Formulation, Optimization, and Evaluation of a pH-Responsive In-situ gel of Tavaborole using Design of Experiments (DOE)

Abstract

Onychomycosis, a chronic fungal infection of the nail, presents challenges in topical treatment due to the nail plate’s dense keratin structure and limited drug permeation. Tavaborole, a boron-containing antifungal agent, offers potent activity against dermatophytes but requires optimized formulations for effective transungual delivery. The present study aimed to formulate and optimize pH-responsive in-situ gels of Tavaborole using a Quality-by-Design (QbD) approach and Design of Experiments (DOE) methodology. Tavaborole was characterized as a clear, colorless liquid with no particulate matter, demonstrating purity and stability. Solubility studies revealed poor aqueous solubility but enhanced solubility in ethanol, methanol, and acetone, confirming its hydrophobic nature. UV-visible spectrophotometry at 272 nm provided a reliable method for quantitative analysis, with a linear calibration curve (R² ? 1.0). Various concentrations of Carbopol 934, HPMC K4M, and Triethanolamine were evaluated, significantly influencing viscosity (3426–4717 cps), gelation time (130–228 sec), pH (6.3–6.6), and drug release profiles. Formulations F12, F2, and F10 demonstrated the highest drug release (up to 92.51%) with suitable gelation and viscosity, ensuring effective and sustained topical delivery. FTIR analysis confirmed stable drug-excipient interactions, contributing to gel formation and controlled release behavior. Minimal bias percentages and predictive regression models validated the formulation process. Overall, the study establishes that the synergistic combination of Carbopol 934, HPMC K4M, and Triethanolamine enables the development of stable, patient-friendly, and efficient Tavaborole in-situ gels for onychomycosis treatment, with potential for improved therapeutic outcomes and patient compliance.

Keywords

Tavaborole; Onychomycosis; pH-responsive in-situ gel; Carbopol 934; HPMC K4M; Drug release

Introduction

Onychomycosis is a persistent fungal infection of the nail unit, primarily caused by dermatophytes, but occasionally by yeasts and non-dermatophyte molds. It is one of the most common nail disorders worldwide, accounting for a significant portion of nail abnormalities seen in clinical practice [1]. The condition not only affects the aesthetic appearance of nails but also poses functional limitations, discomfort, and in some cases, secondary infections, particularly in immunocompromised individuals and patients with diabetes [2]. Treatment of onychomycosis remains challenging due to the dense keratinized structure of the nail plate, which acts as a formidable barrier to drug penetration, and the slow growth rate of nails, which prolongs treatment duration [3]. Conventional systemic antifungal therapies, although effective, are often associated with hepatotoxicity, drug–drug interactions, and other systemic side effects [4], whereas topical treatments typically suffer from poor nail penetration and insufficient retention time, limiting their therapeutic efficacy. Tavaborole, a novel boron-containing antifungal agent, has emerged as a promising candidate for topical treatment due to its potent activity against dermatophytes, favorable safety profile, and ability to disrupt fungal protein synthesis [5]. Despite these advantages, achieving sustained drug release and sufficient permeation through the nail plate remains a significant challenge [6]. This has led to growing interest in innovative drug delivery systems that can enhance drug bioavailability, prolong residence time, and improve patient adherence. In-situ gel systems have garnered considerable attention as a potential solution to these limitations [7]. These formulations are applied in a liquid or semi-liquid state and undergo a sol-to-gel transition in response to specific physiological stimuli such as pH, temperature, or ionic strength. pH-responsive in-situ gels are particularly suitable for topical applications in the nail environment, as the formulation can transform into a gel upon contact with the slightly alkaline pH of the nail bed [8]. This transition not only increases the contact time of the drug at the target site but also facilitates controlled and sustained drug release, improving therapeutic outcomes. Additionally, in-situ gels can enhance patient compliance due to their ease of application, minimal irritation, and non-invasive nature [9]. The development of an optimized pH-responsive in-situ gel requires a systematic approach to formulation design and evaluation. Quality-by-Design (QbD) is an established framework that emphasizes a thorough understanding of formulation and process variables to ensure the desired product quality [10]. Coupled with Design of Experiments (DOE), QbD enables the systematic investigation of critical factors affecting formulation performance, such as polymer type and concentration, viscosity, gelation time, and drug release profile. By applying this structured methodology, it is possible to identify optimal formulation parameters, reduce variability, and ensure reproducibility. This study aims to formulate and optimize a pH-responsive in-situ gel containing Tavaborole for the treatment of onychomycosis, leveraging the principles of QbD and DOE. By integrating advanced formulation strategies with a systematic optimization approach, this study seeks to address the limitations of conventional topical antifungal therapies and provide an effective, patient-friendly solution for onychomycosis management. The findings are expected to contribute to the development of clinically relevant, optimized transungual delivery systems that can enhance drug efficacy, reduce treatment duration, and improve overall patient outcomes.

