INTRODUCTION:

Acne is a skin disease that often occurs in women and men starting from puberty, which can reduce the sufferer's self-confidence. Acne is a skin disease in the sebaceous glands¹. Acne can occur due to increased keratinization, excess sebum, and sebaceous gland hyperplasia, bacterial hypercolonization, and inflammatory processes associated with the adaptive immune system². Bacteria that cause acne include Propionibacterium acnes (P. acnes), Staphylococcus epidermidis (S. epidermidis), and Staphylococcus aureus (S. aureus)³. P. acnes and S. epidermidis was the most dominant bacteria causing acne compared to S. aureus⁴.

P. acnes is a gram-positive bacterium that lives naturally in the sebaceous glands. P. acnes will stimulate sebum production⁵. The excess sebum will increase the production of proinflammatory cytokines, such as IL-6, IL-8, IL-1α, and tumor necrosis factor-α in epithelial cells, lymphocytes, and neutrophils that can cause an inflammatory reaction and cause acne lesions⁶. The proliferation of P. acnes bacteria can be prevented by using antibiotics such as doxycycline, tetracycline, clindamycin, clarithromycin, erythromycin, and azithromycin⁷.

In this study, Clindamycin HCl was used as an anti-acne. Clindamycin HCl can reduce the population of P. acnes in sebaceous glands by inhibiting protein synthesis on the 50S ribosomal subunit^8-10. However, using Clindamycin HCl as an anti-acne has obstacles related to its bioavailability. Based on previous studies, clindamycin HCl has a bioavailability ranging from 0.7-12.9% of the dose used within 24hours¹¹. This low bioavailability will impact the effectiveness of the drug where the mechanism of the acne itself is in the sebaceous glands, which require deeper penetration. To increase the bioavailability of clindamycin HCl, it is necessary to develop drug delivery systems such as ethosomes.

Ethosomes are lipid bilayer vesicles modified from liposomes containing high ethanol concentrations up to 45%¹².The high ethanol concentration in the formulation will help solubilize the drug and make the lipid structure permeable, so it can quickly enter through the gaps between skin cells¹³. In addition, the presence of phospholipids will also help penetrate the drug topically, where the lipids in the ethosomes will fuse with lipids in the skin¹⁴. Ethosom clindamycin HCl will be further formulated into a topical gel preparation.

A gel is a topical preparation containing up to 90% water concentration. Gels have advantages over other topical acne preparations because they do not contain oils that can aggravate acne conditions¹⁵. The gelling agent is an essential ingredient in gel that can affect the characteristics of the resulting preparation. This study used a combination of gelling agents, namely Hydroxypropyl methylcellulose (HPMC) and Carbopol 940. Carbopol 940 is known to have a suitable viscosity effect on the gel, easy solubility in water can form a clear gel, and has acidic properties¹⁶. While HPMC has a promising drug release effect, does not irritate the skin, and has a stiffer texture¹⁷. The combination of HPMC and Carbopol 940 can cover the deficiency of HPMC, which results in a stiff gel, and cover the lack of Carbopol 940, which is acidic, causing skin irritation¹⁸.

Based on the description above, in this study, clindamycin HCl ethosomal gel preparations with variations in the concentration of HPMC and Carbopol 940 will be made using a 2² factorial design. The gel is then characterized to determine the optimum formula. The optimum formula was then tested for stability and anti-acne activity against P. acnes bacteria in vitro by calculating the diameter of the inhibition zone and the minimum inhibitory concentration value.

MATERIALS AND METHODS:

Materials:

The materials used were clindamycin HCl (gift from PT. Dexa Medica, Indonesia), phospholipone 90G (Lipoid, USA), ethanol 96% (Bratahem, Indonesia), carbopol 940 (Bratachem, Indonesia), hydroxypropyl methylcellulose (Bratachem, Indonesia), triethanolamine (Bratachem, Indonesia), propylene glycol (Bratachem, Indonesia), methylparaben (Smartlab, Indonesia), propylparaben (Smartlab, Indonesia), Butyl Hydroxy Toluene (Bratachem, Indonesia), aquadest (Bratachem, Indonesia), Propionibacterium acnes (culture from Sriwijaya University), Nutrient Agar (NA) (Himedia, Indonesia), and Nutrient Broth (NB) (Himedia, Indonesia).

