Abstract
Onychomycosis is a fungal infection of the fingernails or toenails caused by dermatophytes, nondermatophytes, moulds, and yeasts. This condition affects around 10–30% people worldwide, negatively influencing patients’ quality of life, with severe outcomes in some cases. Since the nail unit acts as a barrier to exogenous substances, its physiological features hampers drug penetration, turning the onychomycosis treatment a challenge. Currently, there are several oral and topical therapies available; nevertheless, cure rates are still low and relapse rates achieves 10–53%. Also, serious side effects may be developed due to long-term treatment. In light of these facts, researchers have focused on improving topical treatments, either by modifying the vehicle or by using some physical technique to improve drug delivery trough the nail plate, hence increasing therapy effectiveness. Therefore, the aim of this paper is to explain these novel alternative approaches. First, the challenges for drug ungual penetration are presented. Then, the chemical and physical strategies developed for overcoming the barriers for drug penetration are discussed. We hope that the information gathered may be useful for the development of safer and more effective treatments for onychomycosis.
Introduction
About 50% of nail diseases are caused by fungal infections. Although several dermatophytes, nondermatophytes, moulds, and yeasts may be involved in these disorders, the main species belong to the genera Trichophyton, Epidermophyton, or Microsporum. These diseases, known as onychomycosis, are characterized by thickening, roughness, and splitting of the nail.1–4 Way beyond cosmetic distress and social concern, the pathology may progress with several symptoms such as discomfort and pain, associated with physical and occupational limitations. As consequence, patients may have reduced quality of life.5,6
Approximately 10–30% of the world population is affected by onychomycosis. Among the cases, 20–25% do not achieve complete cure, and relapse rates achieves 10–53%.6,7 These data are particularly worrying to patients with high risk for development of opportunistic infections, which may have the worsts outcomes.8,9
In clinical practice, onychomycosis treatments are focused on surgical avulsion, systemic drug therapy, and topical drug therapy alone or in combination with physical methods. Currently, terbinafine is the gold standard to oral treatment, followed by itraconazole. Along with it oral fluconazole, ketoconazole, and griseofulvin are being used,10 but treatment duration can be extended to about one year, what may lead to significant systemic side effects such as liver and cardiac damage.11–15 Therefore, topical therapy is greatly requested to promote drug delivery to the site of action, minimizing adverse events. Nevertheless, topical onychomycosis treatments present low efficiency due to several difficulties related to drug penetration into the nail plate.
In this way, this review aims to present the challenges for drug ungual penetration and development of new strategies, discussing the main therapies under research.
Search strategy and selection criteria
The review was based on literature search using the Endnote software and Web of Science Database. First, a broad search was performed including the term ‘onychomycosis,’ which retrieved 1640 results. From these, the first 500 references, comprehending publication from 2010 to 2015, were briefly analyzed, and the novel strategies for treating onychomycosis listed. A second search included the terms ‘Onychomycosis + topical,’ which retrieved 122 results from 1977 to 2015. Studies referring to novel physical enhancement techniques, chemical modifications of drug molecule or proposal of innovative formulations were considered in this review. Then several other searches were performed using the strategy term listed in the first phase + the terms onychomycosis or nail (e.g., ‘iontophoresis + onychomycosis’ and ‘iontophoresis + nail’). The most relevant references are discussed along the text or referenced in the tables. Clinical studies reporting the topical efficacy of conventional formulations are not included in this review.
The human nail and different types of onychomycosis
The nail unit protects the terminal phalanges of fingers and toes from trauma, amplifies fine touch perception, and helps to grab and hold small objects. The nail plate is a tough structure that rests over a thin glabrous epidermis, known as nail bed. After new cells are produced, they differentiate, keratinize, and force the old ones toward the dorsal surface. Since basal cells of the nail matrix are continuously divided by mitosis, the plate grows unceasingly throughout life.16,17 Thereby, fingernails and toenails are completely replaced in about 6 and 12–18 months, respectively.5
Onychomycosis can be classified in different types depending on the site in the nail where fungal colonization occurs, along with clinical signs and symptoms.18 The main classes are Distal and Lateral Subungual Onychomycosis (DLSO); Proximal Subungual Onychomycosis (PSO); White Superficial Onychomycosis (WSO); and Candida Onychomycosis. The term Total Nail Dystrophy (TDO) is used to describe the end stage of any kind of the pathology.2,3,5–7 Infection peculiarities are related to the sort of opportunist microorganism, patient's health and nail characteristics.6–8
Barriers of topical treatment to onychomycosis
In its normal condition, the human nail acts as a protective barrier against penetration of foreign material. In fact, for this reason, effective topical treatment of onychomycosis is also hampered.19,20 This feature occurs due to nail's composition (80% protein, 7–12% water, less than 1.5% lipid, and trace amounts of minerals and electrolytes) and its physiological and morphological properties 19–21 (Figure 1).
Schematic representation of topical therapy of human nail. Description of human nail barrier and drug properties that affect drug permeation. Reproduced with permission 18. This Figure is reproduced in color in the online version of Medical Mycology.
Following topical administration, the drug has to overcome different challenges to enter the nail plate, diffuse through its layers and reach the nail bed. First of all, the nail plate has approximately 25 tightly bound layers of dead flattened cells, filled with α-keratins, which are cross-linked with disulfide bonds.6,22 However, in this highly keratinized and compact structure, certain permeability is observed. Yet, it is worthy to note that it usually occurs in low concentrations and low drug flux.23 Among other effects, this is related to nail plate's behavior like a concentrated hydrogel rather than a lipophilic membrane.24 Hence, it is expected that similarly as occurs to hydrogel, the penetration into the nail may be favored to molecules smaller than the pore size, in comparison to large molecules.22,23,25 Thus, drug permeation may occur via convection, diffusion, or partition.20,23 The mechanisms are affected by hydrophobic and electrical interactions between the drug and the keratin fibers.23 Therefore, physicochemical properties of the drug and formulations characteristics influence the permeation.
Drugs characteristics for topical application
The main drug properties that influence the nail permeability are molecular weight, lipophilicity, affinity to keratin, ionization, pH, and sublimability.
Molecular weight (MW) is one of the most important properties that control drug permeability through the nail plate.22,25 Therefore, smaller molecules, which are able to easily pass through nail pores, present enhanced permeation.22 Indeed, in vitro studies observed that tolnaftate (307.41 Da) has a lower nail permeability compared with 5-fluorouracil (130.01 Da).26 Along with the molecules lipophilicity, MW is primordial to determine the permeation.
Regarding a nail's pores, hydration may be used to increase the nail plate porosity.27,28 It has been reported that rising ambient relative humidity from 15 to 100% leads to threefold increase of ketoconazole permeation.27 Also, after immersion in aqueous media, important changes such as swelling and softening of the nail occur,26 which points to the use of hydration for enhancement of ungual permeability.
The importance of drug lipophilicity has been evidenced on studies employing molecules with the same MW.20,22,25,29 Such studies have shown that extreme drug lipophilicity is a very disadvantageous characteristic for nail penetration.30 This may be explained by the great keratin affinity related to highly lipophilic drugs. However, keratin binding is often reversible; thus, keratin layers may act as reservoir extending the antifungal effect.22
Another significant feature is the isoelectric point (pI) of keratins, which is in the range of 4.0–5.0. In consequence, the nail carries a negative charge at pH above its pI.22 Whenever molecules and the nail bear opposite charges, it is expected that permeability increases.21,22,31 On the other hand, drug molecule ionization decreases its nail permeability from an aqueous vehicle. Indeed, when the permeability coefficients of benzoic acid and lidocaine were determined for nonionic and ionic forms, neutral molecules presented about 10 times higher permeability compared to ions. This effect could be related to apparent MW increase (around 100 times) caused by ion hydration.22,25
Drug delivery may also be enhanced by pH changes. Pre-treatments or co-treatments of the nail plates with extremely alkaline pH (pH > 11) or extremely acidic pH (pH < 3) solutions could be useful.31 It is just important to consider that the antifungal activity of some molecules may also be modified by the pH. It was reported that the in vitro activity of naftifine against Trichophyton mentagrophytes decreases as pH decreases from pH 7 to pH 4.32 Similar results were found on in vitro antifungal activities of amphotericin B, ciclopirox, fluconazole, ketoconazole, voriconazole, posaconazole, itraconazole, and clotrimazole against Candida albicans vaginal isolated with reduced susceptibility to fluconazole.33
Some drugs have the property of changing directly from solid to gas phase without transition through the intermediate liquid phase. For example, amorolfine, other morpholine derivates and terbinafine have the ability to sublimate at 23–25 °C and at 37 °C. Amorolfine hydrochloride is also capable to sublimate from its galencial forms Loceryl® nail lacquer and Loceryl® cream.34 This ability may lead the drug to overcome air cavities present in mycotic lesions and reach tissue layers on the other side of these cavities. Seeking this property, Polak et al.35 investigated other antimycotic agents (amphotericin B, nystatin, fucytosine, miconazole, clotrimazole, bifonazole, ketoconazole, fluconazole, itraconazole, voriconazole, naftifine, caspofungin and ciclopiroxolamine); however, the results showed that the studied drugs are not sublimable.
