In Vitro Release Testing of Acyclovir Topical Formulations Using Immersion Cells

Abstract

The objective of the study was to reinforce the applicability of the immersion cells for the in vitro release testing (IVRT) of topical formulations by using marketed acyclovir 5% cream formulation (Cream 1) as a model. The method employing the immersion cells was optimized by studying the effect of variables, such as membrane type, media temperature and volume, agitation speed, and cell size, on acyclovir release from the formulation. The in-house formulation similar to the qualitative and quantitative composition of Cream 1 and the other trial formulations with variable compositions were prepared and studied by using the immersion cells. Various other brands of acyclovir topical formulations available in the Indian market were also subjected to IVRT by using the optimized method. An increase in the media temperature from 32°C to 37°C and the stirring speed from 50 to 100 to 150 rpm led to an increase in the drug release. As the immersion cell size increased (0.5, 2 and 4 cm² surface area), the release rate also increased. Nitrocellulose membrane showed the highest drug release and Fluoropore^™ the least. The optimized IVRT method could establish the differences in the drug release rates among the formulations with the altered compositions. The method could also prove its discriminatory potential for various marketed formulations. The immersion cell method could serve as a simpler, facile, and reliable aid during product development and also as a quality control tool in assessing stability, aging, and batch-to-batch uniformity of semisolid formulations.

Introduction

In vitro release testing (IVRT) is an important quality and performance parameter for evaluation of the topical semisolid formulations. Similar to the solid oral dosage forms, IVRT of semisolids can help during product development stages in identifying the critical formulation and manufacturing variables. The release profile of the active ingredient from the formulation allows for optimization of the physical characteristics of the formulation during product development. A well-established, discriminatory IVRT method can also provide support during the stages of clinical development by establishing sameness as minor formulation changes are implemented during the course of clinical assessment. Besides aiding in product development, the IVRT can serve as a quality control tool to confirm batch-to-batch uniformity of the product. It helps in comparing the in vitro release profiles of test and reference products, though it is not expected to correlate or be predictive of in vivo bioavailability or bioequivalence. With the establishment of in vitro in vivo correlation for the developed method, the prediction of in vivo bioequivalance could also be possible.

A number of apparatuses, including vertical diffusion cells (VDCs), immersion cells, inverted immersion cells, flow-through cells, paddle over disk apparatus, and rotating cylinders apparatus, have been recommended for the IVRT of topical semisolid and transdermal formulations.^1–3 The IVRT has been made mandatory by the U.S. FDA since 2013 as one of the quality and performance parameters for the testing of semisolid formulations. As per the guidance of the U.S. FDA, the IVRT should be performed in a manner described in USP General chapter<1724> Semisolid Drug Products.⁴ The USP indicates the use of one of the three types of cells—VDCs, commonly known as Franz diffusion cells; immersion cells, also known as Enhancer cells; and Flow-through cells or USP Type 4 apparatus. Of these, VDCs are explored the most for developing and validating IVRT of formulations of many drugs; whereas flow-through cells have been explored the least for this purpose. The immersion cell method employs the routine USP Type 2 dissolution apparatus and needs only a set of flat-bottom flasks of 200 mL capacity and mini paddles to conduct the IVRT. Uniform and accurate loading of the semisolid formulation in the immersion cells is easily possible without the incorporation of any air bubbles. The immersion cells are rugged and easy to handle. The prepared cells are placed at the bottom of the flasks, receptor medium is then added to the flasks, and finally the IVRT is carried out in a manner similar to that of the solid oral dosage forms. Alternatively, the prepared cells could also be placed in the flasks already filled with the receptor medium. Sampling of the receptor medium can be performed accurately without any errors. Moreover, a higher dissolution volume (up to 200 mL) allows for better maintenance of sink conditions. In spite of these advantages, both industry and academia show limited use of the immersion cell technique for the IVRT of semisolid formulations. Ophthalmic ointments of cyclosporine⁵ and acyclovir,^6,7 topical formulations of drugs such as tramadol,⁸ mupirocin,⁹ ibuprofen,¹⁰ hydrocortisone acetate,¹¹ and triamcinolone acetonide¹² are a few examples that have been evaluated by using immersion cells.