Production of Clindamycin HCl Ethosomes:

Ethosomes of clindamycin HCl were prepared by the thin layer hydration method using glass beads¹⁹. The ethosomal formula refers to the previous research conducted by Apriani et al.²⁰, namely, the concentration of clindamycin HCl was 1%, Phospholipone 90G was 2%, ethanol was 40%, and phosphate buffer pH 7.4 ad 100%. Phospholipone 90G was dissolved in dichloromethane and ethanol in a ratio of 2:1. The mixture was then evaporated to form a thin layer. The thin layer was then hydrated using a hydroethanolic solution containing clindamycin HCl, 96% ethanol, and phosphate buffer pH 7.4 to 100mL.

Freeze Drying of Clindamycin HCl Ethosomal Suspension:

The ethosomal suspension was dried using the freeze-drying method at a temperature of -50^oC for five days before being formulated into a gel.

Production of Clindamycin HCl Ethosomal Gel:

The clindamycin HCl gel formula was obtained through a 2² factorial design so four formulas were obtained. The independent variables used were HPMC and Carbopol 940. The design of the gel formulation formula can be seen in Table 1. HPMC was dispersed in distilled water at 90^oC, while carbopol was dispersed in distilled water and added three drops of TEA until it swelled. The HPMC and carbopol bases were mixed and ground until homogeneous. Methylparaben and propylparaben were dissolved in propylene glycol and incorporated into the gel mass. Butyl hydroxytoluene and ethosomal clindamycin HCl were added to the gel mass and ground homogeneously. After that, aquadest was added to a volume of 100mL²¹.

Table 1: Design formula of clindamycin HCl ethosomal gel

No	Ingredients	Formula (%)
No	Ingredients	F1	F2	F3	F4
1	Clindamycin HCl Ethosomes Powder	1	1	1	1
2	Carbopol-940	0.5	1	0.5	1
3	HPMC	0.5	0.5	1	1
4	TEA	3 gtt	3 gtt	3 gtt	3 gtt
5	Propyleneglycol	10	10	10	10
6	Methylparaben	0.02	0.02	0.02	0.02
7	Propylparaben	0.18	0.18	0.18	0.18
8	Buthyl Hydroxy Toluene	0.1	0.1	0.1	0.1
9	Distilled water ad	100 mL	100 mL	100 mL	100 mL

Characterization of Clindamycin HCl Ethosomal Gel

Organoleptic:

Organoleptic observations consisted of observing the shape, color, and odor of the gel made visually.

Homogeneity:

The homogeneity test was carried out by applying the preparation to a glass object.

pH:

The pH test was carried out using a pH meter.

Viscosity Test:

The viscometer is used as a viscosity measuring device. Place 30mL of gel in a beaker into the viscometer at 60 rpm. Observations were made by looking at the instrument needle leading to the viscosity scale number. Viscosity values are indicated by stable needle movement²².

Spreadability:

The test was carried out by placing 0.5grams of the preparation on a transparent glass and closed for about 1 minute. Then calculate the area given by the preparation. After that, it was covered again with transparent glass, given a load of 50 and 100grams, respectively, and left for 1 minute. Then, the increase in the area produced by the preparation was calculated²³.

Adhesion:

A total of 0.25g of the preparation was placed between two glass objects, then pressed with a load of 1kg for 5 minutes. The load is lifted from the object glass. Then the object glass is mounted on the test equipment with a load of 80g. The time of the release of the preparation from the object glass was recorded²³.

