Evaluation of Efficacy and Safety of Topical Nanoliposomal Amphotericin B 0.4% Gel as a Potential Treatment for Onychomycosis: An Interventional Pilot Clinical Study

. Onychomycosis is a frequent fungal nail disease that is hard to treat and, in most cases, needs long-term therapy with oral agents. Traditionally, oral agents are favored over topical agents, but it should not be overlooked that they come with broad adverse efects and concomitant drug interactions, which can be unsuitable for many individuals. Terefore, alternative approaches need to be addressed by the medical team for numerous cases. On the other hand, local administration of antimicrobials can come as advantageous because of having a more selective site of activity, avoiding of-target systemic adverse efects, and rapid administration at the site of infection. In this study, we are evaluating a new topical delivery method of amphotericin B. To investigate the efcacy and safety of topical nanoliposomal amphotericin B 0.4% as a possible therapeutic option, this pilot, single-group, before-after clinical study was conducted on 15 onychomycosis patients. Te evaluation was processed during 36weeks of follow-up and on three endpoints of week 12, week 24, and week 36, for both clinical and mycological responses. Tree patients were excluded; of the remaining 12, 50% showed a complete cure, 16.66% had an efective clinical response, 16.66% had a partial clinical response, and 16.66% showed no response at week 12. Mycological cure was calculated as 50% at week 12. At week 24, our measurements were calculated as 91.66% for complete cure, 8.33% for no response, and 91.66% for mycological cure. One patient reported nail plate detachment at week 2 but continued the topical application; follow-up between weeks 2 and 24 showed a complete cure and regrowth of healthy nails. No other adverse efects were detected. Overall, our study suggests that topical nanoliposomal amphotericin B 0.4% is an efective treatment, which is accessible, afordable, and user-friendly, has minimum adverse efects, and could be regarded as an alternative treatment for those ineligible for systemic therapy. Tis trial is registered with IRCT20150101020514N18.


Introduction
Onychomycosis is an umbrella term commonly used to describe all fungal nail infections [1].It is responsible for more than 50% of nail disorders.Onychomycosis generally afects 5.5% of the population worldwide and has an international spread [2].It is the most common nail complaint at dermatology clinics.Age of more than 70 years, immune defciency, and diabetes are some of the known risk factors for developing onychomycosis [3][4][5].
Furthermore, diabetic patients are 2.77 times more likely to develop onychomycosis compared to control [6]; nevertheless, the disease can present itself at younger ages and among people with normal immune system status.Clinical presentation of onychomycosis usually follows an interdigital palmar and/or plantar fungal infection that has progressed to nailbeds.Dermatophyte fungi, nondermatophyte flamentous fungi, and yeasts are the most common causes of onychomycosis [1].Trichophyton, Epidermophyton, and Microsporum among dermatophyte species (spp.);Aspergillus and Fusarium among nondermatophyte flamentous spp.; and Candida, Malassezia, and Trichosporon among yeast spp.are the leading pathogens causing onychomycosis [1].Clinically speaking, Trichophyton rubrum and Trichophyton interdigitale from the dermatophyte family are the main responsible fungi for the majority of the cases.
