JUGLONE COMPLEXES OBTAINED WITH p-CYCLODEXTRIN

Technical Field of the Invention

The present invention relates to antibacterial/antifungal products containing a juglone/p-cyclodextrin inclusion complex. In particular, the present invention relates to a drug containing the juglone/p-cyclodextrin inclusion complex. The present invention also relates to a surface coating material comprising the juglone/p-cyclodextrin inclusion complex.

Background Art

Bacterial/fungal infections are a global problem that causes serious diseases all over world, which are becoming increasingly difficult to treat due to the increasing number of antibiotic-resistant species. There is a steady decrease in the number of new antibiotics that are being developed and approved over the past decade. Therefore, there is a need to provide new materials having antibacterial/antifungal mechanisms different than those of the traditional antibiotics. For this purpose, it has become important to re-evaluate existing antibiotics or antimicrobial agents by using them in combination with different carrier systems and combinational therapies.

As a result of an evaluation of molecules such as secondary metabolites, which are derived from natural sources and known for their antimicrobial properties, in combination with new generation carrier systems in the prevention and/or treatment of bacterial/fungal diseases, it has been found that the effectiveness of these compounds is increased (Arasoglu et al., Journal of applied microbiology, 2017. 123(6): p. 1407-1419, DOI: 10.1111/jam.13601; Arasoglu et al., Journal of agricultural and food chemistry, 2016. 64(38): p. 7087-7094, DOI: 10.1021/acs.jafc.6b03309; Gomes et al., Journal of Food Science, 2011. 76(2): p. N16-N24, DOI: 10.1111/j.l750-3841.2010.01985.x; Toti et al., Biomaterials, 2011. 32(27): p. 6606-6613, 10.1016/j.biomaterials.2011.05.038; Abdollahi and Lotfipour, Biomed. Int, 2012. 3(1), 1-11; and Misra et al., Nanomedicine, 2009. 4(5): p. 519-530, DOI: 10.2217/nnm.09.28).

A rapid increase in the use of antibiotics in recent years results in the selection of antibiotic-resistant microorganisms. Therefore, it is now attempted to solve the resistance problem by using many new methods/techniques, including the discovery of new antibiotics for the elimination of all microorganisms that cause infection, especially bacteria/fungi that develop resistance. Particularly, the bacteriophage therapy, combinational antimicrobial chemotherapy, use of vaccines with antibiotics, alternative medicine approaches, and plant-based antibiotic therapies aimed at increasing the biological effectiveness of the existing antimicrobial agents, and nanotechnological/molecular/biotechnological approaches aimed at increasing the bioavailability of the antimicrobial agents have been widely increased in recent years. The use of nano-sized carrier systems such as conjugation with linear polymers, polymeric nanoparticles, metallic nanoparticles, and inclusion complexes for this purpose has allowed the re- evaluation of plant-based secondary metabolites having antimicrobial properties. One of them, juglone, an amber-colored pigment that is available in the roots, leaves, stems and barks of walnut trees, is the main compound responsible for the biological activity of walnut. Herbal preparations of walnut are often used in traditional medicine in the treatment of fungal infections, bacterial, viral, and helminthic infections, hypoglycemia, hypotension, diarrhea, ringworm and even cancer.

The use of the juglone molecule, known for its antibacterial/antifungal properties, is highly limited due to its low water solubility. Therefore, it is very important to obtain a higher biological activity at lower concentrations by increasing the water solubility of juglone. The lowest concentration of a chemical/drug that prevents the apparent growth of microorganisms has been named as the "minimum inhibition concentration" ("MIC"). As a result of antimicrobial studies, the MIC values being quite high due to low water solubility highlights the need to increase the effectiveness of the respective agent with new formulations. The MIC value of the free juglone molecule on S. aureus ATCC6538 strain is high, being 37.5 pg/mL. (Wu et al., "Inhibition effect of juglong on several food deterioration microorganisms." China Brew 8 (2009): 76-78).

Juglone (5-hydroxy-l,4-naphthoquinone) is an allelochemical. It has antimicrobial properties; but factors such as low solubility in water and low stability limit the possibility to benefit from these properties of juglone. There are studies on the encapsulation of juglone into polymeric nanoparticles to improve its antimicrobial effects (Arasoglu et al., Journal of applied microbiology, 2017. 123(6): p. 1407- 1419, DOI: 10.1111/jam.13601; Arasoglu et al., Journal of agricultural and food chemistry, 2016. 64(38): p. 7087-7094, DOI: 10.1021/acs.jafc.6b03309).

