INTRODUCTION:

Psoriasis is a chronic inflammatory skin disease affecting approximately 2–3% of the population in the USA and over 125 million people worldwide. The disease is characterized by scaly, erythematous skin plaques, which display inflammatory cell infiltration and neovascularization resulting from hyperprolifera- tion of the epidermis with incomplete differentiation of keratino- cytes and abnormal formation of horn cells¹.

An estimated 25% of sufferers have contemplated suicide as a result of having to suffer from the disease itself and also the social stigma of visible and unsightly symptoms².

The cause of psoriasis is unknown, although it appears to be an autoimmune disease with the likelihood of genetic predisposition³.

Acitretin (ACT) is an oral retinoid effective in the treatment of psoriasis. It is the major metabolite of etretinate with the advantage of a much shorter half-life when compared with etretinate.

Acitretin have antineoplastic, chemopreventive, anti-psoratic, and embryotoxic properties. acitretin activates nuclear retinoic acid receptors (RAR), resulting in induction of cell differentiation, inhibition of cell proliferation, and inhibition of tissue infiltration by inflammatory cells. This agent may also inhibit tumor angiogenesis²⁴. Chemical structure of acitretin is shown in fig. 1.

Fig. 1. Structure of Acitretin

New therapies incorporating nanotechnology, and an increased understanding of psoriasis, have brought us closer to the goal of a safe and efficacious treatment of the disease³. Novel topical carriers have been evaluated for enhancing skin penetration of ACT exemplified by microemulsion^8,9, nanogel¹⁰, niosomes¹¹, liposomal hydrogel¹², deformable liposomes^5,6 and solid lipid nanoparticles (SLNs)^7,13. However, they exhibit limitations of poor drug encapsulation efficiency, expulsion of drug during storage and high-water content in the formulation¹⁴. Nanostructured lipid carriers (NLC) have been explored largely in topical and cosmetic preparations, which overcome to a large extent the drawbacks associated with other nanocarriers^15–17. It is a new-generation SLN formulation composed of a solid lipid matrix entrapping variable spatially incompatible liquid lipid nano-compartments and stabilized by surfactants. NLC has an imperfect crystal structure, which prevents the expulsion of encapsulated drug thereby increasing drug loading. Furthermore, topically they impart cosmetic advantages by increasing skin hydration leading to enhanced emollience and permeation.

The current study was aimed to prepare nanogel formulation of acitretin and aloe emodin . This process will involve two steps in which nanoemulsion will be prepared and it will be incorporated in gel formulation.the nanoemulsion will be prepared by mixing of oil and appropriately screened ratio of surfactants and cosurfactants, there by generating the isotropic solution in which, droplets will be having diameter in nano range. The size reduction will be attributed by the spontaneous homogenization of the mixture. The nanoemulsion offer advantages of dose reduction and decrease in side effects by increasing the rate of permeation.

Characterization of Acitretin:

UV spectrophotometric analysis of acitretin drug sample:

Ten mg acitretin drug sample was accurately weighedand transferred to a 100ml volumetric flask to obtained a stock solution of 100µg/ml solution. An aliquot of above solution was diluted with 10 ml ofmilli Q water to obtained 20µg/ml solution and the sample was scannedbetween 200-600nm on a double beem UV/visible spectrophotometer shimadzu 1700. The UV spectrum of acitretin sample is shown in fig.11

Fig. 2: UV spectrum of acitretin in milli Q water

Melting point of acitretin:

Melting point of acitretin sample had determine by open capillary method. Sample of drug has filled into capillary which was previously sealed from one end. Capillary has placed in thiel’s tube, and filled with the liquid paraffin, along with thermometer, tube has heated and melting point has recorded. Experiment was performed in triplicate and average reading was noted.

FTIR spectral analysis of acitretin drug sample:

The FT-IR analysis of acitretin sample had performed and spectrum of acitretin had obtained between wave number regions of 400-4000 cm-1 o a FTIR spectrophotometer (Shimadzu ® IR Affinity-1) and shown in fig 3.

Table 1: Interpretation of FTIR spectra of acitretin drug sample

S. No.	Wave no.(cm^-1)	Interpretation
1.	1355	C-H bending
2.	1210	C-O streching
3.	1700	C=O
4.	1294	C=C

Differential scanning calorimetry (DSC) of acitretin:

DSC analysis acitretin drug sample was performed on differential scanning calorimeter (Perkin Elmer 6000). Approximate 3mg of acitretin drug sample was placed and sealed aluminum pans and heated from 50°C to 300ºC in a dry nitrogen atmosphere at a heating rate of 10ºC/min. A DSC thermogram of acitretin drug was obtained is shown in fig. 4.

