Formulation of Topical Itraconazole Nanostructured Lipid Carriers (Nlc) Gel for Onychomycosis

Taranjeet Kukreja¹, Swarnlata Saraf ^*

¹University Institute of Pharmacy, Pt. Ravi Shankar Shukla University, Raipur - 492010, Chhattisgarh, India

Abstract:

Itraconazole is a triazole antifungal agent that is synthesised. Itraconazole has been manufactured into a variety of pharmacological formulations and administered by a variety of ways. Itraconazole pills are used to treat pulmonary fungal infections that can spread throughout the body. Because Itraconazole is not yet officially listed in any pharmacopoeia, only a few procedures for quality control and stability testing in pharmaceutical formulations have been published. The development of itraconazole-loaded nanostructured lipid carriers (ITZ-NLC). Itraconazole NLC have been successfully developed. Itraconazole topical NLC developed for the treatment of onychomycosis does not exist, according to my survey.The development and evaluation of stable itraconazole topical gel formulations proved successful. Itraconazole NLC was produced utilising the microemulsion method, indicating the viability of adopting this method as a continuous manufacturing tool for NLC formulation. Further, the ITZ- NLC was incorporated in the gelling agent and we evaluated it under specific parameters.

Keywords: Nanostructured lipid carriers, Itraconazole, Nail delivery, Onychomycosis.

Introduction

Common fungal nail infections include onychomycosis (OM) (Olbrich et al.,2022). Dermatophytes typically cause onychomycosis, a nail fungal infection. However, infections that cause nail disease include yeasts and non-dermatophyte moulds (NDM). (Haghani et al., 2022). Tinea unguium is the term used to describe onychomycosis brought on by dermatophytes. The word "onychomycosis" includes diseases caused by yeasts, saprophytic moulds, and dermatophytes in addition to dermatophytes. One kind of dystrophic nail is an aberrant nail that is not brought on by a fungal infection. Both fingernails and toenails can have onychomycosis, but the toenail infection is significantly more common. We'll talk about the prevalence of the condition, clinical subtypes, staging, diagnosis, and treatment options for toenail onychomycosis (Bodman., 2022). About half of all nail illnesses are onychomycosis, which is brought on by non-dermatophyte moulds (NDMs), a dermatophyte species. New epidemiologic studies could aid in the treatment and prevention of onychomycosis as the disease becomes more prevalent. (Razavyoon et al., 2022; Navarro-Bielsa., 2022).

The most typical fungus infection of the nail, onychomycosis, affects the area beneath the fingertips and toes. Onychomycosis can currently be treated with oral and topical medications, either separately or in combination. Poor drug bioavailability and potential gastrointestinal and systemic side effects have been linked to oral antifungal medication. (Dehari et al., 2022).

Reviewing the literature on topical OM therapies used in clinical trials, evaluating their effectiveness, and talking about potential barriers to their success were the study's main goals. First, a thorough search of papers published in the MEDLINE database (PubMed) between January 2004 and July 2022 was carried out, with a particular focus on medications being tested in clinical settings for the topical treatment of OM. It was evident that none of the papers chosen had taken into account the fungi used NLCs treatment therapy. As a result, we thought about various key OM factors, including nail structure and fungal invasion mechanisms. (Costa et al., 2022).

Physicians can determine the causes of nail disorders with the use of a detailed grasp of nail anatomy. A diagnosis can be made by noting changes to the nail. Physical examination, dermoscopy, and, occasionally, nail biopsy should all be part of the patient evaluation process. The most prevalent nail ailment in the world, onychomycosis, needs to be recognised from other nail illnesses with comparable symptoms. Onychomycosis empiric treatment without confirmatory tests has been suggested, but studies have shown that testing to avoid ineffective therapy is cost-effective. (Mervak., 2022).

There are few available choices for treatment; oral administration of antifungal drugs typically requires lengthy regimens, which are linked to serious side effects, medication interactions, and high recurrence rates (NogueirasNieto et al., 2011; van Hoogdalem et al., 1997).

Although topical therapies are more practical, their efficiency is limited by the nail plate's rigidity and cohesiveness, which don't offer a different channel for medication penetration as skin appendage structures (Nair et al., 2009).

