INTRODUCTION:

Topical drug delivery systems are dosage forms involves drug transport to viable epidermal and or dermal tissues of the skin for local therapeutic effect while a very major fraction of drug is transported into the systemic blood circulation. Topical administration of therapeutic agents offers many advantages over conventional oral and invasive methods of drug delivery. Several important advantages of transdermal drug delivery are limitation of hepatic first pass metabolism, enhancement of therapeutic efficiency and maintenance of steady plasma level of the drug.

Liquid Crystalline Phases which help to incorporate and control release of drug with varying size and polar characteristics are used great interest as novel dosage forms, due to their considerable solubilizing capability for both oil and water-soluble compounds. Use of lipid help to biodegradation and make it interesting drug delivery system for various routes. Topical application help to minimize the first pass metabolism of drug and lowering the dose of drug. The presence of wound, tine, acne etc. having water content on that region, due to presence of water help to diffusion of drug from liquid crystalline phases¹. The liquid crystalline materials possess the crystalline aspect that is both orientation and position due to these molecules is not locked rigidly in a lattice; instead they exhibit liquid like diffusion and cross the stratum corneum as a barrier. Therapy can easily terminate by wash-out if toxicity occurs. Various phases of low viscosity to high viscosity help to release of drug at controlled drug release to sustained drug release. High thermodynamic stability is important properties of liquid crystalline phase².

A GMO, an absorption enhancer, has promoted reversible disruption of the lamellar lipid bilayer in the stratum corneum, thus boosting the intercellular lipid fluidity.as per the reported literature a GMO forms a cubic phase gel with other excipients resulted in a higher transdermal flux Moreover, the cubic phase gel is more viscous compared with lamellar phase gel. The cubic phase forms a biological membrane-like structure that was suggested to promote a strong bioadhesive property to the skin, thus enabling the fusion of GMO with the lipophilic SC layer where drug can be released from the curved bicontinuous lipid bilayer^3-4.

ITZ molecule is highly anisotropic with a rod like structure. Nematic and smectic liquid crystalline phases of ITZ were previously achieved via controlled cooling of melted crystals. However, a nematic phase can easily be misinterpreted as isotropic (amorphous) using X-ray diffraction due to similarity in diffraction patterns of both phases^5-7. The story of co-crystals begins in 1844 with Friedrich Wohler‘s synthesis of quinhydrone from hydroquinone and quinone. The co-crystals are a crystalline structure made up of two or more components in definite stoichiometric ratio, which each component is defined as either a atom, ion or molecules. The structure exhibit long range order and components interact via non-covalent interactions such as hydrogen bonding, ionic interactions and van der Waals interactions⁸.

The objectives of present investigation were to evaluate performance of ITZ co-crystals in liquidcrystalline formulation for in-vitro and in-vivo parameters. as the literature has been reported on use of co-crystals for improvement in solubility, dissolution rate and stability, work focused on evaluation of performance in various dosage forms is very limited reported.

MATERIALS AND METHODS:

Materials:

Glycerylmonooleate was purchased from TCI Japan. Itraconazole was a gift sample by Glenmark Pvt. Ltd. Mumbai. Poloxamer 407 was purchased from (BASF). And other chemicals and organic solvents were of analytical grade.

Preparation of Co-crystals:

Co-grinding cocktail co-crystallization method was used to prepare co-crystals in short itraconazole (705.05 mg, 1 Mmol) and different co-crystal formers like Succinic acid and Serine (1 Mmol) were weighed and transferred to mortar and pestle. Samples were co-grinded for 45 minutes ⁹.

Formulation of ITZ and Co-crystals loaded Liquid Crystalline gel:

This gel was prepared, containing glycerylmonooleate, as an oil phase and the aqueous phase containing poloxamer 407, isopropyl myristate, preservative and water. The Liquid Crystalline gel was prepared by heating the oil phase containing 1% itraconazole and the aqueous phase kept apart at 60^oC. After that the oil phase was added drop wise to the aqueous phase with constant stirring. And follow the same procedure for co-crystal loaded liquid crystalline gel. The ingredients are shown in table 1.