Juglone is an allelochemical that belongs to the naphtoquinone subgroup of quinones and is a secondary metabolite produced by the walnut tree Juglandaceae). (Strugstad and Despotovski, Journal of Ecosystems and Management, v. 13, n. 3, jan. 2013. Available at: <https://jem- online.org/forrex/index.php/jem/article/view/119/473> Date accessed: 25 may 2020). It is reported that juglone has anticancer (Xu et al., Bioorganic 8i medicinal chemistry letters, Volume 23, Issue 12, 15 June 2013, Pages 3631-3634, 10.1016/j.bmcl.2013.04.007; Zakavi et al., Antibacterial effect of Juglansregia bark against oral pathologic bacteria, DOI: 10.1155/2013/854765), cytotoxic and genotoxic (Kiran et al., Cell biology international, 2009, Cell Biology International, 33: 1039-1049, DOI:10.1016/j.cellbi.2009.06.018; Paulsen and Ljungman, Toxicology and applied pharmacology, 2005. 209(1): p. 1-9, DOI: 10.1016/j.taap.2005.03.005), antioxidant (Zakavi et al., DOI: 10.1155/2013/854765; Chobot and Hadacek, J Chem Ecol 35, 383-390 (2009). DOI: 10.1007/S10886-009-9609-5; Yigit et al., Turk Mikrobiyol Cem Derg, 2009, Vol 39, Issue 1-2, P: 007-011; Tan et al., "Synthesis and antibacterial activity of juglone derivatives", J. Chem. Chem. Eng. 6 (2012) 84-89), antiviral (Montenegro et al., Chemico-biological interactions, 2010. 184(3): p. 439-448, DOI: 10.1016/j.cbi.2010.01.041), antibacterial (Xu et al., Bioorganic 8i medicinal chemistry letters, 2013; Zakavi et al., DOI: 10.1155/2013/854765; Tan et al., "Synthesis and antibacterial activity of juglone derivatives", J. Chem. Chem. Eng. 6 (2012) 84-89; Dama et al., Journal of Ecotoxicology 8i Environmental Monitoring, 1998. 8(3): p. 213-215; Albayrak, A., Ceviz Yaprak Ozutleri ve Juglonun Bazi Mikroorganizmalar Uzerine Etkisi, Biyoloji Ana Bilim Dali. 2006, Dumlupmar Universitesi: Kutahya. p. 47; Oliveira et al., Food and Chemical Toxicology, 2008. 46(7): p. 2326-2331, DOI: 10.1016/j.fct.2008.03.017; Clark et al., Phytotherapy Research, 1990. 4(1): p. 11-14, DOI: 10.1002/ptr.2650040104) and antifungal (Clark et al., Phytotherapy Research, 1990. 4(1): p. 11-14, DOI: 10.1002/ptr.2650040104; Bonjar et al., Journal of Biological Sciences, 2004. 4(3): p. 405-412, DOI: 10.3923/jbs.2004.405.412; Bonjar, J Biol Sci, 2004. 4 : p. 212-5, DOI: 10.3923/jbs.2004.212.215; Nahrstedt et al., Planta Medica (Germany, FR), 1981 Aug;42(4):313-32, PMID: 7280092; Sharma et al., Analytical Letters, 2009. 42(16): p. 2592-2609, DOI: 10.1080/00032710903202055) effects. However, its application and bioavailability are limited due to its low water solubility resulting from its hydrophobic character.

Due to its low solubility in water, the solubility problem of the juglone molecule may be overcome by using organic solvents such as ethanol, methanol, acetone, ethylacetate that are known to be toxic effects, but with an inevitable increase in toxicity.

As an alternative to organic solvents, there are studies in the literature in which juglone is evaluated with various carrier systems in order to improve its properties such as increased water solubility, cytotoxicity, and chemical instability. In the state of art, there are three different studies on the solution of the low water solubility problem of juglone and including a carrier system: PLGA (Arasoglu et al., Journal of applied microbiology, 2017. 123(6): p. 1407-1419; Arasoglu et al., Journal of agricultural and food chemistry, 2016. 64(38): p. 7087-7094), wherein a micellar system of a Poloxamer 188/phospholipid mixture (Jin et al., International journal of pharmaceutics, 2016. 515(1-2): p. 359-366, DOI: 10.1016/j.ijpharm.2016.10.027) is used. In order to provide a controlled release, spherical and amorphous nanoparticles were produced in both systems.

For the reasons listed above, there is still a need to provide an antimicrobial drug that has high solubility and high stability against changes in temperature and pH.

Objects of the Invention

The main object of the invention is to provide solutions to the problems mentioned in the prior art.