Fig. 4: DSC thermogram of of acitretin drug sample

Preformulation Studies:

UV-Spectrophotometric Calibration Curves of Acitretin:

Preparation of calibration curve of acitretin in milli Q water:

Precisely weigh 10mg of acitretin, transfer it to a 100 mL volumetric flask, dissolve it with an appropriate amount of Milli-Q water, and dilute to 100mL to obtain a stock solution of 100μg/mL. Make appropriate dilutions of stock solutions in the concentration range of 0-100µg/ml. The resulting solution was scanned at a wavelength of 243nm on a dual-beam UV-Vis spectrophotometer. The observations are recorded in Table 6 and shown graphically in Figure 5.

Table 2: Absorbance data for calibration curve of acitretin in milli Q water

S. No.	Concentration (µg/ml)	Absorbance (Mean± S.D., n=3)
1.	0	0±0
2.	20	0.275±0.005
3.	40	0.589±0.003
4.	60	0.899±0.008
5.	80	1.226±0.003
6.	100	1.478±0.002

Fig.5: Calibration curve of acitretin in milli Q water

Preparation of calibration curve of acitretin in phosphate buffer pH 7.4

Accurately weigh 10mg of Acitretin, transfer it to a 100 ml volumetric flask, dissolve it in an appropriate amount of pH 7.4 phosphate buffer, and dilute to 100ml to obtain a stock solution of 100µg/ml. Dilute the above stock solution to a concentration of 0-10µg/ml with pH 7.4 phosphate buffer and scan the resulting solution at a wavelength of 243nm on a dual beam UV-Vis spectrophotometer. The observations are recorded in table 7 and graphically represented in fig 6.

Table 3: Absorbance data for calibration curve of acitretin in phosphate buffer pH 7.4

S. No.	Concentration (µg/ml)	Absorbance (Mean± S.D., n=3)
1.	0	0±0.00
2.	2	0.223±0.003
3.	4	0.373±0.005
4.	6	0.569±0.002
5.	8	0.768±0.005
6.	10	0.985±0.003

Fig.6: Calibration curve of acitretin in phosphate buffer pH 7.4

Solubility Determination of Acitretin Different Media

Solubility was determined in DM water. phosphate buffer (pH 7.4), ethanol, methanol, excess quantity of the drug was added into vials containing 10ml of media. The vials were sealed with the aluminum caps and placed on the incubator shaker at 25ºC temperature for 24 hr and then kept undisturbed for next 24 hr. The samples were filtered through whatman filter paper, suitably diluted and analyzed spectrophotometrically using UV- Visible spectrophotometer (shimadzu® 1700) at 243nm. The results are recorded in table 4.

Table 4: Solubility of acitretin in different media

S. No.	Solvent	Amount of drug dissolved (mg/ml)	Inference
1.	Water	2.0	Very slightly soluble
2.	Methanol	1.2	Very slightly soluble
3.	Acetone	0.8	Very slightly soluble
4.	Ethanol	1.0	Very slightly soluble
5.	Aqueous buffer	4	Very slightly soluble
6.	DMSO	1.5	Very slightly soluble
7.	Phosphate buffer pH 7.4	3	Very slightly soluble
8.	Tetrahydrofuran	5	Very slightly soluble
.9.	Dimethyl formamide	0.2	Practically insoluble

Selection of oils:

The nanogel was formulated using nanoemulsion which consist of one or more surfactants and drug dissolved in oil. This is an isotropic mixture which should be a clear, monophasic liquid at ambient temperature, and should have good solvent properties to allow presentation of the drug in solution. The solubility of acitretin and aloe emodin in various surfactants and oils were determined. These components are soluble in each other to form monophasic liquids.

Evaluation of Developed Gel Formulation:

Viscosity:

The viscosity of the formulated gel and marketed product was measured by dial viscometer (Brookfield^® LVT) viscometer. The dial readings were noted using all T spindles and at each spindle speed (sequentially). The appropriate dial readings were considered for calculations of viscosity in cps by multiplying the dial reading with the factor (specified according to the spindle number and the spindle speed). The observations are recorded.

Calculations:

Dial reading for spindle E was found to be stable and repeatable.

Factor for Spindle E is 4680/N

N= RPM

Viscosity = Dial reading × Factor

Table 5: Viscosity of developed formulation and marketed gel

S. No.	Formulation	Viscosity (cps)
1.	Developed formulation	16000
2.	Marketed product (oxalgin gel)	18000

Prediction of Optimized Acitretin and Aloe Emodin Nanogel Formulation:

The data were statistically analyzed by design expert software and the criteria for optimization was set based on the desired characteristics of the final formulation i.e. minimum particle size, minimum PDI, maximum flux and required drug release pattern (slow release to obtained sustained effect). 26 solutions were suggested by the software, one solution with maximum desirability of 0.683 was selected as the optimized formulation and the targeted nanogel were prepared as per the composition of selected batch. The prepared nanogel were characterized and the results obtained were compared with that predicted by the software.

