoculum of Microsporum canis (LM 216) was measured after exposure to the MICs of terpinolene and terbinafine at 28°C. After the incubation period (4h and 8h), the extracellular K+ concentration was measured by turbidimetric procedures using K+ assay kits in a UV 5100 spectrophotometer. Control experiments without the test drugs were performed similarly. Sodium dodecyl sulfate (SDS) at 2% was used as a reference compound capable of inducing K+ release (Souza et al. International Journal of Food Microbiology, 2010). All test drugs increased K+ release when compared to the control (absence of drugs) (p<0.05, unpaired t-test). Terbinafine had a similar effect to SDS (p>0.05, unpaired t-test). The results are shown in Figure 1: K+ ion release rate of Microsporum canis LM 216 in the absence (control) and presence of SDS, terbinafine, and terpinolene.These results demonstrate the antifungal potential of terpinolene by disrupting the functionality of the fungal membrane. Petition 870190026589, dated 03 / 20 / 2019, pages 16 / 21 8 / 10 Example 3: Study of the modulating effect of terpinolene on the antifungal action of terbinafine.

[022] To evaluate whether the test drugs modulate the action of antifungals against the tested strains, the method proposed by Coutinho et al. (Chemotherapy, 2008) was used. The MIC values ​​of terbinafine were determined in the absence and presence of subinhibitory concentrations of terpinolene (1 / 4 MIC) or chlorpromazine (1 / 4 MIC), using the microdilution technique. Chlorpromazine was used as a positive control, as it is an efflux pump inhibitor. The plates were sealed and incubated at 28°C for up to 7 days for reading. The assay was performed in triplicate. The MIC of terbinafine decreased in the presence of terpinolene, except for the Trichophyton interdigitale (H6) strain. Chlorpromazine selectively modulated the resistance of M. canis LM 216 and T. interdigitale H6 to terbinafine, since it is an efflux pump blocking drug. This was not evidenced with the T. strain.interdigitale Δ mdr2, possibly because it does not express genes that encode efflux pumps and confer resistance to antifungals. These results demonstrate that terpinolene increased the sensitivity of fungi to terbinafine, enabling its use as a pharmacological adjuvant in the treatment of dermatophytoses.

[23] Table 2: Minimum inhibitory concentration (MIC) values ​​of terbinafine alone and combined with terpinolene (1 / 4MIC) and chlorpromazine (1 / 4MIC) against dermatophytes. Strains CIM terbinafine CIM combined terbinafine (pg / mL) Petition 870190026589, dated 03 / 20 / 2019, pages 17 / 21 9 / 10 isolated (pg / mL) Terpinolene Chlorpromazine Microsporum canis (LM 216) 1 0.0625 0.25 Trichophyton interdigitale (H6) 0.5 0.5 0.25 Trichophyton interdigitale (Δ mdr2) 0.5 0.0625 0.5 *Modal values ​​from three experiments. Example 4: Study of the association between dihydrojasmone and terbinafine

[024] The effect of the association of terpinolene with terbinafine was determined by the checkerboard technique, using the Microsporum canis strain (LM 216). From this technique, the fractional inhibitory concentration index (FICI) was determined by the sum: CIFa + CIIFb, where A represents terpinolene and B terbinafine. CIFa = CIMa combined / CIMa alone, while CIIFb = CImb combined / CIMB alone. The FICI is interpreted as follows: synergism (< 0.5), additivity (0.5-1.0), indifference (> 1.0 and < 4.0) or antagonism (> 4.0) (Lewis et al. Antimicrobial Chemotherapy, 2002). The MIC values ​​decreased when the drugs were combined; terbinafine decreased its MIC to 0.125 pg / mL and terpinolene to 128 pg / mL. The final effect was synergistic from the combination of terpinolene and terbinafine (ICIF = 0.25), meaning that together these drugs are more potent than when used separately.These results. Petition 870190026589, dated 03 / 20 / 2019, pages 18 / 21 10 / 10 confirm the possibility of using subinhibitory concentrations of drugs for protection against mycoses, reducing drug costs, potential side effects, and the emergence of fungal resistance.

Claims

CLAIMS 1. Process and production of antifungal compositions composed of terpinolene as a potentiator of the action of conventional antifungal drugs, characterized by containing isolated terpinolene or mixtures of different concentrations of terpinolene and terbinafine capable of preventing fungal growth.

2. Process and production of antifungal compositions composed of terpinolene as a potentiator of the action of conventional antifungal drugs, according to claim 1, characterized by terpinolene at a minimum concentration of 1024 pg / mL.

3. Process and production of antifungal compositions composed of terpinolene as a potentiator of the action of conventional antifungal drugs, according to claim 1, characterized by comprising proportions of the drugs, preferably of at least 128 pg / mL of terpinolene and 0.125 pg / mL of terbinafine in the final mixture of the composition.

4. Process and production of antifungal compositions composed of terpinolene as a potentiator of the action of conventional antifungal drugs, according to claim 3, characterized by comprising the use of dimethyl sulfoxide, preferably as an emulsifying agent, respecting the final volume of the terpinolene solution in distilled water.

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