Table 1. Formulation batches

Formula-tion code	Drug (mg)	GMO: Water (%)	Poloxame 407	Iso-propyl myristate	Methyl Paraben	ITZ cocrystals (mg)
L1	50	15:85	0.85	0.25	0.02	-
L2	50	30:70	0.85	0.25	0.02	-
L3	50	45:55	0.85	0.25	0.02	-
L4	50	60:40	0.85	0.25	0.02	-
CL1	-	15:85	0.85	0.25	0.02	64
CL2	-	30:70	0.85	0.25	0.02	64
CL3	-	45:55	0.85	0.25	0.02	64
CL4	-	60:40	0.85	0.25	0.02	64

Evaluation of Gel Formulations:

The prepared gel formulations were evaluated for appearance, homogeneity and pH by using a pH meter (Systronics µ pH system 362)

Examination under Polarized light microscope:

The physical investigation of prepared gel formulations were carried out by using polarized light microscopy (Nikon, Kanagawa, Japan), in order to determine type of phases a small amount of sample was spread on a clean glass slide. The existence of birefringence was verified by observation under cross polars employing magnification of 20 X. The photomicrographs of the sample were taken.

Differential scanning calorimetry measurements:

This measurement was performed on drug, as well as prepared gel using instrument (SDT Q600 V20.9 Build 20 Universal V4.5A TA instrument). Temperature range was kept (0-200^oC). 2-4mg weight of sample was taken and the heating rate was 10^oC/min.

FTIR analysis:

The IR spectra of ITZ, Drug loaded gel, co-crystal loaded gels were recorded using IR spectrophotometer (FT/IR-4100 JASCO UK). The analysis was performed with spectra measures over the frequency range 400-4000.

Determination of Drug content:

The drug content of the prepared gels was carried out by dissolving accurately weighed quantity (0.5g) of gel equivalent to 10 mg of drug in 10 ml of phosphate buffer pH 7.4:methanol (60:40%), the volume was made up to100 ml and 1 ml of the above solution was further diluted to 10 ml with same media. After suitable dilution absorbance of the solution was recorded by using UV/ visible spectrophotometer (Shimadzu-1601 Tokyo, Japan) at 254 nm.

Rheological study:

The ITZ gel prepared with liquid crystal vehicle thought to have intermediate behavior between solid and liquid. This study was carried out for prepared gel formulations by using (R/S plus Rheometer, Brookfield Engineering Lab Inc. USA).

In-vitro drug diffusion study:

The In-vitro drug release study was carried out through cellophane membrane (surface area 2.1 cm²Sigma, Inc. Mo, USA), and human cadaver skin. Keshary-Chien diffusion cells were used for these studies. Cellophane membranes were mounted on a Keshary-Chien diffusion cells, which was kept at 37ºC by circulating the water in outer compartment. The known quantity (500mg) was placed in donor compartment. The receptor compartment was filled with 20 ml of phosphate buffer pH 7.4, from which samples of 0.5 ml were collected at regular intervals during 12 hours and replaced with the same amount of buffer to maintain sink condition. The samples were analyzed by using UV-Spectrophotometer (Shimadzu-1601 Tokyo) at 254 nm.

Drug Release kinetics:

The mechanism of drug release from the gels were studied by subjecting in-vitro diffusion studies in to different kinetic equations like Zero order, First order, Higuchi, Hixson Crowell, Korsemeyer-Peppas models.

In-vitro antifungal Activity:

This study was carried out by using Agar-cup diffusion method and Candida albicans culture was used. The nutrient agar broth was used as a media. The subculture of Candida albicans (Obtained faculty of allied sciences microbiology and biotechnology, KIMSDTU, Karad) was spread in petri-dish. On solidification, 1cm holes were made and filled with a formulation. The one plate hole filled with pure ITZ solution, second plate hole filled with ITZ loaded gel and third plate hole filled with co-crystal loaded gel and fourth plate filled with 1% drug with hydroxypropyl cellulose gel for comparison.