As it may be more complicated to substitute or modify a certain molecule without affecting its antifungal and toxicological properties, most of the researches have been focusing on altering the nail plate barrier.
Topical delivery strategies
Currently, topical onychomycosis treatments are focused on formulations containing agents such as amorolfine, ciclopirox, tioconazole, efinaconazole, and tavaborole. However, condition improvement is usually achieved in less than 30% of the cases, with complete cure rates below 20%.1,36
Since the nail properties make topical therapy a challenge, different approaches such as chemical strategies and physical methods have been studied to circumvent the physiological barriers and improve treatment efficacy (Fig. 2).
Chemical and physical strategies to improve onychomycosis topical treatment. This Figure is reproduced in color in the online version of Medical Mycology.
Chemical strategies
Chemical strategies use or incorporate chemical compounds into the topical formulation to enhance penetration of drugs through the nail.20,26,28,37
Different mechanism can be involved to promote drug penetration. Some penetration enhancers such as urea, sodium salicylate, salicylic acid, and papain are keratolityc agents. They act disrupting the protein tertiary structure and secondary linkages.26,37 These substances may create new ‘pores’ that are interconnected by transport channels.
Although some reports state that keratolytic agents were not effective to enhance the permeation of drugs in topical nail delivery, Nair et al.38 showed that there was a moderate increase in terbinafine permeation over 24 h of passive delivery when nail plates were kept immersed in salicylic acid solution (4 mg/ml) for keratolysis. In this study, terbinafine permeation was significantly increased when keratolityc agents were used combined with iontophoresis.
Combination of different compounds may lead to best results. In fact, great enhancement effect was observed when using N-(2-mercaptopropionyl) glycine combined with urea when compared to its isolated use.37
Thiol compounds, such as N-(2-mercaptopropionyl) glycine, cleave the disulphide bonds in the nail plate increasing the permeation flux of drugs.20,28,39–42In vitro studies demonstrate a marked swelling and softening of the nail plate when these compounds are used. It was observed that N-acetyl-L-cysteine and 2-mercaptoethanol increased the permeation flux of tolnaftate (lipophilic drug) and 5-fluorouracil (hydrophilic drug).26N-acetyl-L-cysteine also significantly prolonged the mean residence time of oxiconazole in upper nail layers.42 Likewise, N-acetyl-L-cysteine solution (10%) increased the delivery of triamcinolone acetonide across hoof membrane.28
Thioglycolic acid was also evaluated as penetration enhancer and the results showed that this compound was efficient to increase the ungual flux of caffeine20,41 and methylparaben.20 Similar results were also found by Hao et al. (2008),40 which showed that thioglycolic acid increased the permeation of both mannitol and urea through the nail plate. Moreover, a greater permeation enhancement was observed with arising concentrations of thioglycolic acid.40 Also, the addition of thioglycolic acid (5%) to nail lacquer improved the permeability of voriconazole by 0.7. In addition, voriconazole nail lacquer was effective in inhibiting the growth of the nail fungi T. rubrum.39
Likewise, studies with the EcoNailTM lacquer containing 2-N-nonyl-1,3-dioxolane showed that the formulation containing this substance delivered 6 times more econazole than the lacquer without the enhancer.43 This product completed phase II clinical studies.22
Although surfactants may be able to denature keratin, Malhotra et al.37 demonstrated that a gel formulation containing 10% of sodium lauryl sulfate did not increase penetration through the nail. They also showed that pyrithione and its derivatives (8-mercaptomenthone, meso-2,3-dimercapto succinic acid and sodium metabisulphite) were all ineffective to enhance ungual penetration.
On the other hand, keratolytic enzymes such as the keratinase (produced by Paecilomyces marquandii) may act on the intercellular matrix as well as on the nail corneocytes resulting in disruption of the nail plate. Thereby, keratinase enzymes may also be used as penetration enhancers.44
Hydrophobins, which are small amphiphilic fungal proteins, were also evaluated as penetration enhancers. They have the ability to reduce surface tension and adhere to hydrophilic or hydrophobic surfaces, which enhanced caffeine penetration in low doses. Further studies are required to define whether hydrophobins can be used as enhancers for biomaterials and medical application to the nail plate.45
Regarding vehicle formulation, solvents such as dimethylsulfoxide (DMSO) may be used to alter the concentration of lipids present in the nail plate and cause conformational changes in keratin structure.45 Trials of samples containing DMSO increased the penetration of radiolabeled urea, salicylic acid, and ketoconazole into the intermediate nail plate (50% higher than saline controls).46
Also, when considering vehicles, it is worth to note that water may physically change the nail plate features.26 Although aqueous vehicles clearly promote ungual permeation, the penetrating enhancing ability of ethanol is likely to be absent in the nail, despite its ability to act as an epidermal enhancer.20,41 Vejnovic et al.45 observed that formulations containing 20% of ethanol did not significantly influence permeability coefficients in comparison with pure water formulations. Even though Naumann et al.30 showed that increasing water content in the vehicles did not increase the penetration rates of the administered drug, it is argued that the presence of water is not important for the penetration as long as the drug is saturated in the solution. Acetone is another solvent which has been reported to have null effects on nail permeability41 and could have significant implications for the development of new topical medications.47
Physical strategies
The use of physical methods has been extensively studied for topical and transdermal drug delivery.48 Although they are usually more expensive due the involved technology and equipment, these methods are proving to be more potent than chemical approaches to enhance nail permeation of topically applied drugs.6
Most of these techniques produce a mechanical impact on the tissue, abrading or creating channels that could allow for even higher MW molecules to penetrate. Some of these techniques are presented below.
Abrasion of the nail plate
From the top of the nail unit, it consists of a dorsal plate, an intermediate plate, and a ventral plate, comprising about 0.5–1.0 mm of densely packed keratin molecules. The plate constitutes the main barrier for topically applied drugs.22 An approach to increase drug permeation is to abrade the nail surface by filing/sanding/debridement, which removes the dorsal layer and diminishes the thickness of the plate.6,49–52
Nail abrasion is one of the oldest methods used to treat hyperkeratosis of the nail plate or partial removal of it.50,53 It is practical and can be performed with sandpaper, which can be connected to a dermabrader device,50 to dental drills,6,52 or even to common nail files.
Though this method presents lower success rates, when compared to other physical ones, it shows some effectiveness. Combination of oral treatment with nail abrasion shows increased efficiency, with reduction in recurrence events.54 A study with diabetic patients with WSO and DLSO was performed to evaluate the effect of combining topical treatment and nail abrasion. Nail surfaces of patients were slightly drilled using a grinder to reduce nail thickness. In this study the cure rate of onychomycosis was lower (16.7%) compared with oral therapy; however, some patients with WSO were cured, and some patients with DLSO showed improvement.55
In other study,56 three treatment groups were submitted to nail abrasion. The first group used oral terbinafine in combination with ciclopirox nail lacquer twice a week. The second one used only ciclopirox nail lacquer twice a week. The third group used ciclopirox nail lacquer 5 days a week, for 12 months, with 6 months posttreatment follow-up. At follow-up, the diseased nail plate area reduced from 50 to 25% for 96% of the patients. Also, culture was negative to 72% in all groups, with no statistical differences among them.
The main benefits of this method over the chemical ones are that it is fast, inexpensive, and the treated area may be accurately selected, not affecting lateral epidermis. In addition, this technique may favor patient compliance, since it is absent of negative odors referred to medicines, presents aesthetic value, and the sanding should cause minimum discomfort. Still, nail abrasion can greatly improve chemical treatments when combined with them. In fact, this technique has been widely applied to enhance the action of antifungal nail lacquers.50,51
Microporation of the nail plate
Microporation consists on drilling individual small holes in the nail plate, without affecting the nail bed. The FDA-approved device for this technique is called PathFormer (Path Scientific, Carlisle, USA), and it was initially developed to drain hematomas.6,49,52,57
Boker and co-workers studied microporation with posterior use of terbinafine cream, aiming to employ this technique to treat onychomycosis.58 They observed enhanced permeation of the applied drug.
