Evaluation of the efficacy and safety of ionic liquids containing ketoconazole in patients with tinea pedis: A randomized controlled clinical trial

Abstract Ionic liquids (ILs) loading ketoconazole (KCZ) have shown better efficacy on rats with tinea pedis than the marketed Daktarin® but clinical studies are still lacking. In this study, we described the clinical translation of ILs containing KCZ (KCZ‐ILs) from the lab into the clinic and evaluated the efficacy and safety of KCZ‐ILs in patients with tinea pedis. Thirty‐six enrolled participants were randomized to receive either KCZ‐ILs (KCZ, 4.72 mg/g) or Daktarin® (control group; KCZ, 20 mg/g) topically twice daily, making the lesion be covered with a thin layer of medication. The randomized controlled trial lasted for 8 weeks including 4 weeks of intervention and 4 weeks of follow‐up. Primary efficacy outcome was the proportion of treatment success responders, defined as patients achieving negative mycological result and ≥60% relative reduction in total clinical symptom score (TSS) from baseline at week 4. Secondary outcomes mainly for evaluating the relapse of disease included the proportion of treatment success individuals at week 8 and fungal recurrence rate at weeks 2, 3, 4, and 8. After 4 weeks of medication, 47.06% of the KCZ‐ILs subjects were treatment successes compared with only 25.00% of those using Daktarin®. Throughout the trial period, KCZ‐ILs induced a significantly lower recurrence rate (52.94%) than that of control patients (68.75%). Furthermore, KCZ‐ILs were found to be safe and well‐tolerated. In conclusion, ILs loading only 1/4 KCZ dose of Daktarin® showed a better efficacy and safety profile in the management of tinea pedis, creating a new opportunity for the treatment of skin diseases caused by fungal infection and is worthy of clinical application.


| INTRODUCTION
Tinea pedis is a common superficial fungal infectious skin disease that exists worldwide. 1 Trichophyton rubrum and Trichophyton interdigitale are reported as two major aetiological dermatophytes responsible for high prevalence and recurrence rates. 2 Although tinea pedis is not fatal, patients have disproportionate itching, pain, and emotional distress, resulting in a significant decline in quality of life.
Ketoconazole (KCZ), an imidazole antifungal agent, is effective for both superficial and deep fungal infections. 3,4 Various reported serious side-effects including liver toxicity, acute renal failure, and dermatological complaints greatly limit the oral administration of KCZ. [5][6][7] In clinical practice, topical preparations containing KCZ have been shown to be helpful in the treatment of tinea pedis, 8 tinea capitis, tinea corporis, 9 tinea nigra, 10 and pityriasis capitis. 11 However, the commonly used 2% KCZ cream was usually prescribed under mild and dramatically improved symptoms, 12,13 or for combined drug administration. 9 In essence, high octanol-water partition coefficient (logP, 4.31), poor solubility (40 μg/ml), and high affinity for keratinized tissues of KCZ lead to the poor transdermal effectiveness and bioavailability, hindering its usage in clinic. 14,15 Despite the availability of several effective KCZ-loaded agents in the topical drug arena, their therapeutic outcome is less than optimal, calling for the development of innovative drug delivery systems.
Ionic liquids (ILs) hold great promise to be optimal active pharmaceutical ingredients or drug carriers owing to enhanced drug solubility, bioavailability, and superior antifungal efficacy. [16][17][18][19][20] Recently, the IL of choline geranate ([Ch][Ger]) has highlighted the potential for effective treatment of tinea pedis. Specifically, our previous study showed [Ch][Ger] had excellent inhibitory activity against T. Interdigitale, a major aetiological dermatophyte of tinea pedis, as well as a significant synergistic antimicrobial effect with KCZ. 2,21 Further, ILs have remarkable permeability, suggesting the ability to clear the dermatophytes in skin more thoroughly. 22 Moreover, the [Ch][Ger]-based formulation in our preliminary research had better performance in treating rats with tinea penis than the marketed Daktarin ® . 21 According to these encouraging results, clinical studies of topical ILs-based formulations for treating microbial infection are worth investigating.
Translation of the formulations from bench to bedside is of great significance, which could be facilitated by scale-up, characterization, stability, mode of action, dosing, preclinical toxicology in animals, and F I G U R E 2 CONSORT diagram of the participants F I G U R E 1 The amount of KCZ delivered into skin after treatment of various formulations. Data are mean ± SD (n = 3), statistics by oneway ANOVA and ****p < 0.0001 compared with Daktarin ® (Control).
finally, human studies on efficacy and safety. 22 Following the first example of clinical translation of ILs technology for the treatment of dermatological conditions in 2020, 22 herein, we demonstrated a small-scale (36 patients enrolled) but successful clinical translation case of KCZ-ILs, a modified aqueous solution of our previously published ILs-based formulation. 21 In this trial, the efficacy and safety of KCZ-ILs and Daktarin ® in patients with tinea pedis were studied systematically, providing a reference to guide more ILs-based clinical transformation in future. To pave the way for ILs from bench to bed, scale-up,   attributing to C O and OH stretch of carboxylic acid were observed. 22 This system yields micelles of 11 nm in diameter determined by dynamic light scattering (DSL, Figure S4), which is similar to those found in other ILs-based micelles. 23 The packaging was performed in brown glass bottles.   KCZ-ILs demonstrated minimal change in stability with 2.9% loss of KCZ and 3.6% loss of geranic acid, respectively ( Figure S5). Although an impurity peak with content of 7.9% appeared in the chromatographic profile of KCZ-ILs, it did not interfere with the determination of key constituents. Additionally, no significant variation in the physical appearance was observed, suggesting that KCZ-ILs can be well preserved for a period of at least 14 months.