Krishnaiah et al.¹³ have reported the use of immersion cells for the IVRT of acyclovir cream formulations, wherein they studied the influence of variables such as emulsification time and temperature, homogenization speed, and pH of aqueous phase on the release of the acyclovir from its formulation. In the present work, we also chose marketed acyclovir 5% cream formulation as a model for the development of the IVRT method employing immersion cells. However, unlike Krishnaiah et al.,¹³ the focus of our work was to understand the influence of variables of the immersion cell method, such as media volume, media temperature, stirring speed, membrane type, and exposed area, on acyclovir release from a standard marketed cream formulation. To our knowledge, all these variables have not been studied collectively for the IVRT of acyclovir cream formulations using immersion cells. We also attempted to study the ability of the developed IVRT method to establish the difference among the in-house acyclovir cream formulations prepared with deliberately varied compositions. The method was further used for comparing the in vitro release rates of various marketed generic topical formulations of acyclovir. Thus, the aim of our work was to reinforce and establish the importance of a simple, facile IVRT method involving immersion cells that could be used commercially as well as in academic research as a quality control and formulation development tool.

Materials

Acyclovir was obtained as a gift sample from Indeus Life Sciences Pvt. Ltd. (Mumbai, India). Nitrocellulose, Fluoropore^™ (polytetrafluoro ethane), and Durapore^™ (polyvinylidene fluoride) membranes of 0.45 μm pore size and 25 mm diameter were purchased from Millipore (Bangalore, India). Boric acid, potassium chloride, cetostearyl alcohol, propylene glycol, sodium lauryl sulfate, white soft paraffin, and liquid paraffin were purchased from Analab Finechem, Pune, India. Poloxamer 407 was obtained as a gift sample from BASF India Pvt. Ltd. (Mumbai, India). Four brands of acyclovir 5% cream formulations and one brand of acyclovir 5% ointment formulation were procured from the local pharmacy after ensuring that none of them had an expiry date before 2021.

Methods

Study of Method Variables

Preparation of the immersion cells

Immersion cell model A (cell size of 2 cm²; Electrolab India Pvt. Ltd.) was used with USP type 2 apparatus (EDT 08lx; Electrolab India Pvt. Ltd.) to determine the in vitro release of acyclovir from the marketed acyclovir 5% cream formulation (Cream 1). A set of flat-bottom dissolution vessels (200 mL) held in place with the aid of holders and mini paddles instead of standard paddles were used in the test. The nitrocellulose membrane (0.45 μm average pore size, 150 μm thickness) was soaked in dissolution medium 30 min before the loading of immersion cells into the dissolution test apparatus. The reservoir of the cell was filled completely and uniformly with the cream formulation by using a spatula. The weight of the cream filled into these enhancer cells was determined by subtracting the weight of empty cell assembly from the weight of cells completely filled with cream. The mean weight of the cream used in the drug release testing was 1.5 g. The soaked nitrocellulose membrane was dabbed on a filter paper and placed over the surface of the sample compartment without the formation of wrinkles in the membrane. The immersion cell components were assembled as specified by the manufacturer.

IVRT using different method variables

The assembled immersion cell was placed at the bottom of the dissolution vessel with the membrane facing up. The paddle height was set at 1.0 ± 0.2 cm above the surface of the membrane. Purified Milli-Q water was used for the preparation of alkaline borate buffer pH 9.2. The buffer was vacuum-filtered through a 0.45 μm filter and stirred vigorously under vacuum for 15 min to ensure that the level of dissolved gases in the buffer was below 5 parts per million. The buffer, 200 mL (pre-heated to 32°C ± 0.5°C) was added to the dissolution vessel containing the loaded immersion cell to start the test. The samples (5 mL) were withdrawn at 0.25, 0.5, 1, 2, 4, and 6 h intervals with simultaneous replenishment with equal quantity of fresh buffer. The amount of acyclovir released in the samples was determined by a validated ultraviolet (UV) spectrophotometry method at the λ_max of 254 nm. The cumulative amount of the drug released per unit surface area of the membrane was plotted against the square root of time to determine the in vitro release rate (slope of the plot) of acyclovir from the formulation. The experiment was carried out at three different paddle stirring speeds, that is, 50, 100, and 150 rpm. A similar IVRT experiment was performed by using Fluoropore (0.45 μm average pore size, 150 μm thickness) and Durapore (0.45 μm average pore size, 150 μm thickness) membranes in place of nitrocellulose membrane. While employing each of these membranes, the effect of varying stirring speeds (50, 100, and 150 rpm) on the drug release was studied. Each experiment was carried out in six replicates.