Several systemic and topical medications are available for the treatment of onychomycosis.Traditionally, oral agents are favored over topical agents by clinicians.Te reason behind this is that topical therapy normally has a lower difusion rate into the nailbeds and hence takes more time to reach a favorable clinical response.Terbinafne, itraconazole, and fuconazole are some of the available oral agents.Among those patients who have completed their treatment cycle, these agents have shown response rates of 76%, 58%, and 48% in clinical studies, respectively [7,8].Oral itraconazole or terbinafne are traditionally recommended as the frst-line therapy by clinicians [8]; nonetheless, it should not be overlooked that due to their broad adverse efects and concomitant drug interactions, both are unsuitable for many individuals.Terefore, alternative approaches need to be addressed by the medical team for numerous cases [9].Multiple topical agents are also at the disposal, such as ciclopirox olamine, clotrimazole, and efnaconazole.Despite their conventional lower potencies and more limited on-site penetration, local administration of antimicrobials can come as advantageous because of having a more selective site of activity, avoiding of-target systemic adverse efects and rapid administration at the site of infection.Furthermore, the recent progress made in the feld of drug delivery such as novel molecular confgurations for the drug or its vector (e.g., liposomal forms), or local laser-based drug delivery with fractional ablative lasers to increase the absorption of topical treatments [10], has ofered new perspectives for clinicians to reconsider topical therapies.Overall, the efcacy of current therapies varies in diferent individuals and independent studies that are conducted at diferent times [7], possibly due to recent fungal drug resistance and nonselective general treatments that fail to target the causative fungi.Terefore, onychomycosis seems to have become more frequent than any time before.Tese observations alarm the need for fnding novel and more efcient therapeutic approaches for the treatment of onychomycosis that are suitable and afordable for all patients while keeping the unwanted systemic adverse efects to the minimum [7].Tis study aims at evaluating the efcacy of a newly manufactured topical agent (topical nanoliposomal amphotericin B 0.4%) as a potential treatment for onychomycosis in the setting of a pilot clinical trial study.
Amphotericin B is an antibiotic produced by Streptomyces nodosus [11] and has been used for a long time for the treatment of systemic fungal infections [12].Its mechanism of action mostly relies on pore formation in the fungal plasma membrane, thus inducing cell death by osmotic lysis.Systemic administration of amphotericin B can come with certain adverse efects, including nephrotoxicity, discomfort at the site of injection, and constitutional symptoms and therefore should be prescribed selectively with prior precaution and indication [13].Liposomal amphotericin B (AmBisome ® ), lipid complex amphotericin B (Abelcet ® ), and colloidal dispersal of amphotericin B are the current available lipid-based formulations of this drug [14].
Nanoliposomal forms of amphotericin B are novel formulations of this drug with minimal toxic efects.Tey are capable of passing natural biological skin barriers by targeting cutaneous macrophage cells in the dermis and epidermis [15].Topical nanoliposomal amphotericin B 0.4% is one of the recently available agents of this group.It has a size of approximately 100 nm and its efciency has been evaluated as promising for the treatment of cutaneous leishmaniasis in previous studies [16].An in vitro study of topical nanoliposomal amphotericin B 0.4% for the treatment of fungal infection has shown compelling results for two clinically important dermatophytes (T.rubrum and T. interdigitale (recently renamed as T. indotineae)) [17], yet the drug has not been clinically assessed among patients.Te purpose of this study is to evaluate the efcacy of topical nanoliposomal amphotericin B 0.4% for the treatment of onychomycosis in a clinical pilot study.We pharmaceutically produced topical nanoliposomal amphotericin B 0.4% gel (SinaAmpholeish ® 0.4%) at Minoo Company in Tehran, Iran, for the upcoming administration in the clinical setting [18].
Specifcally, the investigators of this study wish to (I) determine the safety and efcacy of this therapeutic approach for the treatment of onychomycosis, (II) determine the required treatment duration to cure the nails and eliminate the fungus, and (III) report posttreatment fndings including treatment failure, reinfection, or recurrence.