As a result, only a very little amount of nail medication can reach deeper layers (middle and ventral nail plate) (Naumann et al., 2014).

Numerous alternatives, including chemical (Brown et al., 2009; Nogueiras-Nieto et al., 2011; Vejnovic et al., 2010) and physical approaches, have been suggested to improve topical nail drug delivery. (Dutet and Delgado-Charro, 2009, 2010; Hao et al., 2009).

Additionally, colloidal carriers may be used alone or in conjunction with other approaches to improve drug penetration to the nail plate. For instance, (Tanriverdi and Ozer., 2013) used liposomes and ethosomes; (Naumann et al.,2014) used nano/microemulsions; (Nogueiras-Nieto., 2013) used cyclodextrins (Chiu., 2015) used polymeric nanoparticles associated with microneedles; and (Naumann et al.,2014) used nanovesicles associated with permeation enhancers (nPEV) (Bseiso et al., 2016). Despite signs they may be very promising methods to boost drug nail penetration, to our knowledge.

Since their lipid matrix can interact with the stratum corneum's lipid membrane and facilitate skin drug administration, lipid nanoparticles including solid lipid nanoparticles (SLN) and nanostructured lipid carriers (NLC) have been widely employed for topical application  (Pardeike et al., 2009; Yoon

et al., 2013). Additionally, the occlusive layer that forms on the skin surface greatly improves medication skin penetration and tissue hydration (Fan et al., 2013; Jenning et al., 2000; Obeidat et al., 2010). Denoting these systems may also be able to increase the amount of moisture in the nail plate, which would facilitate medication penetration.

In reality, one of the most significant elements that affects the physical characteristics of nails is nail hydration (Wessel et al., 1999). Nail has a network of holes that allows molecules to permeate when it is moistened, acting like a hydrogel (Hao et al., 2008; Nogueiras-Nieto et al., 2011). In fact, according to (Benzeval et al., 2013), the nail plate can absorb between 0.3 and 0.5 g of water for every gramme of dry nail, giving it a softer and more flexible structure. Additionally, hydration has been shown to enhance the size and number of nail pores (Nogueiras-Nieto et al., 2011).

Objective

Conventional oral antifungal medications for onychomycosis (OM) frequently fail to completely cure the disease and may be linked to side effects, medical interactions, and compliance concerns that limit their usage in a large population of patients. Although topical therapy can avoid systemic side effects, it is nevertheless constrained by the nail plate's physical barrier (Ortner., 2022). The purpose of treatment is to get rid of the disease-causing organism and make the nail look normal again (Uemura., 2022). Onychomycosis treatment is difficult, and the best treatment approaches have been well-documented in the literature. On the other hand, little is known about how onychomycosis is really managed in primary care. Information on practise is crucial since it can show a lot, including how closely national recommendations are implemented and which demographic groups seek or refuse treatment. (Sajeed et al., 2021).

Materials and Method

Itraconazole was bought from Nosch laboratories Pvt. Ltd., Tween 80 was bought from Loba chemie Pvt. Ltd., Stearic acid was bought from Loba chemie Pvt. Ltd., Methanol Himedia laboratories Pvt. Ltd., Carbapol 940P Himedia laboratories Pvt. Ltd., Coconut oil was purchased of Dabur Parashute, Oleic Acid was purchased from Loba chemie Pvt. Ltd., water used was distilled water prepared in the laboratory. All other chemicals and reagents were of analytical grade or superior.

Solid and liquid lipid selection

The choice of solid lipid was made based on itraconazole's solubility, which produces a visibly clear solution in lipid that melts when observed with the unaided eye in normal light. A lipid is Stearic acid, cetyl palmitate, and cholesterol were the materials used in this study. Itraconazole (10mg) When different amounts of chosen lipids were heated past their melting point in a 15 ml glass vial temperature-controlled water bath. After Under ordinary light, the solubility of itraconazole was seen after melting the lipid in vials. (Shah et al., 2007; Sharma et al., 2009).

Itraconazole's behaviour during partitioning in different lipids

A 10 mg dose of Itraconazole was dissolved in a hot distilled water and 1 g of melting lipid mixture (1ml). In a hot water bath set at 65°C for 24 hours, the mixture was shaken. After cooling, the drug content was spectrophotometrically examined after the aqueous phase was separated by ultracentrifugation (Bhalekar et al., 2009).