Another object of the invention is to provide an antimicrobial agent having enhanced efficiency.

Another object of the invention is to provide a juglone-containing drug with increased solubility in water, and improved stability against pH changes and temperature.

Another object of the invention is to provide a surface coating material containing an antimicrobial agent having enhanced efficiency.

Summary of the Invention

The present invention provides an antibacterial/antifungal drug containing a juglone/p-cyclodextrin inclusion complex. With the said complex form, the solubility of the juglone in water, its stability against temperature and pH changes are improved. In the said complex, juglone provides antibacterial/antifungal activity. This results in the biological activity of the complex produced. Beta cyclodextrin (0-CD) acts as a carrier to increase the solubility of the biologically active molecule in water. This is provided by -OH groups in the structure of 0-CD.

Detailed Description of the Invention

In the scope of the present application, a crystalline inclusion complex using cyclodextrin is provided. The controlled release mechanism of the said complex is different from the other two carriers in the literature.

Betacyclodextrins are widely used in the fields of agriculture, health, chemical and environmental engineering. A molecular encapsulation with beta-cyclodextrins, which can be used as an effective solubilizer, stabilization enhancer, taste masking agent, and is available in a full range of dosage forms from solid to liquid, is an accepted and useful means for pharmaceutical formulators with many pharmaceutical products containing cyclodextrin complexes in the market (He et al., Colloids and Surfaces B: Biointerfaces, 2013. 103: p. 580- 585, DOI: 10.1016/j.colsurfb.2012.10.062; Nicolescu et al., Preparation and Characterization of Inclusion Complexes Between Repaglinide And 0-Cyclodextrin, 2-Hydroxypropyl- p-Cyclodextrin and Randomly Methylated 0-Cyclodextrin, Farmacia, 2010. 58(1): p. 78-88; Choi et al., Carbohydrate Polymers, 2017. 163: p. 118-128, DOI: 10.1016/j.carbpol.2017.01.073; Chaudhary and Patel, 2013, Cyclodextrin inclusion complex to enhance solubility of poorly water soluble drugs: A review, International Journal of Pharmaceutical Sciences and Research, 4(1), p.68. -76; Li et al., Food Chemistry, 2017. 221: p. 296-301, DOI:

10.1016/j.foodchem.2016.10.040; Yang et al., Asian Journal of Pharmaceutical Sciences, 2017. 12(2): p. 187-192, DOI: 10.1016/j.ajps.2016.08.009; Carneiro et al., International journal of molecular sciences, 2019. 20(3): p. 642, DOI: 10.3390/ijms20030642; and httDs://www.roauette.com/Dharma-and- nutraceuticals-beta-cvclodextrin). In the state of art, no study is available referring to any complexes of 0- cyclodextrin with juglone molecule or their inclusion complexes.

In the study being the subject of the present application, many properties of the juglone molecule (such as increased solubility or dispersibility in water, increased stability against temperature and pH changes) have been improved by forming an inclusion complex with 0-cyclodextrin (0-CD) molecule. In the systems available in the literature, the solubility of the juglone molecule loaded into spherical nanoparticles by encapsulation could only be increased to a limited extent compared to that of the improvement of the present application.

Cyclodextrins (CDs) of the present application are generally used as active agent carriers to increase the solubility of the water-insoluble active ingredients. CDs are widely preferred host molecules for molecular encapsulation by forming inclusion complexes of low molecular weight hydrophobic molecules (guest molecules). Non- covalent interactions change the physical, chemical, and biochemical properties of encapsulated molecules (He et al., Colloids and Surfaces B: Biointerfaces, 2013. 103: p. 580- 585, DOI: 10.1016/j.colsurfb.2012.10.062; Nicolescu et al., Preparation and Characterization of Inclusion Complexes Between Repaglinide And 0-Cyclodextrin, 2-Hydroxypropyl-0-Cyclodextrin and Randomly Methylated 0- Cyclodextrin, Farmacia, 2010. 58(1): p. 78-88; Choi et al., Carbohydrate Polymers, 2017. 163: p. 118-128, DOI: 10.1016/j.carbpol.2017.01.073).

CD inclusion complexes have numerous advantages, such as increased stability (against pH, oxygen, sunlight, thermal and storage), increased solubility and bioavailability, controlled release of the encapsulated molecule and reduced toxicity. 0-cyclodextrin is preferred over other cyclodextrin types (alpha or gamma) in host-guest type inclusion complexes due to the easy of accessibility and low cost techniques (Chaudhary and Patel, 2013, Cyclodextrin inclusion complex to enhance solubility of poorly water soluble drugs: A review, International Journal of Pharmaceutical Sciences and Research, 4(1), p.68. -76; Li et al., Food Chemistry, 2017. 221: p. 296-301, DOI: 10.1016/j.foodchem.2016.10.040; Yang et al., Asian Journal of Pharmaceutical Sciences, 2017. 12(2): p. 187-192, DOI: 10.1016/j.ajps.2016.08.009).