Determination of Optimum Formula:

The optimum formula is based on the desirability value close to 1. If the desirability is getting closer to the value 1, then the program to produce the desired formula is perfect. The preparation must be homogeneous as a requirement to meet the homogeneity test, have a pH in the range of 4.5-6.5²⁴, a viscosity value in the range of 2000-4000 cP²⁵, has a spreadability of 5-7 cm²⁶, and adhesion by more than 4 seconds²².

Antiacne Activity Test:

The antiacne activity test measured the minimum inhibitory concentration using the liquid dilution method²⁷. The clindamycin HCl ethosomal gel and clindamycin HCl gel used were 80, 70, 60, 50, and 40 µg/mL²⁸. A test tube adds a certain amount of test solution with NA media and bacterial suspension. The mixture was vortexed for 10 s. The MIC value was obtained by looking at the turbidity compared to the positive control containing NA media and bacterial suspension and the negative control, DMSO solution. The smallest concentration that looks clear without any microbial growth is defined as the Minimum Inhibitory Concentration (MIC).

Data Analysis:

Data analysis was carried out using the Design Expert version 13 to see the influence of factors and factor interactions on the response by observing the R2, R2-adjusted, R2-predicted, Adequate precision, p-value, coefficient, and percent contribution.

RESULT:

Clindamycin HCl Ethosomes:

The resulting clindamycin HCl ethosomal suspension was milky white and no precipitation occurred. The ethosomal formula refers to the research of Apriani et al.²⁰, namely 2% phospholipone and 40% ethanol. The ethosomal suspension was then dried by the Freeze-drying method to obtain a drier form (Figure 1). However, the drying results obtained are still in the form of a paste because ethanol has a freezing point at a temperature of -114.1^oC so that when using a freeze dryer temperature of -50^oC, the resulting ethosomal suspension is not completely dry²⁹.

Figure 1. Clindamycine HCl Ethosome Solution (A) and Dried (B)

Clindamycin HCl Ethosomal Gel:

The preparation of the clindamycin HCl ethosomal gel was characterized by the preparation and the results were obtained as shown in Table 2 and Figure 2. The results of the gel preparation characteristics of the four formulas indicate that the resulting gel has good characteristics because it is in accordance with the gel requirements, namely the preparation must be homogeneous, has a pH in the range of 4.5-6.5, a viscosity value in the range of 2000-4000 cPs, spreadability of 5-7cm, and adhesion of more than 4 seconds.

Table 2: The Characteristics of Clindamycin HCl Ethosomal Gel

Parameter	Formula
Parameter	F1	F2	F3	F4
Organoleptic	White, thick and odorless	White, thick and odorless	White, thick and odorless	White, thick and odorless
Homogeneity	Homogeneous	Homogeneous	Homogeneous	Homogeneous
pH	5.13 ± 0.04	5.15 ± 0.03	5.21 ± 0.03	4.57 ± 0.01
Viscosity (cPs)	2689.36±168.10	2912.23±275.58	3090.53±294.18	2511.06±147.09
Spreadability (cm)	5.80 ± 0.14	6.10 ± 0.13	6.30 ± 0.13	5.80 ± 0.08
Adhesion (s)	210.19 ± 1.77	230.07 ± 1.62	249.54 ± 2.08	211.46 ± 1.83

Table 3: The results of the analysis of the response model of pH, viscosity, spreadability, and adhesion

	Response	Parameter
	Response	p-value	R²	Adjusted R²	Predicted R²	Adequate precision
R1	pH	< 0.0001*	0.9888	0.9846	0.9748	32.8585
R2	Viscosity	0.1413	0.4757	0.2791	-0.1796	3.5564
R3	Spreadability	0.0093*	0.7451	0.6495	0.4265	5.4913
R4	Adhesion	< 0.0001*	0.9872	0.9823	0.9711	30.4051

* indicates that the response has a significant effect (p<0.05)

Optimum Formula of Clindamycin HCl Ethosomal Gel:

The optimum formula for clindamycin HCl ethosomal gel was determined using the Design Expert 12® application. The system in the application will determine the optimum formula based on the desirability value of the desired response criteria. The response criteria in this study are pH in the range 4.5-6.5, spreadability in the range 5-7, and maximized adhesivity. The concentration of the formula with the desirability value getting closer to 1 indicates that the formula is getting closer to the desired response criteria³¹. The optimum concentration obtained in this study for HPMC and Carbopol 940 was 1% and 0.5%, respectively, with a desirability value of 0.994.