| Translational aspects of ILs
Dosing was determined by the in vitro permeation test. The permeability of KCZ in ILs-based aqueous solutions was assessed to determine the optimal formulation for the following clinical study.
Among them, 1/4 and 1/16 KCZ-ILs were obtained by diluting the KCZ-ILs with water 4 and 16 times, respectively. As shown in  Besides the body-mass index, the characteristics of the patients at baseline did not differ significantly between the two groups (Table 1).

| Total clinical symptom score
The change in TSS after 4 weeks of treatment was À4.94 (2.51) (p < 0.001) in the KCZ-ILs group and À4.44 (2.34) (p < 0.001) in the Daktarin ® group. The symptoms began to improve after only 1 week of treatment, and the effect lasted for the whole study period which could be visualized directly from representative photographs of patients with TSS ranged from 8 to 12 ( Figure 3). The difference in TSS between the two groups was statistically significant (F = 58.58, p < 0.001), mainly contributed by weeks 2 and 3 (Table S2).
In order to clarify the specific effect of both formulations, seven signs including lesion size, maceration degree, erosion area, exudation degree, scale, keratosis, and pruritus, directly attributable to tinea pedis at the lesion site were evaluated. The results showed that KCZ-ILs had a significant improvement on lesion area, scaling, keratinization, and pruritus while Daktarin ® tended to relieve scaling and pruritus symptoms (Figure 4, Tables S3 and S4).

| Mycological eradication rates
Mycological eradication rates evaluated by direct microscopic examination were presented in Table 2

| Overall efficacy outcomes
As the primary efficacy variable, overall efficacy was derived by integrating with mycological results and TSS improvement. A four-grade scale (cured, markedly effective, effective, and ineffective) with proper modification of previously published categories was used in this study. 24 Approximately 53% of patients achieved efficacy, 41% attained markedly efficacy, and 6% achieved a complete cure in the KCZ-ILs group, compared to 19% for inefficacy, 56% for efficacy, 19% for markedly efficacy, and 6% for complete cure in the control group. The primary efficacy outcome was the proportion of treatment success (cured and markedly effective) responders. As shown in Table 3 and Figure S6,

| Safety results
No patients applying the KCZ-ILs to lesion sites reported adverse reactions. One patient (6.25%) in the control group experienced transient mild erythema in the lesions 1 week after application of the The proportion of patients changed from negative to positive was calculated as the ratio of the number of new negative to positive cases to the total number of negative cases in the previous period.
drug, which did not affect the continuation of treatment and the determination of efficacy.