In the literature, in order to avoid the water solubility problem of the juglone molecule, the encapsulation of the juglone molecule into the nanoparticles formed with a PLGA polymer, i.e., encapsulation of the juglone molecule into a carrier polymeric system was previously carried out (Arasoglu et al., Journal of applied microbiology, 2017. 123(6): p. 1407-1419, DOI: 10.1111/jam.l3601; Arasoglu et al., Journal of agricultural and food chemistry, 2016. 64(38): p. 7087-7094, DOI: 10.1021/acs.jafc.6b03309). However, although the water solubility of the juglone- loaded nanoparticles produced by the study in the said references is higher than the free juglone, the water solubility of the resulting system is still limited and the MIC values are lower than the free juglone in the said study, but higher than the complex of the present application (Table 1).

Yet, the water solubility values achieved in the jug lone- Betacyclodextrin complex which is the subject of the present application are surprisingly improved in the relevant technical field. This improvement in solubility is reflected in the MIC values, and the MIC values of the complex of the present application are extremely low as seen in Table 1. For comparison, the MIC values of the free juglone and juglone loaded into PLGA nanoparticles are taken from the literature. In the three cases, the antimicrobial activity of juglone was tested on E. coli, S.aureus and C. albicans.

MBC (minimum bactericidal concentration) values of the free juglone and juglone provided in the form of a 0-CD inclusion complex are presented in Table 2 below.

Table 1. MIC values of the systems formed by loading the juglone molecule into different carriers. MIC values (pg/mL)

Microorganism: E. coii S. aureus C. albicans

Juglone* 62.5 17.5 62.5 p-CD inclusion complex <5.2 <5.2 <5.2

Juglone loaded PLGA nanoparticles* 62.5 17.5 31.25

*: (Arasoglu et al., Journal of applied microbiology, 2017. 123(6): p. 1407- 1419, DOI: 10.1111/jam.13601; Arasogiu et a!., Journal of agricultural and food chemistry, 2016. 64(38): p. 7087-7094, DOI: 10.1021/acs.jafc.6b03309)

Table 2. MBC values of the juglone molecule in free state and in p-CD inclusion complex.

MBC values (pg/mL)

Microorganism: E. coii S. aureus C. albicans

Juglone 10.4 10.4 10.4 p-CD inclusion complex <5.2 <5.2 <5.2

The juglone/p-cyclodextrin inclusion complex which is the subject of the present application may be included in a drug as an antimicrobial ingredient. Said drug may be provided as a topical ointment and/or a gel formulation for use, e.g., in the treatment of local infections.

In addition, the juglone/p-cyclodextrin inclusion complex can also find non-medical uses. For example, the juglone/p-cyclodextrin inclusion complex may be included in a polymeric architectural coating material (e.g., paint formulation) for floors and/or walls; it may provide an antimicrobial surface on floors and walls where such a material is applied (e.g., hospital floors and walls).

Thus, the present invention provides a juglone/p-cyclodextrin inclusion complex, a formulation containing same, and a drug having this formulation, preferably in the form of an ointment or a gel. Alternatively, the present invention provides antibacterial paint formulations having said formulation. It is clear that a chemist specialized in pharmaceutics or antimicrobial construction paints, obtaining the knowledge from the present application that the juglone/p-cyclodextrin inclusion complex has high water solubility and high biological activity, will have no difficulty in designing the respective formulations for the intended use. In the context of present invention, since the solubility of juglone in water is improved, it is preferred that each of the said formulations are aqueous. A chemist with knowledge and experience in preparing inclusion complexes will have no difficulty in preparing the juglone/p-cyclodextrin inclusion complex suitable for use in the said formulations.

In the light of the above, the present application provides the following:

- A juglone/p-cyclodextrin inclusion complex;

- A formulation containing the juglone/p-cyclodextrin inclusion complex; said formulation preferably containing water;

- In one embodiment of the invention, said formulation is an antibacterial or antifungal drug formulation; said formulation is preferably in the form of an ointment or a gel for topical application;

- In an alternative embodiment of the invention, said formulation is an antibacterial and/or antifungal paint formulation for hospital floors or walls; said formulation preferably containing water.