Microemulsions technique

Table 1 lists the SLN, NLC, and surfactant compositions. We modified the microemulsion process to create the formulations. Oleic acid and stearic acid were combined using a magnetic stirrer, then melted at 75 ⁰C to create a homogeneous and transparent oil phase. The temperature of the aqueous phase, which consisted of mixing the surfactant Tween-80 with double-distilled water, was kept at 75 ⁰C. A microemulsion was created by adding the oil phase drop-by-drop to the aqueous phase and agitating the mixture for 20 minutes at 75 ⁰C at 600 rpm. (NLC) To create NLC, this warm NLC was diluted in cold water (2–3 ⁰C) while being mechanically stirred. then filtered for 15 minutes using Whatman filter paper. The experiment was among the manufacturing parameters that were tested in a systematic investigation.

Table 1: Formula table for preparation of blank NLCs.

The formulation of Itraconazole loaded NLC Gel

In distilled water, disperse. Wait 15 minutes while gently agitating the polymer to soak it out thoroughly. Pour 35 ml of glycerin in. The itraconazole solution received 10 ml of itraconazole-loaded NLC. To create uniform hydro gel, the dispersed mixture was agitated at 1000 rpm for 30 minutes. To neutralise the itraconazole, slowly add 0.5 ml of triethanolamine. 15-20 minutes for the mixture to fully gel. Carbopol was used as the gelling agent due to its compatibility with the NLC dispersion and spreadability. Table 2 lists the components of several gel formulations.

Table 2: Composition of NLC-based gel formulations

Characterization of NLCs systems

Dispersion stability studies

Dispersion stability tests were done to solve the formulation's metastability issue. A few formulations underwent a 30-minute centrifugation at 3000 rpm. The heating and cooling cycle was used with the formulations that didn't exhibit any phase separations (freeze thaw cycle). In a hot air oven, six cycles between 4°C and 45°C were completed, with storage at each temperature lasting at least 48 hours. For future research, the formulations that withstood the stability tests at these temperatures were chosen.

Drug content and entrapment efficiency (%EE) 

The ethanol:dichoromethane (1:3) solvent mixture was used to dissolve 1 mL of the produced NLCs, and the solutions were then immediately injected into the cuvate of the UV system following the proper dilutions with mobile phase. The solutions were then tested for the presence of drugs. NLC was centrifuged at 20 ⁰C for 30 min. (Eppendorf Centrifuge 54 30R) at 25,000 rpm to calculate the percent entrapment efficiency (percent EE). A mixture of ethanol and dichloromethane (1:3) was used to dissolve the pellets, and the amount of itraconazole that was trapped inside was then measured using the UV Spectroscopy method before the pellets were appropriately diluted with mobile phase. Table 3 shows the percent entrapment efficiency.

The E.E. was calculated using the following equation:

EE(%)= total amount of Itraconazole-free amount of itraconazole x 100

                                 Total amount of itraconazole

Table 3: Entraptment Efficacy

Result and Discussion

Appearance

The hydrogel was observed to be free from greatness and translucent that conforms the equal distribution catalase loaded NLC inside the gel matrix.

pH study

Because gel is used directly to the nails and can prevent irritation issues with the skin and nails, a pH analysis of the gel is crucial. The pH of the skin is about between 6 and 7, while the pH of the nail surface ranges from 5.1 to 5.6. This pH is suitable for topical use. A digital pH metre was used to measure the pH of the generated plain gel, plain NLC loaded gel, and itraconazole loaded NLC gel formulations at room temperature. The generated unloaded NLC, itraconazole-loaded NLC, and plain gel all had pH values of 5.2, 5.4, and 5.5, respectively.

Drug Content Study

In a 25 mL volumetric flask containing 15 mL of methanol, NLC loaded gel equivalent to 25 mg of ITZ was added, and the mixture was agitated for 30 minutes. Methanol was used to bring the volume up to 25 mL. To obtain 20 g/mL, 0.2 mL of the solution was further diluted using 4.8 mL of methanol and 5 mL of 0.1 N HCl. Through a 0.45 membrane filter, the final solution was purified. Using a spectrophotometric instrument (Shimadzu UV-1800), the solution's absorbance was calculated at 258 nm.