Also, in a recent study, Chiu et al. used a dermaroller (Infinitive Beauty, Birmingham, UK) to produce micropores on the nail surface.59 They applied polymeric nanoparticles loaded with a lipophilic fluorophore and observed its sustained delivery into deeper regions of the treated nails when compared to nonporated nails. These promising results are an incentive for further studies.
Etching the nail surface
Etching consists on the formation of profuse microporosities by chemical exposure. These pores increase the surface area and promote reduction of contact angle, enhancing wettability. Therefore, drug permeation through the nail plate is facilitated while a bonding of polymeric delivery system follows on the nail surface.51,60
Repka et al.61 showed that roughness scores increased twofold when the dorsal nail plate were treated with phosphoric acid gel compared to control. This strategy was effective to improve nail permeability of ketoconazole. It was demonstrated that the treated nail showed sixfold higher ketoconazole permeation than the nonetched nail. Moreover, it is reported that ketoconazole in hot-melt extruded films can promote extended release.60
Vaka et al.62 pretreated human nail with chemical etchants, 1% or 10% phosphoric acid, or 10% lactic acid gels, for a period of 60 seconds. Although lactic acid failed to enhance the drug permeation, phosphoric acid, in both concentrations, promoted modifications in the nail that lead to enhanced permeation of 5-fluorouracil and terbinafine hydrochloride.
Iontophoresis
Iontophoresis is a noninvasive technique based on the application of a mild electric field to enhance the permeation of molecules through biological membranes.48 The system is composed of two electrodes and a power source. The ionized drug is placed in contact with the electrode bearing the same charge, while the counter-electrode is placed apart in another place of the body. Once the electric field starts, in physiological conditions, cations flow from the anode towards the cathode, whereas anions move on the contrary.63
Although the first in vivo study involving ungual iontophoresis was published in 1986,64 only from the last decade this approach was retaken in an attempt to treat ungual infections.65 The results have shown that iontophoresis highly improves drug delivery compared to the passively applied drug.
In most iontophoretic studies to treat onychomycosis, terbinafine is used, as it is the most potent agent (MIC = 3–6 μg/ml) against dermatophytes.66,67 Since this drug exhibits a pH-dependent solubility (pKa = 7.1), it is commonly iontophoretically delivered at acidic pHs (∼pH 3) to ensure that the drug is fully ionized, favoring its electromigration.
In vitro and ex vivo experiments have shown remarkably higher drug diffusion into nail bed when iontophoresis is applied, compared with passive topical drug delivery, or even oral treatment.12,67–71
A clinical study using iontophoresis to deliver terbinafine applied for 4 weeks a 5 days-a-week treatment, using a current density of 100 μA/cm2. The study showed that 40% of the patients presented improvement in their condition vs. 11% of those treated with the drug passively applied 72. Additionally, 8 weeks after completion of treatment, percentage of patients having fungal elements in nail decreased from 53% in the passive group to only 16% in group that received iontophoresis.72
Iontophoresis can also be used in combination with other techniques. Nair et al.38 showed that abrasion of dorsal layer combined with iontophoresis enhanced the permeation of terbinafine hydrochloride compared to abraded nails without iontophoresis or untreated nails with iontophoresis.
Trans-ungual delivery of drugs using iontophoresis can be possible by the use of small devices, what makes it a more affordable technique.72 Also, unlike other methods, iontophoresis does not impact nail structure, as demonstrated by Benzeval and co-workers.73 In addition, it is important to highlight that, during clinical studies, patients only reported a tingling sensation with current application,68 demonstrating the potential patient acceptance of the technique.
Nevertheless, safety of the routine clinical use of iontophoresis is being pointed as a limitation, due cutaneous adverse effects.74 Therefore, more studies need to be performed to confirm long-term use safety of this technique.
Lasers
Laser is an acronym for “light amplification by stimulated emission of radiation.” Since 1983, photoselective damage to pigmented structures of fungus has been observed,75 and since then, lasers have been used to treat onychomycosis. As the treatment is targeted, surrounding tissue is not affected by photochemical, photothermal, and photomechanical effects.76–78
To achieve efficiency, laser light has to be absorbed by the target area, and sufficient energy to damage microorganisms is needed. However, the treatment should be either in a pulsed mode or be delivered at a moderate energetic level, so that generated heat can be dissipated by patient's tissue, preventing cutaneous damage and associated pain.79 In this way, several laser parameters (e.g., wavelength, pulse duration, frequency, spot size, and energy fluency) and treatment parameters (e.g., duration, number of treatments, intervals) may also affect the therapy efficacy.76,80
Laser sources used to treat onychomycosis can vary, including neodymium-doped yttrium aluminum garnet (Nd:YAG), titanium sapphire, and diode lasers.81,82 An in vitro study with different lasers tested in six species of fungi demonstrated complete pathogen growth impairment when temperatures were measured above 50 °C, which could be achieved with lasers wave-length above 980 nm.83
The efficacy of 1064 nm Nd:YAG laser systems with wavelengths from 1064 to 1444 nm in the treatment of onychomycosis has already been demonstrated in clinical trials, and it is the most studied so far.81,82,84
Clinical and mycological efficacy of Nd:YAG laser vs. topical terbinafine to treat onychomycosis has been recently evaluated. Patients received four sessions of Nd:YAG laser or topical terbinafine twice a day for six months. After this period, patients treated with laser showed improvement, with 80% presenting microbiological clearance, whereas only 50% of the patients treated with terbinafine showed some improvement.78 Other clinical study applied 0.65-millisecond (ms) pulsed 1064-nm laser, and it was observed that seven of eight patients treated over two or three sections with laser had negative post-treatment cultures.85 A similar study showed that about 10% of patients achieved a complete cure, and 90% had good treatment outcomes.77
A larger clinical study involving 82 toenails clinically diagnosed with onychomycosis was performed with 1,064-diode laser employed twice a week every 8 weeks, with significant improvement and/or good clinical improvement to about 41% of the patients. Patient satisfaction was also evaluated in this study and about 60% of the treated patients were completely satisfied.86
This technique has shown good tolerance by patients,85 especially to those for whom medicament treatments could cause some risks,78 with the benefit of presenting only mild or no discomfort.76,77,79,86 However, in spite of the great number of successful in vitro and clinical successful studies, further investigations are still demanded to determine the long-term clinical and microbiological effects of the treatment.81 Until the moment, the only physical device approved by regulatory agencies to topical treatment of ungual diseases is a laser system. It was approved by the FDA, however, for “temporary increase of clear nail in onychomycosis”, that is, as a cosmetic outcome that differs from the medical efficacy approvals granted to topical and oral antifungal agents.76
Photodynamic therapy (PDT)
PDT is a photochemical process, which involves the combination of nontoxic photosensitizer (PS) and the exposure of light that, together, produce reactive oxygen species (ROS). The chain reactions lead to cell inactivation.87–90 PDT mechanism is summarized at Figure 3.
Schematic representation of PDT mechanism. First, the light excites the photosensitizer (PS) to singlet state that could lead to the formation of triplet state after relaxation. The triplet state reacts with physiologic oxygen (reactions type 1 or 2) leading to the formation of reactive oxygen species (ROS) and 1O2, which could act in different biomolecules. Adapted from Yin et al (2013) 89. Reproduced with permission 18. This Figure is reproduced in color in the online version of Medical Mycology.
In the early 1900 s, scientists observed that hyperproliferative cells, such as tumor cells, could selectively internalize PS.87,91 But in 1999, the PS 5-aminolevulinic was approved by FDA to treat premalignant skin lesions.87 Recently, researchers suggest that PDT could also be used to treat fungal and bacterial infections due to the fast growth rate of these cells92 and the rapidly and selectively bind of PS to them.91 In comparison to systemic delivery of antifungal drugs, PDT technique provides dual selectivity, since the PS and the light sources could be locally applied to the superficial fungal infection for hair, skin and nail treatments.88
The selection of the PS is one of the most important steps of PDT. Table 1 demonstrates the most commonly used PS for onychomycosis treatments.
Names and chemical structures of photosensitizers commonly used to treat onychomycosis in photodynamic therapy.
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Names and chemical structures of photosensitizers commonly used to treat onychomycosis in photodynamic therapy.