The effects of temperature of the dissolution medium, volume of the dissolution medium, and immersion cell size on the acyclovir release rate were studied (Supplementary Table S1).

Preparation of Acyclovir Cream Formulations

Acyclovir cream formulations were prepared by using the compositions listed in Table 1. The aqueous phase comprising poloxamer 407, propylene glycol, sodium lauryl sulfate, and purified water was heated to 60°C. The drug was dispersed in the aqueous phase at 60°C with continuous stirring for 15 min. The oil phase was prepared by melting the mixture of white soft petrolatum, liquid paraffin, and cetostearyl alcohol at about 60°C for 15–20 min. The aqueous phase was then added to the oil phase with constant stirring until the resulting cream attained room temperature. The cream was subjected to homogenization for 30 min at 5,000 rpm (L5M-A; Silverson, UK). Formulation F1 was prepared by using the same qualitative and quantitative composition as the reference listed formulation (Cream 1). Formula F2 did not contain propylene glycol, the cosolvent for acyclovir, whereas formulation F3 contained a higher concentration of propylene glycol (60% instead of 40% in F1). Formulation F4 was prepared by altering the quantity of liquid paraffin and white soft paraffin. In formulations F5 and F6, polyethylene glycol 200 and polyethylene glycol 4000 replaced the propylene glycol. The creams thus prepared were filled in multiple-dose, lacquered aluminum tubes (approximate 25 g in each tube) and stored at 25°C/60% relative humidity until further use.

Table 1.

In-House Trials of Acyclovir Cream Formulations

Ingredients	Quantity (% w/w)
	F1	F2	F3	F4	F5	F6
Acyclovir	5	5	5	5	5	5
Cetostearyl alcohol	6.75	6.75	6.75	6.75	6.75	6.75
Sodium lauryl sulfate	0.75	0.75	0.75	0.75	0.75	0.75
Liquid paraffin	5	5	5	12.5	5	5
White soft paraffin	12.5	12.5	12.5	5	12.5	12.5
Poloxamer 407	1	1	1	1	1	1
Propylene glycol	40	—	60	40	—	—
Polyethylene glycol 200	—	—	—	—	40	—
Polyethylene glycol 4000	—	—	—	—	—	40
Purified water	q.s.	q.s.	q.s.	q.s.	q.s.	q.s.

Evaluation of Cream Formulations

Visual examination

The prepared creams were examined visually for their color, appearance, homogeneity, and consistency.

pH

The pH of the in-house formulations and Cream 1 was checked by using a pH meter (pH11; Systronics, Mumbai, India).

Viscosity

The creams were subjected to viscosity determination by using a viscometer (Brookfield-DVE LV, USA). The viscosity measurements were conducted at room temperature (25°C) for each formulation in triplicate by using spindle no. 64 at the shear rate of 50 rpm. The readings were noted after applying the shear rate for 2 min. The viscosity of Cream 1 (marketed formulation) was also determined in a similar manner. The viscosities were compared by one-way analysis of variance (ANOVA) at p ≤ 0.05 followed by post hoc Tukey test by using Graph Pad Prism software (version 7).

Texture analysis

A texture analyzer (CT3; Brookfield, USA) was used to compare the texture properties of the cream F1 and Cream 1. Approximately 30 g of the cream formulation was filled in a cup that was fixed on the base plate of the texture analyzer. A 40-mm (diameter) probe was compressed into the cream and redrawn at the speed of 1 mm/s. Three replicate analyses were performed at room temperature for each formulation, providing the same conditions for each measurement. Parameters such as hardness, adhesive force, deformation, and adhesiveness were determined from the resultant force-time plot and compared by using Student's t test at p ≤ 0.05.

Assay

Cyberlab LC 1700, USA; high-performance liquid-chromatography (HPLC) system was used for the analysis of the drug content in the cream formulations as per the method specified in USP36.³ The system was equipped with a 4.6 × 25 mm C18 column (Kromasil Nuryon, India), UV detector, and binary solvent pump. Glacial acetic acid solution (0.5% v/v) was used as a mobile phase. The flow rate was adjusted to 3 mL/min. The standard solution of acyclovir 0.1 mg/mL was prepared in 0.1 N sodium hydroxide solution and injected into the HPLC system equipped with a 20 μL loop, and the eluting acyclovir solution was detected at 254 nm. For assay preparation, an accurately weighed quantity of acyclovir cream equivalent to 10 mg of acyclovir was transferred to a 100 mL volumetric flask and a sufficient quantity of 0.1 N sodium hydroxide solution was added to the flask. The dispersion was sonicated in the bath sonicator containing cold water for about 30 min, filtered by using Whatman filter paper, and 20 μL of the filtrate was injected into the column. The assay was done in triplicate for all the prepared formulations.