Spreadability Study

Spreadability of the gel formulation may affect a topical formulation's effectiveness and serve as a key indicator of patient compliance. The spreadability of the unloaded gel with no NLC. NLC gel with itraconazole was observed as. For topical formulation, this is okay. 4.1 cm, 4.3 cm, and 5.1 cm, which are suitable for topical application, respectively.

Viscosity study

Fig 1: Brookfield Viscometer

At 25°C, the viscosity of the formulation was measured using a Brookfield viscometer (Model DV-I). A 250 mL beaker containing 175 g of gels was used to determine the viscosity. Spindle T 95 was used to measure the viscosity of each gel.

Fig 2: Viscosity of gel in RPM

In vitro Release study

Utilizing egg membrane, in vitro release tests were conducted on nanostructured lipid carriers (NLCs) loaded with itraconazole. The investigations showed that 80.0% of the medication release was visible after 24 hours.

Fig 3: In Vitro Release Profile for ITZ Solution And ITZ-NLC Formulation.

Conclusion

The results of the current study suggest that microemulsion technologies could be used to successfully manufacture itraconazole's NLCs. The results of this investigation also suggest that the entrapment efficiency is strongly influenced by the type and quantity of liquid lipid. The selection of materials for the development of the NLC formulation should take into account whether or not each component is pharmaceutically acceptable, non-irritating, and does not cause skin sensitization. The greatest amount of ITZ solubility was discovered among the chosen oils that were screened.

NLCs can be used in a variety of ways to administer medications to patients, but their topical use has drawn more attention recently.

It is well recognised that if drug molecules have any issues with solubility or bioavailability along the GI tract, they are candidates for other routes of administration, and if the drug acts topically instead of or in addition to the GI tract, the results are better.

The final Itraconazole-loaded gel formulation was tested for appearance, pH, spreadability, drug content, viscosity, and invitro release, all of which yielded satisfactory results.

Acknowledgment

I am thankful to the University Institute of Pharmacy (UIOP), Pt. Ravishankar Shukla University, Raipur, Chhattisgarh, India for my practical support. I also want to thank Prof. Swarnlata Saraf, University Institute of Pharmacy (UIOP), Pt. Ravishankar Shukla University, Raipur, Chhattisgarh, India for her valuable suggestions and guidance. Thanks are due to Nosch laboratories Pvt. Ltd., India for providing Itraconazole drug sample.

References:

Benzeval, I., Bowen, C.R., Guy, R.H., Delgado-Charro, M.B., 2013. Effects of iontophoresis, hydration, and permeation enhancers on human nail plate: infrared and impedance spectroscopy assessment. Pharm Res 30, 1652-1662.

Bhalekar MR, Pokharkar V, Madgulkar A, Patil N, Patil N. Preparation and Evaluation of Miconazole Nitrate-Loaded Solid Lipid Nanoparticles for Topical Delivery. AAPS PharmSciTech. 2009; 10: 289–296. http://dx.doi.org/10.1208/s12249-009-9199-0

Bodman MA, Krishnamurthy K. Onychomycosis. 2022 May 1. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2022 Jan–. PMID:28722883.

Brown, M.B., Khengar, R.H., Turner, R.B., Forbes, B., Traynor, M.J., Evans, C.R., Jones, S.A., 2009. Overcoming the nail barrier: A systematic investigation of ungual chemical penetration enhancement. Int J Pharm 370, 61-67.

Bseiso, E.A., Nasr, M., Sammour, O.A., Abd El Gawad, N.A., 2016. Novel nail penetration enhancer containing vesicles “nPEVs” for treatment of onychomycosis. Drug Delivery 23, 2813-2819.

Chiu, W.S., Belsey, N.A., Garrett, N.L., Moger, J., Price, G.J., Delgado-Charro, M.B., Guy, R.H., 2015. Drug delivery into microneedle-porated nails from nanoparticle reservoirs. J Control Release 220, 98-106.

Costa PS, Mendes V, Veiga FF, Negri M, Svidzinski TIE. Relevant insights into onychomycosis' pathogenesis related to the effectiveness topical treatment. Microb Pathog. 2022 Jun 15;169:105640. doi: 10.1016/j.micpath.2022.105640. Epub ahead of print. PMID: 35716926.