Skin Irritation study:

Study includes use of rats (220-300 g) obtained from TKCP Warnanagar of either sex were used acclimatized for period of 24 hours to ensure their suitability for Study. Two test animals were kept within a limited access rodent facility with environmental conditions set to a temperature of 25 ± 2°C, a humidity of 60-90% RH and a 12-h light / 12-h dark cycle. The animals were maintained on standard animal feed and had free access to food and water. The day before the experiment, the hairs from the back of the rats were carefully removed with a hair removing cream and a razor without breaking the skin. An area of 4.25 cm² was marked on dorsal skin of rats. The gel was applied (2 g/rat) for 30 minutes and washed off with tap water and occluded with gauze and bandage, the site was observed for any sensitive reaction if any for 24 hours. [Approved letter no. IAEC/TKCP/2014/12]

Stability Study:

The stability study was performed for the formulation L2 and CL2 at room temperature. The reason behind for selection of these batches was the formulations had shown better results in terms of in-vitro and ex-vivo studies. The stability study of L2 and CL2 batches were performed at room temperature for 3 months. The formulations were analyzed for appearance, pH, Phase separation, consistency. The results are shown in table 4.

RESULTS AND DISCUSSION:

Physical investigation and drug content:

After preparation the formulations were examined for their performance related to physical characteristics. The pH value of ITZ loaded gel was found to be 6.8, when the co-crystals were incorporated in the gel, the pH was 6.5. Formulations were white, smooth, opaque, and found homogeneous nature with no any coarse particles and phase separation. After application of the gel on the skin, the skin was not greasy or tacky and the gel was washable with water. The pH of skin varies from 4.5 to 6. So prepared gel formulations were suitable for topical application. The drug content of all the formulations was found in the range of 80±1.12 - 94±1.45 %⁷.

Placebo gel

ITZ loaded gel

Co-crystal baded

Figure 1. Polarized light microscopic images of formulations

Examination under polarized light microscope:

The placebo, ITZ loaded gel and Co-crystal loaded gel was analyzed under polarized light microscopy. Figure 1 shows photographs of liquid crystals. The birefringence that is characteristics of concentric cubic liquid crystal was observed¹⁰. The placebo gel and drug loaded gel shows the formation of cubic liquid crystals, because viscosity was maintained by solvent addition. But in case of Co-crystal loaded gel showed slightly changes in crystal pattern because co-formers and drug inserted into the hydrophobic hydrocarbon matrix of the cubic phase¹¹.

Differential scanning calorimetry measurement:

The pure Itraconazole showed sharp endothermic peak at 180⁰C as shown in figure 2. The drug loaded liquid crystalline gel formulation showed endothermic peak at 96.71°C. Liquid crystalline gel containing co-crystals were showed endothermic peak at 99.2°C. In case of DSC the peak values were showed depression of melting point due to solubilisation of drug into liquid crystalline matrix. Additionally, one of the endothermal (liquid crystalline) transitions (TLC1) in ITZ originally located at 74°C was not observed, which indicates that the presence of other ingredients influenced the ability of ITZ to undergo the smectic A transition⁵.

FTIR analysis:

The FTIR spectra of prepared drug loaded and co-crystal loaded liquid crystalline gel shown in figure 3. In case of L2 batch the peaks were observed at 2857cm^-1 and 2926cm^-1 assigned to the aromatic C-H, N-H stretching bonds, 1639 cm^-1for C=O-N-H bands, 1459 cm^-1 due to C-N stretching and 1284 cm^-1 due to -O- ether respectively. While in case of Cl2 batch the peaks were observed at 2856 cm^-1and 2926 cm^-1 may be assigned to the aromatic C-H, N-H stretching bands, 1638 cm^-1 for C=O-N-H bands and 1459 cm^-1 due to C-N stretching and 1251cm^-1 due to -O- ether respectively. The result revealed that there was no interaction between drug as well as co-crystal, also confirming the compatibility of drug and co-crystals with other excipients ^7,9.