METHODOLOGY

1. 1.  Pre-formulation Studies
      1. Organoleptic Evaluation

The organoleptic characteristics of Tavaborole, including its color, odor, and physical appearance, were observed visually under adequate lighting conditions. The drug was examined for any unusual coloration, particulate matter, or odors that might suggest contamination or degradation. These observations provided initial qualitative information about the drug's purity and physical state [11].

1. 1. 2. Solubility Studies

Solubility studies were conducted to determine the solubility of Tavaborole in various solvents such as distilled water, ethanol, methanol, phosphate buffer (pH 5.5 and 7.4), and acetone. An excess amount of drug was added to 10 mL of each solvent in separate stoppered conical flasks and shaken continuously in a shaking water bath at 25?±?2°C for 24 hours. The solutions were then filtered through Whatman filter paper, and the filtrate was analyzed spectrophotometrically to determine the amount of drug dissolved in each solvent [11].

1. 1. 3. UV Spectrophotometric Analysis and λmax Determination

A stock solution of Tavaborole was prepared by dissolving an accurately weighed amount of the drug in phosphate buffer (pH 5.5) and scanning the solution in the UV range of 200–400 nm using a UV-Visible spectrophotometer. The wavelength at which the drug exhibited maximum absorbance (λmax) was identified and recorded. This wavelength was used for subsequent quantitative analysis [11].

1. 1. 4. Standard Calibration Curve of Tavaborole

To construct the calibration curve, a series of standard solutions of Tavaborole were prepared in phosphate buffer (pH 5.5) at concentrations ranging from 2 to 20 µg/mL. The absorbance of each solution was measured at the previously determined λmax using a UV-Visible spectrophotometer. A calibration curve was plotted by taking absorbance on the Y-axis and concentration on the X-axis. The curve was assessed for linearity and the correlation coefficient (R² value) was calculated to confirm reliability for further quantitative estimations [11].

1. 1. 5. Fourier-Transform Infrared Spectroscopy (FTIR) Compatibility Studies

FTIR spectroscopy was used to evaluate possible interactions between Tavaborole and the selected polymers. The spectra of the pure drug, individual excipients, and physical mixtures of the drug with polymers were recorded using an FTIR spectrometer. Each sample was mixed with potassium bromide (KBr), compressed into a pellet, and scanned over a range of 4000–400 cm?¹. The presence, absence, or shifting of characteristic peaks was analyzed to identify any potential chemical interactions [11].