| DISCUSSION
Clinical trials are conducted in humans to test new treatments, which are typically evaluated over several phases to determine their efficacy and safety. Phase 1 trials focus on the safety and tolerability, generally with a small number of healthy participants (e.g., 20-80). Phase 2 trials are the first time that new treatments are taken in target patients to observe the preliminary efficacy and safety, and to assess whether they have meaningful benefits to warrant further investigation in phase 3 trials. As phase 3 trials are usually multicenter, large-scale, and long-period trials, phase 2 trials acting as pioneers are of great significance. 25 It is now established that the sample size of phase 2 trials ranges from tens to hundreds, of which a few hundred is more common. 25  Relapse is a result of the re-appearance of the same original strain, which is easy to occur in many diseases and can be influenced by detection methods of post-treatment testing. [33][34][35] The detection rate for strains was reported to differ widely between polymerase chain reaction and culture. 35 Thus, patients that appear to be cured using culture or simple mycological test could actually be harboring residual fungi. Throughout the trial period, KCZ-ILs induced a significantly lower recurrence rate (52.94%) than in control patients (68.75%). Therefore, KCZ-ILs were more effective in fungal clearance and preventing fungal relapse than Daktarin ® .
In terms of safety, one patient in the control group experienced transient mild erythema, but none discontinued the treatment. Meanwhile, KCZ-ILs were found to be safe and well-tolerated.
The limitations of this study include the small sample size and short follow-up period. Thus, long-term maintenance of efficacy and long-term adverse events could not be assessed.

| CONCLUSION
With reference to phase 2 trials, this study was designed to be a ran-

| Formulations and treatment
KCZ-ILs (KCZ, 4.72 mg/g) prepared as described above was used in the experiment group while the commercial Daktarin ® (KCZ, 20 mg/g; Xi'an Janssen Pharmaceutical Co., Ltd.; State Drug Quantifier H20043171) was adopted in the control group. After cleaning the identified lesions, the patients topically self-applied the test product using a cotton swab applicator, where the applied amount was able to make the lesions be covered with a thin layer of medication. The product was applied topically twice daily for 4 weeks.

| Assessments
The primary efficacy outcome was the proportion of treatment success responders, 32 defined as patients achieving negative mycological result and ≥60% relative reduction in TSS from baseline at week 4. Assessments including clinical TSS records and mycological analysis of skin samples, were performed at weeks 0 (baseline), 1, 2, 3, 4, and 8 (endpoint). All the scored lesions on the target foot were evaluated using the criteria incorporating seven signs and symptoms (lesion size, maceration degree, erosion area, exudation degree, scale, keratosis, and pruritus) directly attributable to tinea pedis ( Table S1). The severity of each symptom was stratified into four levels (0 = absence of sign to 3 = worst severity of sign). 30 Skin scraping smears were collected from the infected sites and submitted to mycological testing where KOH examination was used for representing mycological results. As the primary efficacy variable, overall efficacy was divided into four-grade scale including cured, markedly effective, effective, and ineffective.
Among these categories, "Cured" and "Markedly effective" were identified as final clinical efficient or treatment successful. 24 Secondary outcomes mainly for evaluating the relapse of disease included the proportion of treatment success individuals at week 8 and fungal recurrence rate at weeks 2, 3, 4, and 8. Besides, TSS changes, mycological eradication rates, and the compliance at each visit were analyzed. Safety was assessed by physical examination, vital signs, and adverse events, especially irritation based on erythema, peeling, papules, and blisters.

| Statistical analysis
Efficacy analyses were based on the per-protocol population, which comprised the patients who had completed the entire trial as prescribed after randomization. All quantitative variables are represented as mean and standard deviation (SD