In-vitro drug diffusion study:

The permeation study was carried out through human cadaver skin with optimized L2 and Cl2 batches for in-vitro diffusion study. The figure 4 and 5 shows permeation profile of drug from liquid crystalline gel through skin. The percent drug diffused of L2 batch was found to be 80.30 ± 0.026% and Cl2 batch shows 83.09 ± 0.024%.The drug diffusion study was carried out to determine the diffusivity of solubilized molecule through the membrane. The mechanism for drug release is diffusional exchange of water from the external media into the matrix, with exchange of drug and water from interior phase to external media¹. The release of drug shown upto 12 hrs. and it revealed the sustained release of drug through formulation. These kinds of results occur due to presence of cubic phase in the formulation generally diffusivity of solubilized drug molecules is reduced by bulk cubic phase¹².

Figure 4. In vitro drug diffusion study of ITZ loaded gels (n=3, Mean± S.D)

In case of co-crystal loaded liquid crystalline gel, sustained release of drug was observed and better results than ITZ loaded gel formulation, because co-crystals are having ability to increase the solubility of drug molecule. Also GMO also increase the solubility of itraconazole. The different drug diffusion pattern was shown by cocrystals due to formation of hexagonal phase in CL3 and CL4 formulations. The increase in the hydrocarbon chain space due to solubilization of co-crystals, might have changed the monoglycerides molecular packing, furthermore cubic phase formation and also CL1 formulation have high viscosity due to increase in the water content may contributed for retardation of drug release^13-15. As shown in figure 5, for first two hours L2 and CL2 batch showed similar drug diffusion, but afterwards Cl2 showed improved permeation through human cadaver skin upto to 10 hours ¹. After 10 hours there might be possibility of phase transformation of co-crystals into pure itraconazole.

Figure 5. In vitro drug diffusion study of ITZ cocrystals loaded gels (n=3, Mean±S.D)

Drug Released kinetics:

Release data of optimized L2 and Cl2 formulations obtained from in-vitro and ex-vivo release test were fitted to various models such as zero order, first order, Higuchi model, Hixson Crowell, Korsemeyerpeppa’s model corresponding to possible release mechanism. Linear regression analysis of each profiles were performed. The Regression coefficients were obtained by plotting the graphs. The R² values for various models are given in table 2. The value of R² for L2 and Cl2 gel formulations were found higher for Higuchi model. Hence the Higuchi release model mostly fitted the in-vitro release profile of itraconazole from liquid crystalline system. Therefore, the drug release rates from liquid crystalline matrices were controlled predominantly by Fickian diffusion^16-17.

Table 2. Drug release kinetic models for in vitro drug diffusion of liquid crystal gel

Formul ation code	Regression coefficient (R²)
Formul ation code	Zero order	First Order	Higuchi Model	Hixson Crowell model	Korsemeyer peppas model
L2	0.8603	0.4785	0.9816	0.8363	0.5591
CL2	0.9001	0.5169	0.9911	0.9011	0.6076

In-vitro Antimycotic activity:

The in-vitro antimycotic inhibitory activity of itraconazole, co-crystals loaded liquid crystalline gel and hydroxypropyl cellulose based standard ITZ gel as a control were conducted using agar-cup method and Candida albicans as a test organism. The inhibition zone values for test and control were presented in table 3. The optimized L2 and CL2 batches showed higher inhibition zone than standard. But Cl2 gel formulation showed higher value than L2. From above results we can conclude that the prepared formulations exhibited better antimycotic activity with improved efficiency^18-19.

Table 3. Zone of inhibition of in vitro antimycotic activity (n=3, Mean ±SD)

Batch Code	Conc. (gm)	Zone of Inhibition (mm± S.D)
Standard ITZ	0.025	18±0.65
L2	0.025	21±0.88
CL2	0.025	25±1.42

Rheological study:

Rheological study was performed on chosen samples by the conventional digital cone plate Brookfield R/S plus Rheometer. The observations revealed that the when decrease the shear stress increases the viscosity, means formulations having good viscoelastic behavior. L2 and CL2 showed increase in viscosity. The figure 6 shows graphical presentation of viscosity. The cubic phase is highly viscous containing water channels surrounded by curved bilayer of amphiphile extended in three dimensional, while the lipophilic drugs are located into lipid bilayer so it controls the release of drug^20-21.