Antiacne Activity of Clindamycin HCl Ethosomal Gel:

The antiacne activity of the clindamycin HCl ethosomal gel and clindamycin HCl gel were seen by measuring the Minimum Inhibitory Concentration (MIC) against P. acnes. The results of the KHM can be seen in Table 6.

Table 6: Minimum Inhibitory Concentration Results

Test Solution	Concentration (mg/mL)
Test Solution	80	70	60	50	40
Clindamycin HCl Ethosomal Gel	-	-	-	+	+
Clindamycin HCl Gel	-	-	+	+	+

Description: - indicates that there is no turbidity, + indicates that there is turbidity

DISCUSSION:

The clindamycin HCl ethosomal gel has a good characteristic because it is in accordance with the gel requirements. The characteristics data was used to analysis model for optimization process.Based on Table 3, the response of pH, spreadability, and adhesion has a good model. But the model is not strong enough for the viscosity response to continuing the optimization process. The viscosity model has an insignificant effect, indicated by a p-value>0.05. If seen in Figure 3, for the viscosity response, it is also noticed that the factor is near from the half normal plot line and also the bar chart in pareto chart was below the Bonferroni limit Therefore, the responses that can continue for the optimization process are pH, spreadability, and adhesion.

The response of pH, spreadability, and adhesion was used to optimization process. The three response was analyzed to see the effect of the HPMC, Carbopol, and their interaction to the response. Based on the response analysis in Table 4, HPMC (A), Carbopol 940 (B), and the interaction of the two factors (AB) had a significant effect on the pH response and adhesion. However, the spreadability response that has a significant effect is the interaction of the two factors (AB), which is marked with a p-value < 0.05. The sign on the coefficients can illustrate the impact of the factors, namely positive (+) and negative (-). The positive (+) sign indicates that the factor has an impact directly proportional to the response value, but if the coefficient sign - then the factor has an inversely proportional effect on the response value. The positive (+) and negative (-) sign can also see in the equation of each response in Table 5.

In the pH response, factors A, B, and AB have a negative effect on the response, meaning that the greater the concentration of A, B, and AB used, the lower the pH value. When viewed from the % contribution, factor AB has the highest % contribution at 41.0033%, followed by factor B at 34.7353% and factor A at 23.1409%. When viewed from the interaction curve of the pH factor in Figure 4, there is a line intersection between factor A and factor B because there is a difference in the direction of the response produced by factors A and B. It indicates that there is an interaction between factor A and factor B. On the contour graph plot, the red area means that the pH of the preparation is getting higher, namely, at the point where the concentration of HPMC is 1% and Carbopol 940 is 0.5%. The more concentration of HPMC used, the pH value will increase, while the more concentration of carbopol used, the pH value will decrease. It is because carbopol has acidic properties^25,32.

In the spreadability response, the interaction between factors A and B (AB) has a negative effect on the response, meaning that the greater the concentration of AB used, the smaller the spreadability value will be. When viewed from the % contribution, factor AB has a % contribution of 70,5882%. The interaction curve also shows that factors A and B have an interaction so that the line intersects between the two. At the same high concentrations of HPMC and Carbopol 940, the spreadability of the preparations will be smaller, as well as when the concentrations of both are equally low. However, the resulting spreadability is higher when combined with HPMC at high concentrations and Carbopol 940 at low concentrations or vice versa. In the contour plot graph, the red area indicates that the spreadability of the preparation is getting higher, namely at the point where the concentration of HPMC is 1% and Carbopol 940 is 0.5%. The viscosity of the resulting preparation strongly influences the results of this spreadability. The more concentrations of HPMC and Carbopol 940 used, the higher the viscosity of the preparation so that the spreadability will be lower. HPMC and Carbopol 940 can expand because they have OH groups, so hydrogen bonds with water will occur and cause swelling³³. The OH group in Carbopol is obtained from the dissociation of acrylic acid in the carbopol 940 structure when in contact with water³⁴.