Study Design and Ethical Statement.
Te study was conducted from December 2021 to October 2022 as a pilot, single-group, before-after study with a sample size of 15 patients who were afected by onychomycosis.Treatment was continued for a period of 12-36 weeks with a primary endpoint follow-up at week (W) 36.Before the study, all patients had a private visit with the medical team in which all of their medical questions were addressed and a full medical and drug history was obtained, including any history of previous antifungal therapy.Key inclusion and exclusion criteria are defned in Table 1.
A consent form was later given to the clinically eligible people to be read and signed.All patients agreed to the study protocol and were fully aware of the therapeutic process and were informed about all of their other treatment options.Tis research was permitted by the Iranian Registry of Clinical Trials, with reference no.IRCT20150101020514N18.

Subjects.
In the beginning, this study included 15 subjects (males: 6 individuals and females: 9 individuals) with an age range of 18-60 years.Patients were entered into the study regardless of their underlying causative fungus and there were patients with either dermatophyte, nondermatophyte, or yeast infection.Onychomycosis was diagnosed clinically and paraclinically using medical assessment along with mycological testing.Fungal nail infection was defned as at least one fngernail or toenail involvement that has been confrmed via direct microscopic examination and positive culture.

Intervention.
Following diagnosis confrmation by the medical team, eligible patients were prescribed 0.4% nanoliposomal amphotericin B gel.Tey were asked to apply the gel topically twice daily on the entire surface of the afected nails and a 6 mm margin around the cuticle.Te time anticipated to reach clinical response was predicted to be 12 weeks for fngernails and 36 weeks for toenails.After the primary visit, 3 subsequent visits were arranged for patients at weeks 12, 24, and 36, respectively.
2.4.Assessment.Te primary assessment was to evaluate the efcacy of nanoliposomal amphotericin B 0.4% gel for the treatment of infected nails based on a comparison of clinical and mycological status before and after receiving the treatment at weeks 12, 24, and 36.On each session, patients were clinically examined at the center by a dermatologist, and their data were recorded in the registry.A visual analog scale (VAS) was used in each session to evaluate patient satisfaction.VAS is a numerical (1-10 score) psychometric satisfaction questionnaire.Patients were then referred to do laboratory tests including direct microscopic potassium hydroxide (KOH) smear and culture every 12 weeks.Furthermore, a photograph was taken of the status of the infected nails at each of the times of the visit to record the gradual possible efects of the therapy.Te fndings of each session were subclassifed into 2 major groups: (i) Mycological assessment scale (MAS), based on the microscopic diagnosis of mycologist: this includes four grades of mild, moderate, severe, and mycological cure (MC).MC is defned as negative direct KOH smear microscopy plus negative culture.(ii) Clinical response, based on the physical examination report of the dermatologist: this includes (1) partial clinical response (PCR), defned as a visible improvement of nailbed involvement which still afects 10%-50% of the target nail plate, and (2) efective clinical response (ECR), defned as a visible improvement of nailbed involvement that afects less than 10% of the target nail plate.
Eventually, complete cure (CC) is defned as total infection-free nail plate + MC.

Safety Evaluation.
Dryness, scaling, erythema, allergic infammatory reactions, burning, pruritus, and some other local adverse efects have been previously reported with the usage of topical antifungal medications.Electrolyte disturbance (potassium and magnesium), anemia, nausea, diarrhea, allergic rash, nephrotoxicity, headaches, and constitutional symptoms have been reported with systemic intravenous therapies with amphotericin B.
Te probable transdermal distribution of amphotericin B into blood circulation has been previously ruled out by Van Bocxlaer et al. [19].Te assumed distribution volume of liposomal amphotericin B is reported to be 0.1-0.44L/kg.Te maximum therapeutic index of this medication is approximately 1-1.5 mg/kg [20].Liposomal formulation of amphotericin B contains 4 mg of active

(A) Key inclusion criteria
Male or female subjects of any race, 18 to 60 years of age (inclusive) Verbal and written informed consent/assent obtained from the subject Good general health, as assessed by the investigator, based on the subject's medical history, physical examination, and safety laboratory tests Target nails for all subjects must have had evidence of nail growth, per subject's report that monthly clipping is needed Subjects are willing to comply with study instructions and return to the vising clinic for all required appointments every 12 weeks for at least 3 visits (B) Key exclusion criteria Male or female who have received oral/IV antifungal therapy within the past 12 weeks prior to screening If topical antifungal medication was used in the past four weeks prior to testing Patients who had a history of immunosuppression or clinical evidence indicating possible immunosuppression Uncontrolled diabetics Patients who have performed a surgical intervention for nail dystrophy in the past Any illness or condition that could have caused nail anomalies or adversely afected the assessment, or the presence of any nail infection other than onychomycosis or in addition to onychomycosis Patients who had received immunosuppressive therapy in the past 3 months prior to screening visit or who had the need for it Females who are pregnant, nursing a child, or planning a pregnancy during the study duration Dermatologic Terapy ingredient per 1 gr of the gel.Assuming that the used amount of the gel at each time of application would be approximately 0.5 gr, there would be a sum of 2 mg of the active ingredient of amphotericin B in each dose of treatment.Considering the apparent volume of distribution, the drug concentration in the plasma of a 70 kg individual will be between 0.06 and 0.28 mg/L, which is considerably below the maximum therapeutic level for this agent (2.26-10 mg/L).All these suggest that it seems unlikely for the topical form of amphotericin B to lead to any of the systemic adverse efects of the injectable form.Consequently, it was estimated that systemic side efects such as nephrotoxicity and those related to higher injectable dosages of amphotericin B would be less likely to happen.Nevertheless, all the patients were monitored for any possibility of drug reactions.
2.6.Antifungal Susceptibility Testing.All isolates were tested for antifungal susceptibility to terbinafne, itraconazole, fuconazole, and voriconazole (all reagents from Sigma-Aldrich) based on the broth microdilution method and the CLSI-M38 3rd ed guideline [21].Minimum inhibitory concentrations (MICs) were determined using the visual examination of fungal growth after 24-72 hours according to the fungal species by an expert mycologist.Te Candida parapsilosis ATCC 22019 was used as the quality control.

Statistical Methods.
Percentage and frequency were used to explain qualitative records.Furthermore, the MIC range and MIC 50 were calculated.All the statistical calculations were conducted using the IBM SPSS ® application, version 22.0.

Participant Demographic Data.
Fifteen patients (9 females (60%) and 6 males (40%)) were included in the study.Te demographic data and clinical characteristics of these 15 patients are shown in Table 2.