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In the early 2000 s, research main focus was to investigate whether commonly used PS in cancer treatments would also accumulate in fungal cells and exert deleterious effects. Hence, in vitro studies were performed applying PS to several fungal species, such as T. rubrum, the most common causative agent of dermatophytosis and onychomycosis. To find the best condition for PDT treatments parameters as type of PS, concentration (or the minimal inhibitory concentration [MIC]), light spectrum, and dose were evaluated (Table 2).
In vitro studies of photodynamic therapy using different photosensitizers and different wavelength of irradiation in fungal cultures.
| PS | Fungus | Results | Reference |
|---|---|---|---|
| Methylene blue, neutral red, and proflavine | Trichophyton mentagrophytes and Microsporum gypseum | Percentages of survival of T. mentagrophytes, after 3 μM of proflavine was 0.56 ± 0.28%; for methylene blue was 35.0 ± 8.0% and for neutral red was 43.0 ± 10.0%. Similar results were obtained for M. gypseum. | Prospst et al.93 |
| 2,2΄:5΄,2″-terthienyl (α-T) and 5-(4-hydroxy-1-butinyl)2,2΄-bithienyl (BBTOH) | T. mentagrophytes, T. rubrum, T. tonsurans, E. floccosum, M. cookei, M. canis, M. gypseum, N. cajetani. | PS (5, 10 and 50 μg/ml) and UV-A (320–400 nm) was applied to Epidermophyton floccosum, which was sensitive to all doses. It was also quite active against Nannizia cajetani. | Romagnoli et al.94 |
| Deuteroporphyrin Monomethylester, Deuteroporphyrin, Phthalocyanine, 5,10,15-tris (4-methylpyridinium)-20-phenyl-[21H,23H]-porphine trichloride (Sylsens B), Hematoporphyrin, | T. rubrum | Sylsens B (4μM) was the most effective PS. For Sylsens B and Deuteroporphyrin monomethylester a concentration of 3 μg/ml or higher was enough to kill T. rubrum in most experiments with a white light (108 J/cm2). For hematoporphyrin this concentration was 10 μg/ml, and for Deuteroporphyrin it was 20 μg/ml. | Smijs et al.95 |
| Haematoporphyrin derivative (HPD), methylene blue (MB) and toluidine blue O (TBO) | T. verrucosum, T. M. canis, Epidermophyton floccosum, T. rubrum, M. gypseum and T. violaceum | Solar simulator (400 W/m2 for 30 minutes) in combination with PS resulted in complete inhibition for spore germination of T. verrucosum, T. mentagrophytes, and M. canis. On the other hand, E. floccosum, T. rubrum, M. gypseum and T. violaceum were less sensitive to irradiation when pretreated with HPD or MB. | Ouf et al.96 |
| Deuteroporphyrin Monomethylester and 5,10,15-tris (4-methylpyridinium)-20-phenyl-[21H,23H]-porphine trichloride (Sylsens B) | T. rubrum | Red light (108 J/cm2) twice much Sylsens B (8μM) was used to have the same fungicidal effect as observed by Smijs et al, 2003. 40 μM of Deuteroporphyrin monomethylester was necessary to obtain the same effect found by Smijs et al, 2003. | Smijs et al.97 |
| 5-aminolevulinic acid (5-ALA) | T. rubrum | White light (128 J/cm2) and 1–10 mmol/l of 5-ALA demonstrated that almost 50% of the fungal growth was inhibited in vitro. The most limiting factor was the fungal 5-ALA uptake. | Kamp et al.98 |
| 5-aminolevulinic acid (5-ALA) | Trichophyton interdigitale | Red light (100 J/cm2) and 10 mM of 5-ALA (6 h of incubation) reduced fungal viability on 42%. 5-ALA penetration in nail plate was evaluated in vitro using Franz diffusion cells. Researchers suggest the improvement of treatment by adding penetration enhancer to the formulation to increase 5-ALA accumulation in the nail plate. | Donnelly et al.99 |
| 5,10,15-tris(4-methylpyridinium)-20-phenyl-[21H,23H]-porphine trichloride (Sylsens B) | T. rubrum | The light dose (108 J/cm2) caused severe disruptions and deformations of fungi cell and emptied fungal elements. | Smijs et al.100 |
| Methylene blue (MB), toluidine blue O (TBO), new methylene blue N (NMBN), and the novel pentacyclic phenothiazinium photosensitizer (S137). | T. mentagrophytes and T. rubrum | 634 nm light at 5, 10 and 20 J/cm2 was applied. S137 showed the lowest MIC. MIC for S137 was 2.5 μM both for fungi evaluated at light dose of 5 J/cm2. NMBN (10 μM and 20 J/cm2) resulted in a reduction of 4 logs in the survival of the T. rubrum and no survivor of T. mentagrophytes was observed. S137 at 1 μM and 20 J/cm2 resulted in a reduction of approximately 3 logs in the survival of both species. | Rodrigues et al.88 |
| Methylene blue | Sporothrix schenckii complex (S. albicans, S. brasiliensis PG1, S. brasiliensis CBS1330, S. globosa, S. mexicana, S. schenckii Ss02, S. schenckii Ss09) | The inocula were irradiated using a diode laser with an output of 35mWat, a wavelength of 685 nm and at a distance of 1 cm for 110 s, resulting in an energy dose of 28 J/cm2. Inactivation of all members of the S. schenckii complex included in the investigation. | Nunes Mario et al.101 |
| PS | Fungus | Results | Reference |
|---|---|---|---|
| Methylene blue, neutral red, and proflavine | Trichophyton mentagrophytes and Microsporum gypseum | Percentages of survival of T. mentagrophytes, after 3 μM of proflavine was 0.56 ± 0.28%; for methylene blue was 35.0 ± 8.0% and for neutral red was 43.0 ± 10.0%. Similar results were obtained for M. gypseum. | Prospst et al.93 |
| 2,2΄:5΄,2″-terthienyl (α-T) and 5-(4-hydroxy-1-butinyl)2,2΄-bithienyl (BBTOH) | T. mentagrophytes, T. rubrum, T. tonsurans, E. floccosum, M. cookei, M. canis, M. gypseum, N. cajetani. | PS (5, 10 and 50 μg/ml) and UV-A (320–400 nm) was applied to Epidermophyton floccosum, which was sensitive to all doses. It was also quite active against Nannizia cajetani. | Romagnoli et al.94 |
| Deuteroporphyrin Monomethylester, Deuteroporphyrin, Phthalocyanine, 5,10,15-tris (4-methylpyridinium)-20-phenyl-[21H,23H]-porphine trichloride (Sylsens B), Hematoporphyrin, | T. rubrum | Sylsens B (4μM) was the most effective PS. For Sylsens B and Deuteroporphyrin monomethylester a concentration of 3 μg/ml or higher was enough to kill T. rubrum in most experiments with a white light (108 J/cm2). For hematoporphyrin this concentration was 10 μg/ml, and for Deuteroporphyrin it was 20 μg/ml. | Smijs et al.95 |
| Haematoporphyrin derivative (HPD), methylene blue (MB) and toluidine blue O (TBO) | T. verrucosum, T. M. canis, Epidermophyton floccosum, T. rubrum, M. gypseum and T. violaceum | Solar simulator (400 W/m2 for 30 minutes) in combination with PS resulted in complete inhibition for spore germination of T. verrucosum, T. mentagrophytes, and M. canis. On the other hand, E. floccosum, T. rubrum, M. gypseum and T. violaceum were less sensitive to irradiation when pretreated with HPD or MB. | Ouf et al.96 |
| Deuteroporphyrin Monomethylester and 5,10,15-tris (4-methylpyridinium)-20-phenyl-[21H,23H]-porphine trichloride (Sylsens B) | T. rubrum | Red light (108 J/cm2) twice much Sylsens B (8μM) was used to have the same fungicidal effect as observed by Smijs et al, 2003. 40 μM of Deuteroporphyrin monomethylester was necessary to obtain the same effect found by Smijs et al, 2003. | Smijs et al.97 |
| 5-aminolevulinic acid (5-ALA) | T. rubrum | White light (128 J/cm2) and 1–10 mmol/l of 5-ALA demonstrated that almost 50% of the fungal growth was inhibited in vitro. The most limiting factor was the fungal 5-ALA uptake. | Kamp et al.98 |
| 5-aminolevulinic acid (5-ALA) | Trichophyton interdigitale | Red light (100 J/cm2) and 10 mM of 5-ALA (6 h of incubation) reduced fungal viability on 42%. 5-ALA penetration in nail plate was evaluated in vitro using Franz diffusion cells. Researchers suggest the improvement of treatment by adding penetration enhancer to the formulation to increase 5-ALA accumulation in the nail plate. | Donnelly et al.99 |
| 5,10,15-tris(4-methylpyridinium)-20-phenyl-[21H,23H]-porphine trichloride (Sylsens B) | T. rubrum | The light dose (108 J/cm2) caused severe disruptions and deformations of fungi cell and emptied fungal elements. | Smijs et al.100 |
| Methylene blue (MB), toluidine blue O (TBO), new methylene blue N (NMBN), and the novel pentacyclic phenothiazinium photosensitizer (S137). | T. mentagrophytes and T. rubrum | 634 nm light at 5, 10 and 20 J/cm2 was applied. S137 showed the lowest MIC. MIC for S137 was 2.5 μM both for fungi evaluated at light dose of 5 J/cm2. NMBN (10 μM and 20 J/cm2) resulted in a reduction of 4 logs in the survival of the T. rubrum and no survivor of T. mentagrophytes was observed. S137 at 1 μM and 20 J/cm2 resulted in a reduction of approximately 3 logs in the survival of both species. | Rodrigues et al.88 |
| Methylene blue | Sporothrix schenckii complex (S. albicans, S. brasiliensis PG1, S. brasiliensis CBS1330, S. globosa, S. mexicana, S. schenckii Ss02, S. schenckii Ss09) | The inocula were irradiated using a diode laser with an output of 35mWat, a wavelength of 685 nm and at a distance of 1 cm for 110 s, resulting in an energy dose of 28 J/cm2. Inactivation of all members of the S. schenckii complex included in the investigation. | Nunes Mario et al.101 |
In vitro studies of photodynamic therapy using different photosensitizers and different wavelength of irradiation in fungal cultures.