In the adhesion response, factors A, B, and AB negatively influence the response, meaning that the greater the concentration of A, B, and AB used, the value of the adhesion will be smaller. When viewed from the % contribution, factor AB has the highest % contribution, which is 80.4869%. When viewed from the interaction curve of the adhesive response in Figure 4, there is a line intersection between factor A and factor B because there is a difference in the direction of the response produced by factors A and B. It indicates that there is an interaction between factor A and factor B. Same as spreadability, when the concentration of HPMC and Carbopol 940 is high, the adhesion of the preparation will be lower because the preparation is stiff. When both concentrations are low, the adhesion will also be smaller because the viscosity is low. In the contour plot graph, the red area indicates that the adhesive strength of the preparation is getting higher, namely at the point where the concentration of HPMC is 1% and Carbopol 940 is 0.5%.

The optimum formula was obtained at the concentration of HPMC and Carbopol 940 was 1% and 0.5%, respectively, with a desirability value of 0.994. The optimum formula then used for antibacterial activity test. Based on Table 6, in this study, the MIC values were obtained for the clindamycin HCl ethosomal gel and clindamycin gel at a concentration of 60 µg/mL and 70 µg/mL, respectively. It indicates that clindamycin HCl ethosomal gel preparations have better antibacterial activity than clindamycin HCl gel preparations. Drug delivery in the form of ethosomes can increase the antibacterial activity of clindamycin HCl. The presence of lipid components in the ethosomes will assist the process of delivering clindamycin into the bacterial cytoplasmic membrane by fusion³⁵. This fusion reaction can occur spontaneously and quickly because it is driven by non-covalent forces such as van der Waals and hydrophobic interactions. The drug contained in it will enter the cytoplasmic membrane quickly³⁶. In addition, the characteristics of ethosomes will also help penetrate the preparation into the skin due to its nanoparticle size.

CONCLUSION:

Clindamycin HCl ethosomal gel has physical evaluation results that meet all test criteria. Based on the factorial design analysis, the concentration of HPMC (A), Carbopol 940 (B), and their interaction affected the pH response and adhesion, while the interaction of the two factors influenced the spreadability. The optimum formula was obtained at a concentration of 1% HPMC and 0.5% Carbopol 940 with a desirability value of 0.994. The optimum formula of clindamycin HCl ethosomal gel showed a minimum inhibitory concentration (MIC) at 60 µg/mL, while the clindamycin HCl gel showed at 70 µg/mL.

CONFLICT OF INTERESTS:

The authors declare no conflict of interest.

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Received on 04.10.2022 Modified on 15.04.2023

Research J. Pharm. and Tech 2024; 17(2):603-611.

DOI: 10.52711/0974-360X.2024.00094

Response	Parameter	Intercept	A	B	AB
pH	Coefficient	5.014	-0.126*	-0.154*	-0.168*
	p-value		<0.0001*	<0.0001*	<0.0001*
	% Contributions		23.1409 %	34.7353%	41.0033%
Spreadability	Coefficient	6	-0.333	-0.333	-0.200*
	p-value		0.4554	0.4554	0.0015*
	% Contributions		1.96078%	1.96078%	70.5882%
Adhesion	Coefficient	225.309	5.1842*	-4.5442*	-14.4858*
	p-value		<0.0001*	0.0001*	<0.0001*
	% Contributions		10.3085%	7.92039%	80.4869%

Response	Parameter
pH	y = 5.01 – 0.1542A – 0.1258B - 0.1675AB
Spreadability	y = 6 – 0.0333A + 0.0333B -0.2000AB
Adhesion	y = 225.31 – 4.54A + 5.18B -14.49AB