In vitro release testing

The prepared cream formulations were subjected to IVRT by loading into immersion cells (2 cm² surface area). The nitrocellulose membrane was used as a barrier between the cream and the dissolution medium comprising 200 mL of degassed alkaline borate buffer pH 9.2 maintained at 32°C ± 0.5°C and stirred at the rpm of 150. Aliquots, 5 mL each, were withdrawn at the time intervals of 0.25, 0.5, 1, 2, 4, and 6 h during the studies and replaced by an equal amount of buffer. Each formulation was studied in six replicates. The drug content in the aliquots was determined by spectrophotometric analysis at 254 nm. The Higuchi plot was constructed by graphing drug release per unit surface area against the square root of time. The slope of the line represented the release rate.

Statistical Analysis

The release rates of all the trial formulations were compared with that of Cream 1 by one-way ANOVA at p ≤ 0.05 followed by post hoc Dunnett's test by using Graph Pad Prism software (version 7).

IVRT of Various Marketed Formulations

Four different brands of acyclovir cream formulations procured from the pharmacy were labeled as Cream 1, Cream 2, Cream 3, and Cream 4 and one marketed ointment was labeled as Ointment 1. The formulations were subjected to assay and IVRT as per the methods described in the Evaluation of Cream Formulations section. Statistical analysis of the release rate data for these formulations was done by one-way ANOVA at p ≤ 0.05 followed by post hoc Tukey test by using Graph Pad Prism software (version 7).

Results

Study of Method Variables

Cream 1 containing 5% w/w of acyclovir was chosen as a standard formulation for studying and comparing the effect of method variables on in vitro drug release. Acyclovir shows the higher solubility in alkaline borate buffer.¹⁴ Hence, alkaline borate buffer, pH 9.2 was chosen as the receptor medium for the IVRT studies, which would ensure sink conditions.¹³ A number of reports in the literature have pointed out that the type of the barrier membrane used during the IVRT has a significant impact on the release of a drug from its semisolid formulation.^13,15–18 The synthetic membrane should provide inert support and least diffusional resistance to the drug. Nitrocellulose, Fluoropore, and Durapore membranes (same thickness and pore size) were selected to study the release of acyclovir from its cream preparation. The effect of the paddle rotation speed on the acyclovir release by using immersion cells has not been previously reported. During the current investigation, drug release was studied at 50, 100, and 150 rpm. The drug release rates were not significantly different (p ≤ 0.05) at different paddle rotation speeds when nitrocellulose or Durapore were used as barrier membranes (Fig. 1A, C). Generally, an increase in agitation leads to a reduction in the thickness of the diffusion layer (at the interface between the receptor phase and the membrane) and provides better mixing. No significant difference would mean that the system was well mixed and the stagnant layer above the membrane had a minimal effect. However, a significant difference among release rates as a result of the change in agitation speed was observed when Fluoropore was used as a barrier membrane (Fig. 1B). Moreover, Fluoropore showed a significantly lower release rate of acyclovir as compared with the other two membranes (Fig. 1D). This observation was in agreement with the IVRT of acyclovir cream formulations using VDCs done by Nallagundla et al.¹⁹ There could have been an interaction between the drug and Fluoropore (polytetrafluoro ethane), hampering its release into the receptor medium. This could also explain the significant influence of agitation speed on the drug release in the case of this membrane. Higher agitation speed could have helped in dislodging the drug from the membrane, thus improving the release rate. A paddle speed of 150 rpm and nitrocellulose as a barrier membrane were selected for further studies.

Fig. 1.

Effect of stirring speed and membrane type on the in vitro release of acyclovir from the marketed cream formulation (Cream 1). In case of nitrocellulose membrane (A) and Durapore^™ membrane (C), the acyclovir release from Cream 1 did not increase significantly with an increase in the stirring rate from 50 to 100 to 150 rpm. However, stirring rates significantly influenced the drug release when Fluoropore^™ membrane was used (B). The drug release was higher from nitrocellulose and Durapore membranes, whereas use of Fluoropore membrane led to highly variable and poor drug release. (D) The immersion cell size used for the study was 2 cm². Data expressed as mean ± SD (n = 6). SD, standard deviation.