Early Efciency Endpoint (W12).
Two participants were excluded from the study at week 12; one of which had become pregnant and the other one had a renal failure exacerbation and a complementary need for a kidney transplant, which was irrelevant to this clinical trial.One was later excluded at W24 and was not included in the analysis.Of the remaining 12 patients, 6 had CC (50%), 2 had ECR (16.66%), 2 had PCR (16.66%), and 2 had no response (NR) (16.66%) at W12. MC was evaluated as 50% (N � 6/12) (Table 4).

Secondary Efciency Endpoint (W24-W36
).At W24, our measurements were evaluated as 91.66%CC (N � 11/12), 8.33% NR (N � 1/12), and 91.66% MC (N � 11/12).All patients with PCR and ECR in the previous appointment had reached the therapeutic milestone (CC) at W24. Monitoring detected a lack of therapeutic progress in one of the patients.Tis patient was a female infected with Fusarium solani fungus and further medical investigation found out that the subject used nail polish during the therapy and that lack of compliance despite patient education was the main reason behind treatment failure.Te patient was excluded from the study at week 24 and was not included in the analysis.Tere was another patient who did not show any therapeutic response in the clinical setting or the mycological evaluation until the end of the study period (W36).Tis patient had an infection with Candida glabrata and more than 50% nail plate involvement at the beginning of the study.He was eventually considered the only NR patient (Table 4).A yellowish-to-brownish change in the color of the target nails was the most common complaint of this study, starting at W4 on the site of the topical application.At W12 (frst follow-up appointment), all of the 12 actively participating patients had reported a change in nail color, which was also detected by the medical team during the examination.2 of the patients that had experienced this discoloration at the beginning of W4 reported that the color change started to resolve after a while and was fully reversed by W12.Tere was medical documentation of this progress via the sent photographs of the patients to the medical team.One patient reported nail plate detachment at W2 but continued the topical application of the drug on the nailbed until W24, and further follow-up of the patient between W2 and W24 showed CC and regrowth of healthy nails.No other adverse efect was detected throughout this study.Figures 1-3 illustrate the clinical response of onychomycosis before and after treatment with topical nanoliposomal amphotericin B 0.4%.