Figure 6. Rheological study of optimized batches

Skin Irritation study:

No allergic reactions like, redness, irritation was observed on the skin of rats up to 24 hrs. The skin irritation test was performed with optimized formulation L2 and Cl2 in two rats. It was found that the both gel formulation causes no irritation or erythema ^22-25.

Stability Study:

The stability study was performed for the formulation L2 and CL2 at room temperature. The reasons behind for selection of these batches were the formulations had shown better results in terms of in-vitro studies. The stability study of L2 and CL2 batches were performed at room temperature for 3 months. The formulations were analyzed for appearance, pH, Phase separation, consistency. The results shown in table 4.

Table 4. Evaluation parameters after stability studies (n=3, Mean ±SD)

Formulation batch code	Appearance	pH	Phase Separation
L2	White	6.8±0.02	No any phase separation
CL2	White	6.5±0.03	No any phase separation

The gel formulation was white in color. The pH was found to be 6.8 and 6.5. There was no phase separation and consistency was good.

CONCLUSION:

Prepared liquid crystalline gel formulations containing itraconazole and its co-crystals are useful as a topical delivery system. Co-crystals loaded formulations showed improved permeation for certain period of time. The formulations were stable, effective and having good rheological properties for topical application. Co-crystals can be incorporated in novel drug delivery system like liquid crystals for enhancement of therapeutic performance.

ACKNOWLEDGEMENT:

Authors are thankful to the management of Krishna institute of medical sciences deemed to be university for providing laboratory facilities and Shivaji University Kolhapur for providing central instrumentation facility.

CONFLICT OF INTEREST:

The authors declare that they have no competing interests.

REFERENCES:

1. Patrick T. Progress in liquid crystalline dispersions: Cubosomes. Current Opinion in Colloid & Interface Science. 2005; 10 (56):274-279.

2. Namdeo A, Jain N. Liquid crystalline pharmacogel based enhanced transdermal delivery of propranolol hydrochloride. Journal of Controlled Release. 2002; 82 (2) :223-36.

3. Chen Y, Lu Y, Zhong Y, Wang Q, Wu W, Gao S. Ocular delivery of cyclosporine A based on glycerylmonooleate/ poloxamer 407 liquid crystalline nanoparticles: preparation, characterization, in vitro corneal penetration and ocular irritation. Journal of Drug Target. 2012; 20 (10): 856–63.

4. Westesen K, Bunjes H, Hammer G, Siekmann B. Novel colloidal drug delivery systems. PDA Journal of Pharmaceutical sciences and technology. 2001;55(4):240–247

5. Mugheirbi NA, Tajber L. Mesophase and size manipulation of itraconazole liquid crystalline nanoparticles produced via quasi nanoemulsion precipitation. European Journal of Pharmaceutics and Biopharmaceutics. 2015; 96:226-36.

6. Heike B, Thomas R. Thermotropic liquid crystalline drugs. J Pharm Pharmacol 2005; 57: 807–816.

7. Nesseem D. Formulation and evaluation of Itraconazole via liquid crystal for topical delivery system. J Pharmaceutical Biomedical Analysis. 2001; 26(3):387-99.

8. Cheney, Miranda L. The Role of Cocrystals in Solid-State Synthesis of Imides and the Development of Novel Crystalline Forms of Active Pharmaceutical Ingredients 2009. Graduate School Theses and Dissertations. Paper 3693.

9. Shete As, Murthy SM, Korpale S, Yadav AV, Sajane SJ, Sakhare SS, Doijad RC. Cocrystals of Itraconazole with Amino Acids: Screening, Synthesis, Solid State Characterization, In Vitro Drug Release and Antifungal Activity. Journal of Drug Delivery Science and Technology. 2015; 28: 46-55.