The experiment was designed to study the effect of different parameters such as media temperature, volume of dissolution medium, and cell size (0.5/4 cm² surface area) on the in vitro drug release from the cream formulation.

Temperature effect

The IVRT of the Cream 1 was carried out at 32°C and 37°C. The release profiles are shown in Figure 2A. The release rate increased significantly as the temperature of the dissolution medium was increased to 37°C. At the end of the IVRT studies, no apparent change was observed in the consistency of the cream loaded in the immersion cells. This ruled out the influence of change in the viscosity of formulation on the drug release. An increase in the release rate could be attributed to the higher diffusion coefficient of the drug at a higher temperature.

Fig. 2.

Effect of test variables on the in vitro release of acyclovir from the marketed cream formulation (Cream 1). (A) Increase in the media temperature from 32°C to 37°C significantly increased (P < 0.05) the in vitro release of acyclovir from the marketed cream formulation (Cream 1). (B) Media volume of 150 mL showed significantly lower drug release (P < 0.05) as compared with 200 mL of the medium. (C) The drug release increased significantly (P < 0.05) as the immersion cell size increased from 0.5 to 2 to 4 cm². Data expressed as mean ± SD (n = 6).

Media volume effect: Lower drug release was observed when the volume of the dissolution medium was 150 mL instead of 200 mL (Fig. 2B). A lower volume could have led to a lower concentration gradient and hence a lesser release of the drug.

Cell size effect

As the cell size increased, the drug release also increased due to greater exposure of the membrane to the medium. The release was maximum from the cells with 4 cm² area, followed by 2 and 0.5 cm², respectively (Fig. 2C).

Preparation and Evaluation of Cream Formulations

An in vitro release test should be appropriately sensitive, capable of distinguishing formulation or process changes that might have an influence on the formulation performance. To test the discriminatory power of the developed method, six different cream formulations were prepared. Formula F1 had the same qualitative and quantitative composition as the reference listed product Zovirax cream.²⁰ The rest of the formulations were prepared with altered qualitative or quantitative compositions to study the ability of the IVRT method in differentiating the drug release rate.

Evaluation of Acyclovir Cream

Visual examination

The prepared cream formulations were examined visually and were found to be white in color, shiny, smooth on application, homogenous, and free of air bubbles.

pH

The pH of the acyclovir cream formulations was in the range of 6.1–6.2 (Supplementary Table S2).

Viscosity

The viscosity values of all the in-house formulations and Cream 1 are reported in Supplementary Table S3. There was no significant difference found in viscosities of formulation F1, F2, F3, and F5 as compared with that of Cream 1 whereas formulation F4 showed lower viscosity, which could be attributed to the changed composition of the oil phase in this formulation. Formulation F6 showed significantly higher viscosity due to the presence of polyethylene glycol 4000 in the formula in place of propylene glycol. (Supplementary Table S2) The semisolid consistency of polyethylene glycol 4000 at room temperature could have attributed to higher viscosity to this formulation.

Texture analysis

Formulations F1 and Cream 1 were subjected to texture analysis studies to compare the properties such as hardness, cohesiveness, adhesive force, and deformation. Formulation F1, having a similar qualitative and quantitative formula as Cream 1, showed similar texture characteristics as the marketed cream (Supplementary Table S2 and Supplementary Fig. S1).

Assay

The drug content of all the trial formulations was found to be in the range of 95 to 105% (Supplementary Table S2).

In vitro release testing

The release profiles of all the formulations were compared with that of Cream 1 (Fig. 3). Cream 1 is one of the well-established marketed generic acyclovir cream formulations available in India. The release rate of formulation F1 was statistically similar to that of Cream 1. Formulation F2 prepared without the propylene glycol showed a significantly lower release rate, which was also the case of formulations F5 and F6 where propylene glycol was replaced with different grades of polyethylene glycol. Formulation F6 showed the least release rate due to the absence of propylene glycol and also due to the higher viscosity of the formulation imparted by polyethylene glycol 4000. The lower viscosity of the formulation F4 compared with the rest of the formulations led to an increase in the drug release rate, which was statistically not significant(Fig. 3). A higher concentration of the propylene glycol in formula F3 (60% instead of 40%) reduced the drug release, which could be due to higher partitioning of the drug in the cream vehicle and reduced thermodynamic activity of the drug.²¹

Fig. 3.