1. 2. Formulation of tavaborole in-situ gel

A 2³ full factorial design was employed using Design-Expert® software to systematically evaluate and optimize the formulation variables. The three independent formulation factors selected were:

• A: Carbopol 934 (% w/v) – levels: 0.1% (Low), 0.7% (High)
• B: HPMC K4M (% w/v) – levels: 0.1% (Low), 2.0% (High)
• C: Triethanolamine (% w/v) – levels: 0.1% (Low), 0.3% (High)

This design resulted in 8 experimental runs, with 4 center point replicates to ensure model validity and reproducibility, giving a total of 12 runs. The in-situ gel formulations containing Tavaborole were prepared using a cold method. Initially, weighed quantities of Carbopol 934 and HPMC K4M were slowly dispersed in distilled water with continuous stirring using a magnetic stirrer. The dispersion was allowed to hydrate for 12 hours at room temperature to ensure complete swelling of the polymers. Separately, Tavaborole was dissolved in a small volume of ethanol to enhance solubility and was gradually added to the hydrated polymer dispersion under gentle stirring to ensure uniform drug distribution. Triethanolamine was then added dropwise to the mixture to adjust the pH to the physiological range (approximately 6.8–7.4), which facilitates sol-to-gel transition upon application to the nail bed. PEG was added to enhance the penetration. The final volume was made up with distilled water, and the formulation was stirred until a clear and homogeneous solution was obtained. Each batch was labeled (F1–F12) according to the factorial design matrix and stored in airtight containers until further evaluation [12].

1. 3. Post-formulation Evaluations
      1. Clarity and Appearance

The clarity and physical appearance of the prepared in-situ gel formulations were visually inspected immediately after preparation. Each formulation was checked for the presence of any suspended particles, turbidity, or discoloration against both black and white backgrounds under natural light. This assessment ensured uniform dispersion of polymers and the drug without any precipitation or incompatibility [12].

1. 1. 2. pH Measurement

The pH of each formulation was measured using a calibrated digital pH meter. Prior to measurement, the electrode was rinsed with distilled water and dried with tissue paper. The electrode was then immersed directly into the formulation, and the pH reading was recorded once stabilized. This step was crucial to confirm that the formulation's pH was within the acceptable physiological range for nail application [12].

1. 1. 3. Viscosity Determination

The viscosity of each in-situ gel formulation was determined using a Brookfield viscometer equipped with a suitable spindle (usually spindle no. 64). The formulation was transferred into a beaker, and the spindle was immersed in the sample. Measurements were taken at a constant speed (usually 10 rpm) at room temperature. The viscosity in centipoise (cps) was recorded, indicating the formulation's flow behavior and ease of application [12].

1. 1. 4. Gelation Time

To determine gelation time, 1 mL of each formulation was added dropwise into a test tube containing 5 mL of simulated tear fluid (pH 7.4) maintained at 37?±?0.5°C. The time taken for the liquid to transform into a gel was recorded using a stopwatch. Gelation was considered complete when there was no flow of the formulation upon tilting the test tube horizontally [12].

1. 1. 5. Drug Content

To determine drug content, 1 mL of the formulation was accurately measured and diluted with phosphate buffer (pH 7.4). The solution was sonicated to ensure complete dissolution of Tavaborole and then filtered through a 0.45 µm membrane filter. The filtrate was analyzed using a UV-visible spectrophotometer at the λmax of Tavaborole. The drug content was calculated using a previously established calibration curve [13].

1. 1. 6. In-vitro Drug Release Study

The in-vitro drug release was assessed using a modified Franz diffusion cell. A pre-soaked dialysis membrane (or cellophane membrane) was mounted between the donor and receptor compartments. The receptor compartment was filled with phosphate buffer (pH 7.4) maintained at 37°C and stirred continuously. 1 mL of the gel was placed in the donor compartment. At predetermined time intervals, aliquots were withdrawn from the receptor compartment and replaced with fresh buffer. The samples were analyzed spectrophotometrically to determine the cumulative percentage of drug released over time [14].

RESULTS AND DISCUSSION

1. 4.  Organoleptic Characteristics of Tavaborole

The organoleptic properties of Tavaborole were evaluated to assess its color, odor, and physical appearance, ensuring purity, stability, and integrity. The drug was a clear, colorless liquid with no visible discoloration, indicating it remained stable and free from degradation. No unusual or off-putting odor was detected, suggesting absence of volatile impurities, microbial contamination, or chemical breakdown. Visual inspection revealed no particulate matter, confirming the formulation is contaminant-free and adheres to sterility standards. The clear, colorless, and odorless characteristics indicate that Tavaborole has not been exposed to environmental factors such as light or heat. These observations confirm the drug’s stability, purity, and quality. Overall, the organoleptic evaluation supports the safety and efficacy of Tavaborole for topical antifungal use, providing assurance of consistent therapeutic performance.