Dehari D, Mehata AK, Priya V, Parbat D, Kumar D, Srivastava AK, Singh S, Agrawal AK. Luliconazole Nail Lacquer for the Treatment of Onychomycosis: Formulation, Characterization and In Vitro and Ex Vivo Evaluation. AAPS PharmSciTech. 2022 Jun 24;23(6):175. doi: 10.1208/s12249-022-02324-7. PMID: 35750993.

Dutet, J., Delgado-Charro, M.B., 2009. In vivo transungual iontophoresis: Effect of DC current application on ionic transport and on transonychial water loss. J Control Release 140, 117-125.

Dutet, J., Delgado-Charro, M.B., 2010. Transungual iontophoresis of lithium and sodium: Effect of pH and co-ion competition on cationic transport numbers. J Control Release 144, 168-174.

Fan, X., Chen, J., Shen, Q., 2013. Docetaxel–nicotinamide complex-loaded nanostructured lipid carriers for transdermal delivery. Int J Pharm 458, 296-304.

Haghani I, Shams-Ghahfarokhi M, Dalimi Asl A, Shokohi T, Hedayati MT. Prevalence, genetic diversity and antifungal susceptibility profiles of F. fujikuroi, F. solani and Fusarium incarnatum-equiseti species complexes from onychomycosis in north of Iran. Mycoses. 2022 Jul 1. doi: 10.1111/myc.13489. Epub ahead of print. PMID: 35775829.

Hao, J., Smith, K.A., Li, S.K., 2009. Iontophoretically Enhanced Ciclopirox Delivery into and Across Human Nail Plate. J Pharm Sci 98, 3608-3616.

Jenning, V., Gysler, A., Schäfer-Korting, M., Gohla, S.H., 2000. Vitamin A loaded solid lipid nanoparticles for topical use: occlusive properties and drug targeting to the upper skin. Eur J Pharm Biopharm 49, 211-218.

Mervak JE. Hair and Nail Conditions: Nail Conditions. FP Essent. 2022 Jun;517:27-34. PMID: 35679470.

Nair, A.B., Sammeta, S.M., Kim, H.D., Chakraborty, B., Friden, P.M., Murthy, S.N., 2009. Alteration of the diffusional barrier property of the nail leads to greater terbinafine drug loading and permeation. Int J Pharm 375, 22-27.

Naumann, S., Meyer, J.P., Kiesow, A., Mrestani, Y., Wohlrab, J., Neubert, R.H., 2014. Controlled nail delivery of a novel lipophilic antifungal agent using various modern drug carrier systems as well as in vitro and ex vivo model systems. J Control Release 180, 60-70

Naumann, S., Meyer, J.P., Kiesow, A., Mrestani, Y., Wohlrab, J., Neubert, R.H., 2014. Controlled nail delivery of a novel lipophilic antifungal agent using various modern drug carrier systems as well as in vitro and ex vivo model systems. J Control Release 180, 60-70.

Navarro-Bielsa A, Gracia-Cazaña T, Robres P, Lopez C, Calvo-Priego MD, Aspiroz C, Gilaberte Y. Combination of Photodynamic Therapy and Oral Antifungals for the Treatment of Onychomycosis. Pharmaceuticals (Basel). 2022 Jun 7;15(6):722. doi: 10.3390/ph15060722. PMID: 35745641; PMCID: PMC9227606.

Nogueiras-Nieto, L., Begona Delgado-Charro, M., Otero-Espinar, F.J., 2013. Thermogelling hydrogels of cyclodextrin/poloxamer polypseudorotaxanes as aqueous-based nail lacquers: application to the delivery of triamcinolone acetonide and ciclopirox olamine. Eur J Pharm Biopharm 83, 370-377.

Nogueiras-Nieto, L., Gomez-Amoza, J.L., Delgado-Charro, M.B., Otero-Espinar, F.J., 2011. Hydration and N-acetyl-l-cysteine alter the microstructure of human nail and bovine hoof: implications for drug delivery. J Control Release 156, 337-344.

Nogueiras-Nieto, L., Gomez-Amoza, J.L., Delgado-Charro, M.B., Otero-Espinar, F.J., 2011. Hydration and N-acetyl-l-cysteine alter the microstructure of human nail and bovine hoof: implications for drug delivery. J Control Release 156, 337-344.