Comparative in vitro release profiles of in-house acyclovir cream formulations and marketed formulation (Cream 1). The IVRT method using immersion cells could establish the similarity between the release profiles of formulation F1 and Cream 1 having a similar composition. The method was also able to distinguish the differences in the release profiles of formulations F2 to F6, which differed in qualitative or quantitative compositions. The parameters used for IVRT: 200 mL of alkaline borate buffer (pH 9.2) maintained at 32°C, paddle speed of 150 rpm, immersion cell size 2 cm², nitrocellulose membrane (0.45 μm pore size). Data expressed as mean ± SD (n = 6). IVRT, in vitro release testing.

IVRT of Marketed Formulations

Nallagundla et al.¹⁹ have compared the in vitro release of acyclovir from various marketed formulations in South Africa and India by using VDCs. However, use of immersion cells for such a comparison has not been reported so far. We compared the drug release from various marketed acyclovir topical formulations by using the immersion cells. The immersion cell method was able to exhibit the sameness as well as differences in the drug release from these formulations. Cream 4 exhibited a significantly lower release rate among all the tested formulations, whereas the rest of the creams showed a comparable release (Fig. 4). Though the formulations have a similar qualitative and quantitative composition, they could vary in their microstructure, which would reflect in the different drug release rates.²² The developed method could very well establish these differences. Ointment 1 was purposely chosen for the study since it is a different dosage form and hence is expected to show different drug release behavior in spite of containing the same concentration of acyclovir. A significantly higher drug release rate from the ointment compared with the cream formulations (Fig. 4) could be attributed to the higher partitioning of the relatively hydrophilic drug into the receptor medium from the ointment base. The drug would partition in oil and aqueous phases of the cream from where the diffusion rates of the drug into membrane may vary, resulting in an overall slower release rate. The work reported by Mekkaway et al.²³ shows similar findings wherein in vitro release of fluconazole was more from the soluble ointment base as compared with the cream base.

Fig. 4.

Comparative in vitro release profiles of marketed generic acyclovir topical formulations. Immersion cells could establish the differences in the marketed generic cream formulations of acyclovir. The acyclovir ointment showed significantly higher (P < 0.05) drug release compared with the cream formulations. The parameters used for IVRT: 200 mL of alkaline borate buffer (pH 9.2) maintained at 32°C, paddle speed 150 rpm, immersion cell size 2 cm², nitrocellulose membrane (0.45 μm pore size). Data expressed as mean ± SD (n = 6).

Discussion

The IVRT is a well-established technique for characterizing and evaluating the performance of semisolid dosage forms. It is a quality control tool to ascertain the batch-to-batch performance of semisolid formulations. It should be a discriminating method that is generally sensitive to physicochemical changes in semisolid drug products. It serves as a valuable tool for the demonstration of comparative in vitro drug release rates among test and reference products. As per the U.S. FDA SUPAC-SS guidance,²⁴ the IVRT can be used to establish product sameness up to Level 2 changes in all the categories. The USP recommends the use of VDCs, immersion cells, and flow-through cells as the tools to test this parameter. Out of these, the VDCs have been used most commonly for IVRT as well as for the in vitro permeability testing of the semisolids^25–29 for the past 50 years. Setting up and validating the method on flow-through cells is a complex and time-consuming exercise demanding skilled and trained operators and hence seldom used in spite of having the highest discriminatory potential.³⁰ The IVRT using immersion cells is relatively new (developed in the early 90s), simple to use, easy to validate, and a less cumbersome technique as compared with VDCs. Immersion cells, being made of Teflon, are easy to handle and have the least interactive potential. Routine USP dissolution test apparatus can be used for the test. The amount of dissolution medium that can be included for the test can vary between 50 mL and up to 4L, which makes it easy to achieve the requirements of total drug release less than 30% and maintenance of sink conditions as per the U.S. FDA SUPAC-SS guidelines.²⁴ The higher amount of sample can be withdrawn without errors, which also lends itself to simpler analytical techniques for the quantification of the drug. Preparation and loading of the cells is done outside the dissolution apparatus, chances of air entrapment between the formulation and membrane are negligible, and a larger amount of sample can be placed in the cell. Cells with a greater surface area (4 cm²) can be used in case of potent drug formulations where quantification of the drug in the dissolution medium could otherwise be challenging. The only drawback of immersion cells is that the temperature equilibrium between the formulation and receptor phase could take finite time, because Teflon is a poor conductor of heat with a small heat transfer coefficient. This requires the cells and the formulation to be stored at the study temperature before use.