| PS | Fungus | Results | Reference |
|---|---|---|---|
| Methylene blue, neutral red, and proflavine | Trichophyton mentagrophytes and Microsporum gypseum | Percentages of survival of T. mentagrophytes, after 3 μM of proflavine was 0.56 ± 0.28%; for methylene blue was 35.0 ± 8.0% and for neutral red was 43.0 ± 10.0%. Similar results were obtained for M. gypseum. | Prospst et al.93 |
| 2,2΄:5΄,2″-terthienyl (α-T) and 5-(4-hydroxy-1-butinyl)2,2΄-bithienyl (BBTOH) | T. mentagrophytes, T. rubrum, T. tonsurans, E. floccosum, M. cookei, M. canis, M. gypseum, N. cajetani. | PS (5, 10 and 50 μg/ml) and UV-A (320–400 nm) was applied to Epidermophyton floccosum, which was sensitive to all doses. It was also quite active against Nannizia cajetani. | Romagnoli et al.94 |
| Deuteroporphyrin Monomethylester, Deuteroporphyrin, Phthalocyanine, 5,10,15-tris (4-methylpyridinium)-20-phenyl-[21H,23H]-porphine trichloride (Sylsens B), Hematoporphyrin, | T. rubrum | Sylsens B (4μM) was the most effective PS. For Sylsens B and Deuteroporphyrin monomethylester a concentration of 3 μg/ml or higher was enough to kill T. rubrum in most experiments with a white light (108 J/cm2). For hematoporphyrin this concentration was 10 μg/ml, and for Deuteroporphyrin it was 20 μg/ml. | Smijs et al.95 |
| Haematoporphyrin derivative (HPD), methylene blue (MB) and toluidine blue O (TBO) | T. verrucosum, T. M. canis, Epidermophyton floccosum, T. rubrum, M. gypseum and T. violaceum | Solar simulator (400 W/m2 for 30 minutes) in combination with PS resulted in complete inhibition for spore germination of T. verrucosum, T. mentagrophytes, and M. canis. On the other hand, E. floccosum, T. rubrum, M. gypseum and T. violaceum were less sensitive to irradiation when pretreated with HPD or MB. | Ouf et al.96 |
| Deuteroporphyrin Monomethylester and 5,10,15-tris (4-methylpyridinium)-20-phenyl-[21H,23H]-porphine trichloride (Sylsens B) | T. rubrum | Red light (108 J/cm2) twice much Sylsens B (8μM) was used to have the same fungicidal effect as observed by Smijs et al, 2003. 40 μM of Deuteroporphyrin monomethylester was necessary to obtain the same effect found by Smijs et al, 2003. | Smijs et al.97 |
| 5-aminolevulinic acid (5-ALA) | T. rubrum | White light (128 J/cm2) and 1–10 mmol/l of 5-ALA demonstrated that almost 50% of the fungal growth was inhibited in vitro. The most limiting factor was the fungal 5-ALA uptake. | Kamp et al.98 |
| 5-aminolevulinic acid (5-ALA) | Trichophyton interdigitale | Red light (100 J/cm2) and 10 mM of 5-ALA (6 h of incubation) reduced fungal viability on 42%. 5-ALA penetration in nail plate was evaluated in vitro using Franz diffusion cells. Researchers suggest the improvement of treatment by adding penetration enhancer to the formulation to increase 5-ALA accumulation in the nail plate. | Donnelly et al.99 |
| 5,10,15-tris(4-methylpyridinium)-20-phenyl-[21H,23H]-porphine trichloride (Sylsens B) | T. rubrum | The light dose (108 J/cm2) caused severe disruptions and deformations of fungi cell and emptied fungal elements. | Smijs et al.100 |
| Methylene blue (MB), toluidine blue O (TBO), new methylene blue N (NMBN), and the novel pentacyclic phenothiazinium photosensitizer (S137). | T. mentagrophytes and T. rubrum | 634 nm light at 5, 10 and 20 J/cm2 was applied. S137 showed the lowest MIC. MIC for S137 was 2.5 μM both for fungi evaluated at light dose of 5 J/cm2. NMBN (10 μM and 20 J/cm2) resulted in a reduction of 4 logs in the survival of the T. rubrum and no survivor of T. mentagrophytes was observed. S137 at 1 μM and 20 J/cm2 resulted in a reduction of approximately 3 logs in the survival of both species. | Rodrigues et al.88 |
| Methylene blue | Sporothrix schenckii complex (S. albicans, S. brasiliensis PG1, S. brasiliensis CBS1330, S. globosa, S. mexicana, S. schenckii Ss02, S. schenckii Ss09) | The inocula were irradiated using a diode laser with an output of 35mWat, a wavelength of 685 nm and at a distance of 1 cm for 110 s, resulting in an energy dose of 28 J/cm2. Inactivation of all members of the S. schenckii complex included in the investigation. | Nunes Mario et al.101 |
| PS | Fungus | Results | Reference |
|---|---|---|---|
| Methylene blue, neutral red, and proflavine | Trichophyton mentagrophytes and Microsporum gypseum | Percentages of survival of T. mentagrophytes, after 3 μM of proflavine was 0.56 ± 0.28%; for methylene blue was 35.0 ± 8.0% and for neutral red was 43.0 ± 10.0%. Similar results were obtained for M. gypseum. | Prospst et al.93 |
| 2,2΄:5΄,2″-terthienyl (α-T) and 5-(4-hydroxy-1-butinyl)2,2΄-bithienyl (BBTOH) | T. mentagrophytes, T. rubrum, T. tonsurans, E. floccosum, M. cookei, M. canis, M. gypseum, N. cajetani. | PS (5, 10 and 50 μg/ml) and UV-A (320–400 nm) was applied to Epidermophyton floccosum, which was sensitive to all doses. It was also quite active against Nannizia cajetani. | Romagnoli et al.94 |
| Deuteroporphyrin Monomethylester, Deuteroporphyrin, Phthalocyanine, 5,10,15-tris (4-methylpyridinium)-20-phenyl-[21H,23H]-porphine trichloride (Sylsens B), Hematoporphyrin, | T. rubrum | Sylsens B (4μM) was the most effective PS. For Sylsens B and Deuteroporphyrin monomethylester a concentration of 3 μg/ml or higher was enough to kill T. rubrum in most experiments with a white light (108 J/cm2). For hematoporphyrin this concentration was 10 μg/ml, and for Deuteroporphyrin it was 20 μg/ml. | Smijs et al.95 |
| Haematoporphyrin derivative (HPD), methylene blue (MB) and toluidine blue O (TBO) | T. verrucosum, T. M. canis, Epidermophyton floccosum, T. rubrum, M. gypseum and T. violaceum | Solar simulator (400 W/m2 for 30 minutes) in combination with PS resulted in complete inhibition for spore germination of T. verrucosum, T. mentagrophytes, and M. canis. On the other hand, E. floccosum, T. rubrum, M. gypseum and T. violaceum were less sensitive to irradiation when pretreated with HPD or MB. | Ouf et al.96 |
| Deuteroporphyrin Monomethylester and 5,10,15-tris (4-methylpyridinium)-20-phenyl-[21H,23H]-porphine trichloride (Sylsens B) | T. rubrum | Red light (108 J/cm2) twice much Sylsens B (8μM) was used to have the same fungicidal effect as observed by Smijs et al, 2003. 40 μM of Deuteroporphyrin monomethylester was necessary to obtain the same effect found by Smijs et al, 2003. | Smijs et al.97 |
| 5-aminolevulinic acid (5-ALA) | T. rubrum | White light (128 J/cm2) and 1–10 mmol/l of 5-ALA demonstrated that almost 50% of the fungal growth was inhibited in vitro. The most limiting factor was the fungal 5-ALA uptake. | Kamp et al.98 |
| 5-aminolevulinic acid (5-ALA) | Trichophyton interdigitale | Red light (100 J/cm2) and 10 mM of 5-ALA (6 h of incubation) reduced fungal viability on 42%. 5-ALA penetration in nail plate was evaluated in vitro using Franz diffusion cells. Researchers suggest the improvement of treatment by adding penetration enhancer to the formulation to increase 5-ALA accumulation in the nail plate. | Donnelly et al.99 |
| 5,10,15-tris(4-methylpyridinium)-20-phenyl-[21H,23H]-porphine trichloride (Sylsens B) | T. rubrum | The light dose (108 J/cm2) caused severe disruptions and deformations of fungi cell and emptied fungal elements. | Smijs et al.100 |
| Methylene blue (MB), toluidine blue O (TBO), new methylene blue N (NMBN), and the novel pentacyclic phenothiazinium photosensitizer (S137). | T. mentagrophytes and T. rubrum | 634 nm light at 5, 10 and 20 J/cm2 was applied. S137 showed the lowest MIC. MIC for S137 was 2.5 μM both for fungi evaluated at light dose of 5 J/cm2. NMBN (10 μM and 20 J/cm2) resulted in a reduction of 4 logs in the survival of the T. rubrum and no survivor of T. mentagrophytes was observed. S137 at 1 μM and 20 J/cm2 resulted in a reduction of approximately 3 logs in the survival of both species. | Rodrigues et al.88 |
| Methylene blue | Sporothrix schenckii complex (S. albicans, S. brasiliensis PG1, S. brasiliensis CBS1330, S. globosa, S. mexicana, S. schenckii Ss02, S. schenckii Ss09) | The inocula were irradiated using a diode laser with an output of 35mWat, a wavelength of 685 nm and at a distance of 1 cm for 110 s, resulting in an energy dose of 28 J/cm2. Inactivation of all members of the S. schenckii complex included in the investigation. | Nunes Mario et al.101 |