10. Aytekin M, Gursoy RN, Ide S, Soylu EH, Hekimoglu S. Formulation and characterization of liquid crystal systems containing azelaic acid for topical delivery. Drug Development and Industrial Pharmacy.2013; 39(2):228-39.

11. Mohamed MI, Abdelbary AA, Kandil SM, Mahmoud TM. Preparation and evaluation of optimized zolmitriptanniosomal emulgel. Drug Development and Industrial Pharmacy.2019; 28:1-11.

12. Shah JC, Sadhale Y, Chilukur DM. Cubic phase gels as drug delivery systems. Advanced drug Delivery Reviews. 2001; 47:229–250.

13. Rizwan SB, Boyd BJ, Rades T, Hook S. Bicontinuous cubic liquid crystals as sustained delivery systems for peptides and proteins. Expert Opinion on Drug Delivery. 2010; 7(10):1133-44.

14. Lu J, Li YP, Wang J, Li Z, Rohani S, Ching CB. Pharmaceutical cocrystals: a comparison of sulfamerazine with sulfamethazine. Journal of Crystal Growth. 2011; 335:110–14.

15. Choi JH, Lee HY, Kim J, Kim Y.C. Monoolein Cubic Phase Containing Hydrophobically Modified Poly (N-isopropylacrylamide). Journal of Industrial Engineering and Chemistry. 2007; 3:380-386.

16. Barakat NS. Evaluation of Glycofurol-Based Gel as a New Vehicle for Topical Application of Naproxen. AAPS Pharm Sci Tech. 2010; 3:1138-1146.

17. Dash S, Murthy PN, Nath L, Chowdhury P. Kinetic modeling on drug release from controlled drug delivery systems. Acta Polania Pharmaceutica. 2010; 67(3):217-23

18. Kokare C. Pharmaceutical microbiology, principles and application. Nirali Prakashan, Pune.2005.

19. Hata K, Kimura J, Miki H, Toyosawa T, Nakamura T, Katsu K. In Vitro and In Vivo Antifungal Activities of ER-30346, a Novel Oral Triazole with a Broad Antifungal Spectrum. Antimicrobial Agents Chemotherapy. 1996; 40(10):2237-42.

20. Thapa RK, Baskaran R, Madheswaran T, Kim JO, Yong CS, Yoo BK. In vitro release and skin permeation of tacrolimus from monoolein-based liquid crystalline nanoparticles. Journal of Drug Delivery Science and Technology. 2012; 22(6): 479-484.

21. Holler S, Clandia V. Effect of selected fluorinated drug in a ringing gel on rheological behavior and skin permeation. European Journal of Pharmaceutics and Biopharmaceutics. 2007; 66(1):120-6.

22. Madheswaran T, Baskaran R, Thapa RK, Rhyu JY, Choi HY, Kim JO, Yong CS, Yoo BK. Design and in vitro evaluation of finasteride-loaded liquid crystalline nanoparticles for topical delivery. AAPS Pharm Sci Tech. 2013; 14(1):45-52.

23. Qin Z, Chen F, Chen D, Wang Y, Tan Y, Ban J. Transdermal permeability of triamcinolone acetonide lipid nanoparticles. International Journal of Nanomedicine. 2019; 8(14)2485-2495.

24. Gaikwad, D., N. Jadhav, Terminalia Arjuna Transdermal Matrix Formulation Containing Different Polymer Components Asian Journal of Pharmaceutical and Clinical Research.2019; 26: 266.

25. Kulkarni, M., V. Hastak, S. Jadhav. Development and characterization of transdermal delivery system of doxazosinmesylate International Journal of Applied Pharmaceutics. 2019; 11: 43.

Received on 19.05.2021 Modified on 15.07.2021

Research J. Pharm. and Tech. 2022; 15(7):3273-3279.

DOI: 10.52711/0974-360X.2022.00549