Fig. 7: Coutour plot showing the maximum desirabilityof acitretin and aloe emodin nanogel formulation

Fig. 8: Three dimesional showing the maximum desirability of nanogels formulation of acitretin and aloe emodin

Validation of Optimized Response Parameters:

ACR-NGs were prepared using the predicted optimized composition and prepared formulation were evaluated. Predicted optimized variables for acitretin nanogel are listed below in table 6.

Table 6: Predicted optimized variable for acitretin and aloe emodin nanogels (ACR-NGs) formulation

S. No.	Variable	Quantity
1.	Smix	0.96
2.	Oil	5.06
3.	Water	99.76

Validity of experimental design was confirmed by plotting a standard error of design graph. The probability value for the determination of statistical significance was acceptable below 0.1, recommended to below 0.05. The predicted optimized nanogels were prepared and evaluated.

Characterization of the Optimized Acitretin And Alon Emodin Formulation:

Particle size distribution:

Mean particle size and PDI were determined using Malvern zetasizer using water as a dispersion medium.

Fig.9: Particle size distribution of optimized nanogels formulation

Microscopy studies:

Samples of the optimized nanogels formulation were examined using microscope (Leica) at a magnification of 100 X under oil immersion lens, the photographs are presented in fig. 10.

Fig.10: Microscopic evaluation of optimized nanogelsformulation

In-vitro release:

The in vitro relase of optimized nanogel formulation was performed and flux rate was calculated.

RESULT:

The flux rate of acitretin and aloe emodin was found to be 0.482 and 0.452.respectively and the obtained result depicted that predicted and observed response were encloseagreement with each other.

SUMMARY AND CONCLUSION:

Acitretin is a member of the retinoid drug class. Chemically, it is an oral retinoid known as Soriatane and Neotigason that is useful in treating psoriasis. Acitretin acts by preventing the keratinization (process by which skin cells thicken due to the deposition of a protein within them) and excessive cell growth that are present in psoriasis. Acitretin induces cell differentiation, inhibits cell proliferation, and prevents inflammatory cells from infiltrating tissue via activating nuclear retinoic acid receptors (RAR). As a result, it lessens scaling, plaque formation, and skin thickness. Aloe emodin is a dihydroxyanthraquinone with a hydroxymethyl group at position 3 and is chrysazin. It has been isolated from Aloe-related plant species. It is an aromatic primary dihydroxyanthraquinone.

The sample of acitretin and aloe emodin was characterized by melting point determination, U.V. spectroscopy, infrared spectroscopy and differential scanning calorimetry. The melting point of drugs was found to be 263˚C and 223°C. The U.V. spectrum, FTIR spectrum and DSC curve were compared with the reported literature and it was found that the drug sample was pure and used for further studies.

In the preformulation studies, calibration curve of Acitretin and Aloe emodin was prepared in the milli Q water and phosphate buffer 7.4. Solubility of acitretin and aloe emodin was determined in various media such as DM water, methanol, acetone, ethanol, hydrochloride acid buffer, phosphate buffer pH 7.4.

Nanogels of acitretin and aloe emodin were prepared using acitretin and aloe emodin as a drug, cottonseed oil as oil, span 80 and tween 80 as surfactant and co surfactant. The pre optimization of formulation was done by pseudoternary phase diagram. For optimization Box-Behnken design using Design Expert 7.1.6 (trial version) software (Stat-Ease Inc., Minneapolis, USA). Prior to optimization by software, pre-optimization studies were performed in order to decide the levels of factors to be used during optimization studies by software. The Box-Behnken design suggested 15 batches for the nanogel formulation. The concentration of oil, smix and water were selected as independent variables and particle size distribution, PDI, and flux rate were selected as response variables for optimization studies. Response surface graphs, contour plots and 3D contour plots were generated and analyzed for each response variable. Amongst the formulation suggested by the software (ACR-1 to ACR-15) the formulation with maximum desirability was selected as optimized formulation of acitretin and nanogels were prepared and experimentally validated. The particle size and PDI of nanoparticles were 115 and 0.239 respectively, which indicate that particles of optimized batch were of small size and narrow size distribution. In-vitrodrug release of targeted formulation was found to be 97.4 % in 6 hours. The rate of permeation was found to be 0.4032 µg/cm²/hour. The results showed that the formulation had similar activity as that of the pure drug and hence the nanogels after reaching the target site will be equally effective as of drug without producing any side effects and redness in skin.

Hence from the above results and findings it was concluded that developed nanogel of acitretin and aloe emodin can be used a potential gel which is expected to provide site-specific anti-psoriatic activity on epithelial cells with longer residence time and less side effects inside the body.

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Received on 01.09.2022 Modified on 04.10.2022

Res. J. Pharma. Dosage Forms and Tech.2023; 15(1):19-24.

DOI: 10.52711/0975-4377.2023.00004