The studies demonstrated in Table 2 were performed in vitro in fungal culture, and most of them did not take into account the biological barriers that the PS would need to transposes to exert its therapeutic effect.
Other researchers concluded that the success of antifungal PDT was attributed to fungal internalization of the PS,98 and molecule parameters as chemical structure and pka/pH influence its cellular uptake. In addition, the own microorganism's membrane may be a barrier for this type of treatment. The co-administration of substances capable of creating pores in the fungal envelops has been proposed to increase PS uptake, as demonstrated by Coleman and co-workers.102
Although some studies have demonstrated the uptake efficiency of PS in different fungal cells, such as C. albicans, fewer researches are found when the focus is on PS efficiencies on dermatophytes species aiming on onychomycosis treatment. Notwithstanding, research has progressed over time, and the latest publications have evaluated PDT in in vitro models or in vivo conditions that are closer to the disease. Currently, there are papers that deals exclusively with the application of PDT in onychomycosis, commonly named as antimicrobial photodynamic inhibition (Table 3).
In vivo studies with photodynamic inhibition for onychomycosis.
| Number of patients | Pre-treatment | PS and formulation | Wavelength, dose applied | Results | Reference |
|---|---|---|---|---|---|
| 1 patient | 40% urea ointment under occlusion for 7 days and removal of the nail plate. | 5-ALA in 160 mg/g cream | 630 nm at 37 J/cm2 | Cultures of nail were negative and the toenails were considered clinically cured with residual mild traumatic onycholysis | Piraccini et al.103 |
| 2 patients | 20% urea ointment was applied to the nail and covered with a piece of plastic film wrap for 10 hours | 20% 5-ALA acid in aqueous cream | 630 nm at 100 J/cm2 | After the irradiation, the lesion was successfully treated. No dermatophyte was detected on KOH or on culture. | Watanabe et al.104 |
| 3 patients | – | Methylaminolevulinate (MAL-PDT) | 633 nm at 37 J/cm2 | Both potassium hydroxide and culture became negative after MAL-PDT. Appreciable nail improvement was still seen 4 months after treatment. | Taborda et al.105 |
| 30 patients | 20% urea ointment under occlusion and the nail plate could thus be removed easily with the use of forceps | 20% 5-ALA | 570–670 nm at 40 J/cm2 | After one year, 13 patients (43.3%) were cured. Only 5 (16.6%) patients showed complete absence of clinical signs, while 8 (26.6%) patients showed residual changes affecting less than 10% of the nail plate and negative mycological laboratory results. The remaining patients had changes compatible with dermatophyte infection covering more than 10% of the nail plate, as well as positive direct microscopic examination | Sotiriou et al.106 |
| 1 patient | 40% urea ointment under occlusion for 12 hours | Methyl-aminolevulinate (MAL) | 630 nm at 37 J/cm2 | Complete clinical and mycological cure. | Aspiroz et al.107 |
| 8 patients | – | Methyl 5-aminolevulinic acid (MAL) | Not reported | Negative results on six of eight patients 12 months after the last treatment session. All subjects complained of quite severe burning pain during irradiation of light source. | Lee et al.108 |
| 1 patient | 20% urea was applied for 10 min and approximately 0.5 mm thick of nails was removed by a micromotor | Haematoporphyrin derivative - 5 mg/ml (Photogem®) | 405 nm at 54 J/cm2 | Seven days after the illumination, there was a clinical evaluation to confirm the response to the treatment. The sessions were repeated once a week, with a total of six sessions of PDT. Complete healing was reported after an irradiation over six weeks and in addition, the microorganism culture indicated negative for fungi. | Silva et al.109 |
| 80 patients | Nail abrasion (rotation abrasive device) | 2% methylene blue aqueous solution | 630 nm at 18 J/cm2 | Group A (40 patients which received PDT treatment) showed a significant response (P < .002) compared with Group B (which received 300 mg oral fluconazole), especially in patients who required nail abrasion (P < .001). The PDT is safe, effective, and well tolerated; it promotes a favorable outcome with good patient adherence and may be considered as a practical and feasible treatment option for toenail onychomycosis. | Souza et al.110 |
| 1 patient | – | Gel with Toluidine blue O (TBO) at 100 μg/ml and propileneglicol (penetration enhancer) | 635 nm at 200 J/cm2 | Three consecutive treatments within1 week were required to induce improvement of nail morphology and showed significant improvement of nail morphology 6 months after. | Mehra et al.111 |
| Data not provided | – | 10 μg/L free curcumin and curcumin encapsulated in nanoparticles | Blue light at 10 J/cm2 | Studies demonstrate reduction of fungal burden after a single treatment using curcumin encapsulated in nanoparticles compared to free drug. | Rosen et al.113 |
| 62 patients | – | Aqueous Solutions of 2% methylene blue and toluidine blue (1:1) and 10% ethanol. | 600 to 750 nm at 18 J/cm2 | The number of sessions ranged from 1 to 22, and sessions occurred once a month. The average number of treatments for complete clearance was five (5.22). Complete clearance was observed in 45% of the patients, while 40% presented partial clearance and 15% presented no change. | Tardivo et al.112 |
| Number of patients | Pre-treatment | PS and formulation | Wavelength, dose applied | Results | Reference |
|---|---|---|---|---|---|
| 1 patient | 40% urea ointment under occlusion for 7 days and removal of the nail plate. | 5-ALA in 160 mg/g cream | 630 nm at 37 J/cm2 | Cultures of nail were negative and the toenails were considered clinically cured with residual mild traumatic onycholysis | Piraccini et al.103 |
| 2 patients | 20% urea ointment was applied to the nail and covered with a piece of plastic film wrap for 10 hours | 20% 5-ALA acid in aqueous cream | 630 nm at 100 J/cm2 | After the irradiation, the lesion was successfully treated. No dermatophyte was detected on KOH or on culture. | Watanabe et al.104 |
| 3 patients | – | Methylaminolevulinate (MAL-PDT) | 633 nm at 37 J/cm2 | Both potassium hydroxide and culture became negative after MAL-PDT. Appreciable nail improvement was still seen 4 months after treatment. | Taborda et al.105 |
| 30 patients | 20% urea ointment under occlusion and the nail plate could thus be removed easily with the use of forceps | 20% 5-ALA | 570–670 nm at 40 J/cm2 | After one year, 13 patients (43.3%) were cured. Only 5 (16.6%) patients showed complete absence of clinical signs, while 8 (26.6%) patients showed residual changes affecting less than 10% of the nail plate and negative mycological laboratory results. The remaining patients had changes compatible with dermatophyte infection covering more than 10% of the nail plate, as well as positive direct microscopic examination | Sotiriou et al.106 |
| 1 patient | 40% urea ointment under occlusion for 12 hours | Methyl-aminolevulinate (MAL) | 630 nm at 37 J/cm2 | Complete clinical and mycological cure. | Aspiroz et al.107 |
| 8 patients | – | Methyl 5-aminolevulinic acid (MAL) | Not reported | Negative results on six of eight patients 12 months after the last treatment session. All subjects complained of quite severe burning pain during irradiation of light source. | Lee et al.108 |
| 1 patient | 20% urea was applied for 10 min and approximately 0.5 mm thick of nails was removed by a micromotor | Haematoporphyrin derivative - 5 mg/ml (Photogem®) | 405 nm at 54 J/cm2 | Seven days after the illumination, there was a clinical evaluation to confirm the response to the treatment. The sessions were repeated once a week, with a total of six sessions of PDT. Complete healing was reported after an irradiation over six weeks and in addition, the microorganism culture indicated negative for fungi. | Silva et al.109 |