Several studies comparing the IVRT of various topical and ophthalmic semisolid formulations using immersion cells and VDCs have reported lower precision, reproducibility and higher variation in IVRT using VDCs..^31–36 The main reasons for this are higher human interface during sample application and sample withdrawal, leading to higher experimental errors.³⁷ A number of studies have been reported in the literature where VDCs have been employed for testing the drug release and/or permeation from acyclovir topical formulations.^38–43 Considering the advantages of immersion cells, we decided to study the IVRT of acyclovir cream formulations by using immersion cells. We initially studied the effect of test variables such as type of membrane, stirring speed, media temperature, media volume, and cell size on the acyclovir release from its marketed cream formulation. We found that each of these variables had a significant effect on the drug release. The nitrocellulose membrane showed the highest release rate of acyclovir at 150 rpm stirring speed when 200 mL of dissolution medium was used. Cell size with 4 cm² surface area and media temperature of 37°C showed a favorable influence on acyclovir release. The impact of all these variables could differ depending on the type of the drug being studied. The IVRT of triamcinolone acetonide topical semisolid formulation using immersion cells showed the highest drug release with polyethylene membrane in comparison with cellulose membrane or rat skin.¹¹

The developed IVRT method could establish the similarity in the acyclovir release rates from the marketed Cream 1 and the formulation F1 prepared with a similar qualitative and quantitative composition as Cream 1. For the drug to be released from the semisolid vehicle and partition into the skin, it needs to be in the solution form. Propylene glycol has been incorporated in the Cream 1 formula as a cosolvent to render the drug in the solution form. The absence of propylene glycol would affect the solubility of the drug in the vehicle, eventually affecting its release. The cream formulations prepared in the lab by altering the qualitative or quantitative composition of the cosolvent when subjected to IVRT by using an optimized method showed distinct differences in release rates. Similarly, a change in the composition of the oil phase showed a remarkable alteration in the formulation viscosity. The developed method could reflect the difference in the drug release rate owing to the alteration in the viscosity. Thus, the IVRT method using immersion cells was able to establish the similarity between the similar compositions; at the same time, it could reflect the differences in release rates of the formulations with varied compositions. Krishnaiah et al.¹³ have earlier used immersion cells to study the effect of process variables such as emulsification time, homogenization speed, emulsification temperature, and pH of aqueous phase on the in vitro release on acyclovir cream; whereas our work focused more on studying the impact of variation in the qualitative and quantitative composition of the formulation on the drug release. We also compared various marketed topical formulations of acyclovir by using the immersion cells, and the method could very well indicate the differences in their drug release rates.

Conclusion

This study has reinforced that the IVRT using immersion cells is a simple, cheaper yet effective technique that can be used during formulation development and optimization stages as well as a quality control tool to assess batch-to-batch uniformity. The tool could also be used to establish product sameness in most cases of scale-up and post-approval changes not ranging beyond Level 2. As per the guidance released by the U.S. FDA in 2016 for the acyclovir cream formulations,⁴⁴ the immersion cell method as an IVRT technique after proper validation could be used in combination with appropriate in vitro permeation testing to offer the waiver to the expensive in vivo testing based on clinical endpoint determination.

Abbreviations Used

ANOVA

analysis of variance

HPLC

high-performance liquid-chromatography

IVRT

in vitro release testing

SD

standard deviation

VDC

vertical diffusion cell

Disclosure Statement

The authors declare no conflict of interest.

Funding Information

A.A.D. would like to acknowledge partial support from the Ola HAWAII Pilot Project Grant (National Institute on Minority Health and Health Disparities [NIMHD] Grant No. U54 MD007601) and Diabetes Centers of Biomedical Research Excellence (COBRE) Pilot Project Grant (National Institute of General Medical Sciences [NIGMS] grant No. P20 GM113134), Hawaii Community Foundation Grant (Grant No. 19ADVC-95449), and IDeA Networks of Biomedical Research Excellence (INBRE) IV Junior Investigator Award (NIGMS grant No. P20 GM103466). This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Supplementary Material

Supplementary Table S1

Supplementary Table S2

Supplementary Table S3

Supplementary Figure S1