Obeidat, W.M., Schwabe, K., Müller, R.H., Keck, C.M., 2010. Preservation of nanostructured lipid carriers (NLC). Eur J Pharm Biopharm 76, 56-67.

Olbrich M, Ernst AL, Beltsiou F, Bieber K, Ständer S, Harder M, Anemüller W, Köhler B, Zillikens D, Busch H, Künstner A, Ludwig RJ. Biodiversity of mycobial communities in health and onychomycosis. Sci Rep. 2022 May 25;12(1):8872. doi: 10.1038/s41598-022-13074-8. PMID: 35614121; PMCID: PMC9133011

Ortner VK, Nguyen N, Brewer JR, Solovyeva V, Haedersdal M, Philipsen PA. Fractional CO₂ laser ablation leads to enhanced permeation of a fluorescent dye in healthy and mycotic nails-An imaging investigation of laser- tissue effects and their impact on ungual drug delivery. Lasers Surg Med. 2022 Aug;54(6):861-874. doi: 10.1002/lsm.23541. Epub 2022 Apr 22. PMID: 35451510.

Pardeike, J., Hommoss, A., Müller, R.H., 2009. Lipid nanoparticles (SLN, NLC) in cosmetic and pharmaceutical dermal products. Int J Pharm 366, 170-184.

Razavyoon T, Hashemi SJ, Mansouri P, Rafat Z, Saboor-Yaraghi AA, Kamali Sarvestani H, Ghasemi Z. The epidemiology and etiology of onychomycosis in 2 laboratory centers affiliated to Tehran university of medical sciences during 2019-2020. Iran J Microbiol. 2022 Apr;14(2):268-275. doi:10.18502/ijm. v14i2.9196. PMID: 35765553; PMCID: PMC9168250.

Sajeed M, Wei L, Murdan S. What can GP data tell us about the treatment of onychomycosis in the UK? Skin Health Dis. 2021 Dec 16;2(1):e84. doi: 10.1002/ski2.84. PMID: 35665209; PMCID: PMC9060066.

Shah KA, Date AA, Joshi MD, Patravale VB. Solid lipid nanoparticles (SLN) of tretinoin: Potential in topical delivery. Int J Pharm. 2007; 345: 163–171. http://dx.doi.org/10.1016/j.ijpharm.2007.05.061PMid:17644288

Sharma P, Ganta S, Denny AW, Garg S. Formulation and pharmacokinetics of lipid nanoparticles of a chemically sensitive nitrogen mustard derivative. Chlorambucil. Int J Pharm. 2009; 367:187–194. http://dx.doi.org/10.1016/j.ijpharm.2008.09.032

Tanriverdi, S.T., Ozer, O., 2013. Novel topical formulations of Terbinafine-HCl for treatment of onychomycosis. Eur J Pharm Sci 48, 628-636.

Uemura EVG, Barbosa MDS, Simionatto S, Al-Harrasi A, Al-Hatmi AMS, Rossato L. Onychomycosis Caused by <i>Fusarium</i> Species. J Fungi (Basel). 2022 Mar 31;8(4):360. doi: 10.3390/jof8040360. PMID: 35448591; PMCID: PMC9027400.

van Hoogdalem, E.J., van den Hoven, W.E., Terpstra, I.J., van Zijtveld, J., Verschoor, J.S.C., Visser, J.N., 1997. Nail penetration of the antifungal agent oxiconazole after repeated topical application in healthy volunteers, and the effect of acetylcysteine. Eur J Pharm Sci 5, 119- 127

Vejnovic, I., Simmler, L., Betz, G., 2010. Investigation of different formulations for drug delivery through the nail plate. Int J Pharm 386, 185-194.

Wessel, S., Gniadecka, M., Jemec, G.B.E., Wulf, H.C., 1999. Hydration of human nails investigated by NIR-FT-Raman spectroscopy. Biochim Biophys Acta 1433, 210-216.

Yoon, G., Park, J.W., Yoon, I.-S., 2013. Solid lipid nanoparticles (SLNs) and nanostructured lipid carriers (NLCs): recent advances in drug delivery. J Pharm Invest 43, 353-362.