| 80 patients | Nail abrasion (rotation abrasive device) | 2% methylene blue aqueous solution | 630 nm at 18 J/cm2 | Group A (40 patients which received PDT treatment) showed a significant response (P < .002) compared with Group B (which received 300 mg oral fluconazole), especially in patients who required nail abrasion (P < .001). The PDT is safe, effective, and well tolerated; it promotes a favorable outcome with good patient adherence and may be considered as a practical and feasible treatment option for toenail onychomycosis. | Souza et al.110 |
| 1 patient | – | Gel with Toluidine blue O (TBO) at 100 μg/ml and propileneglicol (penetration enhancer) | 635 nm at 200 J/cm2 | Three consecutive treatments within1 week were required to induce improvement of nail morphology and showed significant improvement of nail morphology 6 months after. | Mehra et al.111 |
| Data not provided | – | 10 μg/L free curcumin and curcumin encapsulated in nanoparticles | Blue light at 10 J/cm2 | Studies demonstrate reduction of fungal burden after a single treatment using curcumin encapsulated in nanoparticles compared to free drug. | Rosen et al.113 |
| 62 patients | – | Aqueous Solutions of 2% methylene blue and toluidine blue (1:1) and 10% ethanol. | 600 to 750 nm at 18 J/cm2 | The number of sessions ranged from 1 to 22, and sessions occurred once a month. The average number of treatments for complete clearance was five (5.22). Complete clearance was observed in 45% of the patients, while 40% presented partial clearance and 15% presented no change. | Tardivo et al.112 |
In vivo studies with photodynamic inhibition for onychomycosis.
| Number of patients | Pre-treatment | PS and formulation | Wavelength, dose applied | Results | Reference |
|---|---|---|---|---|---|
| 1 patient | 40% urea ointment under occlusion for 7 days and removal of the nail plate. | 5-ALA in 160 mg/g cream | 630 nm at 37 J/cm2 | Cultures of nail were negative and the toenails were considered clinically cured with residual mild traumatic onycholysis | Piraccini et al.103 |
| 2 patients | 20% urea ointment was applied to the nail and covered with a piece of plastic film wrap for 10 hours | 20% 5-ALA acid in aqueous cream | 630 nm at 100 J/cm2 | After the irradiation, the lesion was successfully treated. No dermatophyte was detected on KOH or on culture. | Watanabe et al.104 |
| 3 patients | – | Methylaminolevulinate (MAL-PDT) | 633 nm at 37 J/cm2 | Both potassium hydroxide and culture became negative after MAL-PDT. Appreciable nail improvement was still seen 4 months after treatment. | Taborda et al.105 |
| 30 patients | 20% urea ointment under occlusion and the nail plate could thus be removed easily with the use of forceps | 20% 5-ALA | 570–670 nm at 40 J/cm2 | After one year, 13 patients (43.3%) were cured. Only 5 (16.6%) patients showed complete absence of clinical signs, while 8 (26.6%) patients showed residual changes affecting less than 10% of the nail plate and negative mycological laboratory results. The remaining patients had changes compatible with dermatophyte infection covering more than 10% of the nail plate, as well as positive direct microscopic examination | Sotiriou et al.106 |
| 1 patient | 40% urea ointment under occlusion for 12 hours | Methyl-aminolevulinate (MAL) | 630 nm at 37 J/cm2 | Complete clinical and mycological cure. | Aspiroz et al.107 |
| 8 patients | – | Methyl 5-aminolevulinic acid (MAL) | Not reported | Negative results on six of eight patients 12 months after the last treatment session. All subjects complained of quite severe burning pain during irradiation of light source. | Lee et al.108 |
| 1 patient | 20% urea was applied for 10 min and approximately 0.5 mm thick of nails was removed by a micromotor | Haematoporphyrin derivative - 5 mg/ml (Photogem®) | 405 nm at 54 J/cm2 | Seven days after the illumination, there was a clinical evaluation to confirm the response to the treatment. The sessions were repeated once a week, with a total of six sessions of PDT. Complete healing was reported after an irradiation over six weeks and in addition, the microorganism culture indicated negative for fungi. | Silva et al.109 |
| 80 patients | Nail abrasion (rotation abrasive device) | 2% methylene blue aqueous solution | 630 nm at 18 J/cm2 | Group A (40 patients which received PDT treatment) showed a significant response (P < .002) compared with Group B (which received 300 mg oral fluconazole), especially in patients who required nail abrasion (P < .001). The PDT is safe, effective, and well tolerated; it promotes a favorable outcome with good patient adherence and may be considered as a practical and feasible treatment option for toenail onychomycosis. | Souza et al.110 |
| 1 patient | – | Gel with Toluidine blue O (TBO) at 100 μg/ml and propileneglicol (penetration enhancer) | 635 nm at 200 J/cm2 | Three consecutive treatments within1 week were required to induce improvement of nail morphology and showed significant improvement of nail morphology 6 months after. | Mehra et al.111 |
| Data not provided | – | 10 μg/L free curcumin and curcumin encapsulated in nanoparticles | Blue light at 10 J/cm2 | Studies demonstrate reduction of fungal burden after a single treatment using curcumin encapsulated in nanoparticles compared to free drug. | Rosen et al.113 |
| 62 patients | – | Aqueous Solutions of 2% methylene blue and toluidine blue (1:1) and 10% ethanol. | 600 to 750 nm at 18 J/cm2 | The number of sessions ranged from 1 to 22, and sessions occurred once a month. The average number of treatments for complete clearance was five (5.22). Complete clearance was observed in 45% of the patients, while 40% presented partial clearance and 15% presented no change. | Tardivo et al.112 |
| Number of patients | Pre-treatment | PS and formulation | Wavelength, dose applied | Results | Reference |
|---|---|---|---|---|---|
| 1 patient | 40% urea ointment under occlusion for 7 days and removal of the nail plate. | 5-ALA in 160 mg/g cream | 630 nm at 37 J/cm2 | Cultures of nail were negative and the toenails were considered clinically cured with residual mild traumatic onycholysis | Piraccini et al.103 |
| 2 patients | 20% urea ointment was applied to the nail and covered with a piece of plastic film wrap for 10 hours | 20% 5-ALA acid in aqueous cream | 630 nm at 100 J/cm2 | After the irradiation, the lesion was successfully treated. No dermatophyte was detected on KOH or on culture. | Watanabe et al.104 |
| 3 patients | – | Methylaminolevulinate (MAL-PDT) | 633 nm at 37 J/cm2 | Both potassium hydroxide and culture became negative after MAL-PDT. Appreciable nail improvement was still seen 4 months after treatment. | Taborda et al.105 |
| 30 patients | 20% urea ointment under occlusion and the nail plate could thus be removed easily with the use of forceps | 20% 5-ALA | 570–670 nm at 40 J/cm2 | After one year, 13 patients (43.3%) were cured. Only 5 (16.6%) patients showed complete absence of clinical signs, while 8 (26.6%) patients showed residual changes affecting less than 10% of the nail plate and negative mycological laboratory results. The remaining patients had changes compatible with dermatophyte infection covering more than 10% of the nail plate, as well as positive direct microscopic examination | Sotiriou et al.106 |
| 1 patient | 40% urea ointment under occlusion for 12 hours | Methyl-aminolevulinate (MAL) | 630 nm at 37 J/cm2 | Complete clinical and mycological cure. | Aspiroz et al.107 |
| 8 patients | – | Methyl 5-aminolevulinic acid (MAL) | Not reported | Negative results on six of eight patients 12 months after the last treatment session. All subjects complained of quite severe burning pain during irradiation of light source. | Lee et al.108 |
| 1 patient | 20% urea was applied for 10 min and approximately 0.5 mm thick of nails was removed by a micromotor | Haematoporphyrin derivative - 5 mg/ml (Photogem®) | 405 nm at 54 J/cm2 | Seven days after the illumination, there was a clinical evaluation to confirm the response to the treatment. The sessions were repeated once a week, with a total of six sessions of PDT. Complete healing was reported after an irradiation over six weeks and in addition, the microorganism culture indicated negative for fungi. | Silva et al.109 |