1. 5. Solubility Studies of Tavaborole

Solubility studies of Tavaborole were performed in distilled water, ethanol, methanol, phosphate buffer (pH 5.5 and 7.4), and acetone to assess its behavior in aqueous, organic, and physiological environments. Excess Tavaborole was added to 10 mL of each solvent, shaken at 25?±?2°C for 24 hours, filtered, and analyzed spectrophotometrically. Tavaborole showed poor solubility in distilled water, confirming its hydrophobic nature. Moderate solubility was observed in ethanol and slightly lower in methanol, indicating better affinity for polar organic solvents. In phosphate buffers, solubility was pH-dependent, being lower at pH 5.5 and higher at pH 7.4, suggesting improved dissolution under near-physiological conditions. Tavaborole exhibited high solubility in acetone, a non-polar solvent, highlighting its lipophilic character. These results indicate that Tavaborole’s solubility is enhanced in organic and slightly basic environments, which is important for topical formulation development. The findings suggest the need for solubilizing agents, co-solvents, or buffered systems to improve drug dissolution and bioavailability. Understanding these solubility characteristics is crucial for designing effective Tavaborole-based topical therapies, ensuring adequate drug delivery to the site of infection and enhanced therapeutic efficacy.

1. 6. Calibration Curve for Tavaborole

To construct the calibration curve for Tavaborole, a series of standard solutions were prepared with concentrations ranging from 2 to 20 µg/mL in phosphate buffer (pH 5.5). The absorbance of each solution was measured at the wavelength of maximum absorbance (272 nm), which had been previously determined using UV-Visible spectrophotometry.

1. 8. Evaluation of tavaborole in-situ gel
      1. Viscosity

Viscosity is a key parameter affecting spreadability, patient comfort, and drug release. The combination of Carbopol 934, HPMC K4M, and Triethanolamine significantly influences gel consistency. F4 exhibited the highest viscosity (4717 cps), indicating a thick gel suitable for sustained and localized drug delivery. Formulations F1, F2, F5, F10, and F12 (3966 cps) showed moderate viscosity, balancing spreadability and retention. F6 had the lowest viscosity (3426 cps), producing a thinner, more fluid gel for easier application and faster absorption. Formulations F3, F4, F5, and F9 (3916–4583 cps) provide intermediate gel thickness, offering a compromise between slow-releasing and fast-absorbing gels. Overall, viscosity variations reflect the impact of polymer content on gel performance and potential clinical applicability.

1. 1. 2. Gelation time

Gelation time is a critical parameter affecting the usability and therapeutic performance of gel formulations. Carbopol 934, HPMC K4M, and Triethanolamine influence gelation time depending on their concentration and ratio. Formulations F1, F2, F10, and F12 gelled quickly (131–133 sec), suitable for rapid drug delivery and immediate local effect. F3 and F4 (161–162 sec) provided moderate gelation, balancing fast formation with extended residence for controlled release. F5, F6, F7, and F8 (207–228 sec) gelled slowly, allowing greater spreadability and longer retention at the application site. F9 (197 sec) showed intermediate gelation, offering a compromise between speed and duration. Gelation time data guide formulation selection based on desired application, drug release profile, and therapeutic requirements. Faster-gelling formulations are ideal for immediate treatments, while slower-gelling gels suit prolonged action.