Discussion
Tis study represents an interim report of the frst assessment of the safety and efcacy of topical nanoliposomal amphotericin B 0.4 for the treatment of onychomycosis and demonstrates efective therapeutic results with minimal adverse efects and no relapse rates.Te defned cure milestones of this study were 12 weeks of treatment for fngernail infection and 24 weeks of treatment for toenail infection, which were achieved for 11/12 of the participants.
Tis experiment included subjects with a broad spectrum of clinical presentations of onychomycosis, including diferent sites of infection and three distinguished clinical subclasses of onychomycosis (DLSO, PSO, and WSO), all of which had an efcient response to the therapy (Table 4).6 of the cases were older than 50 years of age.Given the prior knowledge that older age is related to probable less efcient blood circulation in small vessels, such as those in nails, it is assumed that a clinical response might take longer time due to slower suppliance for nail regrowth.Yet all of these patients (except for one (patient ID: 8) whose case is discussed in detail later in this section) also reached the therapy milestones as anticipated (fngernail at 12 weeks and toenail at 24 weeks) as well as other participants.
Tere are two previous studies about the usage of topical amphotericin B for the treatment of onychomycosis, one with amphotericin B in 30% dimethyl sulfoxide and the other with 0.2% topical amphotericin B in 50% dimethyl sulfoxide.Both of these had promising results with MC of 80% (8/10) plus the clinical cure of 70% (7/10) over 72 weeks for the frst study and MC of 87.5% (7/8) over 52 weeks for the latter [22,23].To our knowledge, this is the frst clinical trial that has evaluated the nanoliposomal form of amphotericin B for the treatment of onychomycosis.
At W12 of this trial, MC and CC were both calculated as 50% (CC: 1/6 toenail (TN), 4/6 fngernail (FN), and 1/6 TN + FN) (Table 4).We had one patient with a toenail infection (patient ID: 4) that reached CC at W12 which was earlier than anticipated (W36); this patient had C. albicans DLSO.Tere was also another subject with mixed TN and FN infection that was completely cured at W12 (patient ID: 1).We deduced that this early response is related to higher penetration levels of the liposomal form medication that infltrates the dorsal nail plate easier and advances mycological clearance and that patients with less involvement are more likely to reach the therapeutic goal sooner than assumed as both of these cases had less than 10% nail plate involvement on each of the target nails.Tere was a considerable increase in MC (91.66%) and CC (91.66%) at W24 compared to the ones at W12.All of the patients with PCR and ECR at W12 reached CC by W24, suggesting that longer durations of treatment (24 weeks) might come with better therapeutic response rates and that the results would continue to increase over time.We had one patient (patient ID: 4) who had FN DLSO with 20% nail involvement and was resistant to the previous oral therapy with fuconazole.Furthermore, she had a MIC greater than 1 mg/mL for fuconazole, confrming resistance as the reason for her previous treatment failure.She reached CC with nanoliposomal amphotericin B 0.4% at W12 with no report of prominent side efects, which according to the recent reports of antifungal resistance can be accounted as a promising result to consider.One of the subjects (patient ID: 8)    did not reach the therapeutic milestone till the end of the study at W36.He was a 54-year-old man with no recorded past medical history of apparent cardiovascular disease nor any other condition that afects nail reperfusion and the healing process.He had previously received oral fuconazole (150 mg once daily for 6 months) but had little to poor response to the treatment.His medical evaluation identifed C. glabrata DLSO in his big toe with over 50% nail plate involvement (this patient had the highest surface involvement among our subjects).Mycological and clinical evaluations at W12, W24, and the fnal appointment at W36 showed no progress.However, he mentioned slight improvement via correspondence with the medical team post-W36, although there is no ofcial documentation of this progress.We attributed this poor response to multiple reasons: frst, this patient had more than 50% surface involvement, and second, the comparison between the fndings obtained from the MIC test (MIC: 0.125 in sessile cells, considered as sensitive) and the clinical outcome (unresponsive) demonstrates that there is a possible additional resistance mechanism in the clinical setting possibly due to bioflm formation of C. glabrata.Except for nail discoloration, we found no other general adverse efects in this study.Compared to the side efects of other treatments with the same efcacy that come with more prominent unwanted oftarget systemic efects, we can say that nail discoloration can be a rather acceptable outcome.Some of the patients reported a normal color recovery after a while; nonetheless, the duration of this study was not long enough to evaluate whether this adverse efect is reversible or not.All subjects were followed up to 6 months after complete cure with nanoliposomal amphotericin B 0.4%.Tere was no relapse.
To better evaluate our clinical results, we decided to also test nanoliposomal amphotericin in an in vitro setting with an antifungal susceptibility test.Te fungicidal activity of nanoliposomal amphotericin B 0.4% is compared with conventional antifungal agents (voriconazole, itraconazole, fuconazole, and conventional amphotericin B) and nanoliposomal vehicle in Table 3. Primarily, the MIC experiment demonstrated that the nanoliposomal vehicle has an independent antifungal activity (MIC range of 0.03-0.25 compared to itraconazole MIC range of 0.03-1 for C. albicans, and MIC range of 0.03-0.5 compared to itraconazole MIC range of 0.03-0.5 for C. glabrata.Itraconazole is considered the standard control treatment).Also, it was found that amphotericin B and the liposomal vehicle have a synergistic therapeutic efect since MIC range and MIC 50 were more ideal in the case of nanoliposomal amphotericin B compound compared to each of the compounds separately (Table 3).Furthermore, nanoliposomal amphotericin B had lower MIC ranges than other oral medications in all species (Table 3), suggesting that it is potentially a quasi-efcient alternative that demands lower doses and that comes in a topical form without the of-target systemic adverse efects.

Conclusions
Overall, our study suggests that topical nanoliposomal amphotericin B 0.4% is an efective treatment, which is accessible, afordable, and user-friendly and comes with minimum adverse efects and it could be regarded as an alternative treatment for patients who are ineligible for systemic treatment.

Study Limitations.
Tis was a clinical pilot study and our main goal was to evaluate the preliminary outcomes of this novel format of amphotericin B for the treatment of onychomycosis; however, we had limitations to compare and comment on the efect of the drug for diferent subtypes of onychomycosis, due to small sample size and lack of randomization.Another limitation of this study was the targeted species; we had two cases that were afected by nondermatophyte molds (F.solani).Unfortunately, both of these cases were excluded during the task of study: one due to pregnancy and the other due to medical emergency, respectively, which restricted us from drug assessment among nondermatophyte subtypes.To achieve both of these goals, future studies with randomized larger sample sizes and several arms should be defned.

Table 4 :
Fungal species, history of antifungal treatment, and outcome of topical nanoliposomal amphotericin B 0.4% in onychomycosis patients.

Table 1 :
Inclusion and exclusion criteria.

Table 3 :
MIC range and MIC 50 of antifungals against fungal species isolated in this study.