| 80 patients | Nail abrasion (rotation abrasive device) | 2% methylene blue aqueous solution | 630 nm at 18 J/cm2 | Group A (40 patients which received PDT treatment) showed a significant response (P < .002) compared with Group B (which received 300 mg oral fluconazole), especially in patients who required nail abrasion (P < .001). The PDT is safe, effective, and well tolerated; it promotes a favorable outcome with good patient adherence and may be considered as a practical and feasible treatment option for toenail onychomycosis. | Souza et al.110 |
| 1 patient | – | Gel with Toluidine blue O (TBO) at 100 μg/ml and propileneglicol (penetration enhancer) | 635 nm at 200 J/cm2 | Three consecutive treatments within1 week were required to induce improvement of nail morphology and showed significant improvement of nail morphology 6 months after. | Mehra et al.111 |
| Data not provided | – | 10 μg/L free curcumin and curcumin encapsulated in nanoparticles | Blue light at 10 J/cm2 | Studies demonstrate reduction of fungal burden after a single treatment using curcumin encapsulated in nanoparticles compared to free drug. | Rosen et al.113 |
| 62 patients | – | Aqueous Solutions of 2% methylene blue and toluidine blue (1:1) and 10% ethanol. | 600 to 750 nm at 18 J/cm2 | The number of sessions ranged from 1 to 22, and sessions occurred once a month. The average number of treatments for complete clearance was five (5.22). Complete clearance was observed in 45% of the patients, while 40% presented partial clearance and 15% presented no change. | Tardivo et al.112 |
It is interesting to note in Table 3 that the majority of PS studied for onychomycosis are methylene blue dye, methyl-aminolevulinate (MAL), and aminolevulinic acid (ALA). Few studies involved the use of new formulations and the evaluation of PS permeation. Also, for the 189 patients evaluated (Table 3), the best results were achieved with pretreatments to increase PS ungual penetration (urea and/or microabrasion).
Although PDT treatments were associated with mild pain, burning, and erythema, the promising results demonstrate a very innovative and prospective field of study.114
Ultraviolet irradiation
The biological effects of ultraviolet (UV) light on the genetic material of cells have been studied since the early 1890 s. This electromagnetic irradiation is classified into four spectral areas: UV-A (315–400 nm), UV-B (280–315 nm), UV-C (200–280 nm), and vacuum-UV (100–200 nm).115 Especially UV-C is highly germicidal due to its strong absorbance by nucleic acid of microbial cells.116
Within this context, Dai et al.115 studied in vitro and ex vivo inactivation of T. rubrum by UV-C light. Results showed that 3–5 logs of cell inactivation in dermatophytes suspensions were produced with 120 mJ/cm2 UV-C irradiation (254 nm). In addition, resistance of T. rubrum to UV-C did not increase after five cycles of subtotal UV-C inactivation in vitro. Nematollahi et al.117 also pointed UV-A, UV-B, and UV-C light as effective in decreasing colony growth of T. mentagrophytes and T. rubrum.
Cronin et al.118 studies also demonstrated that in vitro exposure at 280 nm with 0.5 J/cm2 was inhibitor to T. rubrum. The study points out, however, that since UV-C wavelengths do not transmit through the nail plate, it would not be totally feasible in onychomycosis treatment. The author discusses that a number of factors such as thickness, cracking, and discoloration of the nail are crucial to determinate results. Nevertheless, the research indicates that a possible application of UV-C could be on decontamination of reservoirs of infection such as the shoes of infected individuals.
In fact, an open-label, prospective pilot study with thirty adults using UV-C for 4 weeks, demonstrated positive outcome to a limited number of patients with mild to moderate onychomycosis.119
These findings suggest the potential application of UV as a noninvasive treatment for superficial onychomycosis and prevention to its reinfection and transmission. Clinical trials are needed, however, to state UV as a safe and effective treatment.
Unresolved challenges
Despite the efforts in the search for new drugs, formulations and approaches,30,120–124 some challenges must be solved in the development of new treatments for onychomycosis.
Some in vitro experiments limitations are described by Elkeeb et al.51 as follow. The use of animal hooves to study drug permeability may not be appropriate to evaluate diffusion, once their composition is quite different from human nails, presenting different permeation to drugs. Also, the modified diffusion cells used to perform the study provides super hydration of the nail matrix resulting in a more soft, flexible, and elastic nail, with increased pore size, which promotes a greater permeation. Therefore, the correlation of in vitro to in vivo studies may not be enough achieved.
Recently, Sleven et al.125 developed a novel in vitro onychomycosis model able to predict both drug penetration and drug activity. It is consisted of a screw vial cap, where the nail is mounted with its opening space positioned above a plate with inoculated growth media, in place of the nail bed. Since the use of water is not necessary, it prevents super hydration, and consequent overrated permeation. The new model was successfully tested for terbinafine, amorolfin, fluconazole, and intraconazole against T. mentagrophytes and T. rubrum, demonstrating potential use for testing novel formulations and onychomycosis drugs.
Regarding the in vivo experiments, a significant challenge is the limited number of patients on past clinical trials, which also limits the power of the studies.89 These data are extremely relevant for decisions on the interruption of ongoing studies, as well as for the improvement of strategies already in use or development of new substances and approaches.
Another important challenge that must be considered is the rising problem of antimicrobial resistance, which increases morbidity and health-care costs. Therefore, it is absolutely essential to establish microbiologic surveillance protocols.8 Although there are increasing research focusing on susceptibility of infectious yeasts, fungi, and mould to antifungal agents, little research has been made on in vitro and clinical fungal resistance.126 In this way, the use of physical therapies, by themselves or in combination with drugs, seems to be an attractive approach. Their effectiveness is more improbable to be associated with the development of microbial resistance due to the rather non-specific nature of the targets in these strategies.89 However, considering especially light-based therapies, it is mandatory to take into account the radiation development of skin cancers due to mutagenic effect on healthy human cells.
Also in this sense, it is important that the concern of adverse effects is considered in the development of any treatment, aiming the selective targeting of microbial cells in preference to host mammalian cells. Thus, the safety of the treatment may be improved by the use of drug delivery technology or more specific physical strategy application.
Throughout above, there are many challenges to be solved and an indication of a clear need for further research on effective and safe therapies to achieve the cure and eradication of onychomycosis.
Conclusion
Onychomycosis prevalence worldwide and the ineffectiveness of conventional treatments have lead current research to focus on novel approaches to enhance drug diffusion trough the nail plate. Many important results have already been achieved either by modifying topical formulations or by applying physical techniques that modify the nail plate itself. However, many challenges still exist and further researches are necessary for the development of effective and safe treatments for onychomycosis.
Acknowledgments
The authors acknowledge the financial support of CAPES (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior, Brazil), CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico, Brazil) and FAPDF (Fundação de Apoio à Pesquisa do Distrito Federal, Brazil).
Declaration of interest
The authors report no conflicts of interest. The authors alone are responsible for the content and the writing of the paper.