Viscosity (cps):

• Predicted Viscosity: 3917 cps
• Experimental Viscosity: 3900 cps
• Bias: 0.44%

The viscosity results show a minor difference between the predicted and experimental values, with a bias of 0.44%. This slight variation indicates that the viscosity is consistent and the formulation behaves as expected in terms of gel strength. The slight reduction in experimental viscosity compared to the predicted value is within an acceptable range and reflects small experimental or environmental factors during formulation.

Drug Release (%):

• Predicted Drug Release: 91.25%
• Experimental Drug Release: 90.45%
• Bias: 0.88%

The drug release values show a very close alignment between the predicted and experimental values, with a bias of 0.88%. This small bias suggests that the formulation is performing within the expected release profile. The slight decrease in experimental release might be attributed to minor factors such as variations in the gel matrix or dissolution conditions during testing. Overall, the release behavior is consistent with the desired characteristics for topical drug delivery.

Gelation Time (secs):

• Predicted Gelation Time: 131 secs
• Experimental Gelation Time: 133 secs
• Bias: 1.5%

The gelation time data shows a minor discrepancy of 1.5% between the predicted and experimental values, with the experimental value being slightly higher than predicted. This indicates that the gel may have slightly slower gelation than expected, which could be due to factors such as the neutralization process or environmental conditions. Despite this, the gelation time remains within a reasonable range for the intended topical application.

pH:

• Predicted pH: 6.44
• Experimental pH: 6.44
• Bias: 0%

The pH values for both the predicted and experimental formulations are identical at 6.44, resulting in 0% bias. This indicates perfect alignment between the expected and observed pH, which is important for ensuring that the gel is compatible with skin or nail pH and maintains stability during application. The results indicate that the formulation of Carbopol 934, HPMC K4M, and Triethanolamine produces a highly consistent product across the predicted and experimental values. The small bias percentages observed in viscosity, drug release, and gelation time suggest that the formulation is robust, with only minor deviations that are well within acceptable experimental variability. These findings highlight the reliability of the formulation and predictive modeling used for designing the gel.

• The viscosity is slightly lower in the experimental formulation compared to the predicted, but this difference is minor and does not significantly impact the gel's performance or stability.
• The drug release also shows a very small reduction from the predicted value, but this is still within a range that suggests effective drug delivery.
• The gelation time is consistent with expectations, although the slight increase is not critical. It may be attributed to environmental factors or small variations in formulation preparation.
• The pH matches perfectly, confirming the formulation’s compatibility with topical application and ensuring that it will not irritate the skin.

The predicted vs experimental comparison of the Carbopol 934, HPMC K4M, and Triethanolamine formulation shows excellent agreement with only minor differences. These results suggest that the formulation is stable, effective, and predictable, with minimal variability in terms of viscosity, drug release, gelation time, and pH. The formulation is suitable for topical drug delivery, offering controlled release of Tavaborole while maintaining stability and patient safety.

CONCLUSION

The study successfully formulated and optimized pH-responsive in-situ gels of Tavaborole for topical application. Tavaborole was confirmed to be pure, stable, and hydrophobic, requiring suitable excipients for effective solubilization. Carbopol 934, HPMC K4M, and Triethanolamine significantly influenced key formulation parameters, including viscosity, gelation time, pH, and drug release. Formulations F12, F2, and F10 demonstrated high and sustained drug release (up to 92.51%), indicating their potential for prolonged therapeutic action. The viscosity and gelation times were optimized to ensure both ease of application and controlled release. FTIR studies confirmed stable drug-excipient interactions, likely contributing to gel formation and drug release behavior. The pH of all formulations was compatible with skin and nail application, ensuring safety and minimal irritation. Regression and bias analyses validated the reliability of the experimental design and formulation consistency. Overall, the results highlight the synergistic effect of the selected excipients in enhancing Tavaborole delivery. These optimized formulations present promising candidates for effective, patient-friendly topical treatment of onychomycosis

Author Contributions

All authors contributed significantly to the conception and design of the study, formulation development, data acquisition, analysis, and interpretation.

Conflict of Interest

The authors declare that there are no conflicts of interest associated with this work.

Funding

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

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