In vitro evaluation of mesalazine enteric-coated tablet dissolution by the reciprocating cylinder method

Abstract

Mesalazine formulations are first-line treatments for ulcerative colitis. However, the drug release mechanisms of currently available mesalazine formulations on the market vary, and different in vitro dissolution methods have been used to characterize their in vivo absorption. Thus, there is an urgent need for more in-depth research on in vitro dissolution methods for mainstream mesalazine enteric-coated tablets. The goal of this study is to determine more scientifically rigorous testing methods to enhance the discriminatory power of in vitro dissolution testing of these products. Dissolution tests were performed using the reciprocating cylinder method with a small 250 ml vessel, a reciprocating frequency of 10 cycles/min, a sample volume of 5 ml, and UV spectrophotometric detection. The absorbance values of the dissolution solutions at different pH values were measured using cuvettes with path lengths of 1 cm and 1 mm and at detection wavelengths of 303 nm and 332 nm, respectively. In pH 1.2, 4.5, 5.5, and 6.0 solutions, the linear concentration range was from 5 to 30 µg/ml. In contrast, the linear concentration range in pH 6.8 media was 90 to 660 µg/ml. Method accuracy was tested at levels of 5%, 50%, 100%, and 120%, and the average recovery rates were 105.8%, 102.8%, 100.9%, and 101.2%, respectively. Moreover, the differences in the dissolution data did not exceed 2% with different instruments or analysts or on different days. Using the reciprocating cylinder method, we continuously measured the dissolution of mesalazine enteric-coated tablets in media with various pH values that simulate different sections of the human digestive system. Furthermore, in pH 6.8 dissolution media, drug release from both the homemade and the reference formulations followed zero-order kinetics. The established reciprocating cylinder method for determining the dissolution of mesalazine enteric-coated tablets is suitable for quality control of this type of product.

Background

Inflammatory bowel diseases are inflammatory conditions that affect the gastrointestinal (GI) tract and are responsible for over 50,000 deaths every year¹. The most common inflammatory bowel diseases are ulcerative colitis (UC) and Crohn’s disease. The clinical symptoms of UC patients include primarily weight loss, abdominal discomfort, diarrhoea, and bloody stool. Mesalazine (5-aminosalicylic acid (5-ASA)) serves as a first-line therapy for mild to moderate UC, has been identified as an active component of sulfasalazine and exhibits anti-inflammatory activity in the GI tract^2,3,4,5,6. As a first-line drug for treating colitis, mesalazine enteric-coated tablets have exhibited significant clinical value in the management of UC and Crohn’s disease. There are a variety of mesalazine preparations available on the international market⁷. The largest market share is held by SALOFALK^®, which is produced by the original manufacturer, Dr. Falk Pharma GmbH. Notably, SALOFALK^® is pH sensitive, allowing for precise release of the tablets in the colon.

Sustained-release granules, capsules, and solutions are also available and have been marketed by many manufacturers; however, the drug release mechanisms of products from different manufacturers are not the same. For example, Adeyinka Abinusawa and Srini Tenjarla⁸ studied a variety of mesalazine preparations on the market and reported that SALOFALK^® (500 mg) enteric-coated tablets are pH-dependent, whereas PENTASA^® (500 mg) is a sustained-release preparation, and some of the drug is released in the stomach. Furthermore, in vivo dissolution studies are key indicators of drug efficacy. Although consistent animal experimental data and in vitro dissolution results have been reported for enteric-coated preparations⁹, external dissolution plays a crucial role in characterizing the in vivo absorption of drugs. However, in vivo methods have drawbacks, such as high costs, and ethical concerns arise with animal and human experiments. Additionally, experiments across different species can lead to bias when extrapolating the bioavailability results to humans due to interspecies differences.

The dissolution of mesalazine extended-release (ER) and delayed-release formulations approved by the FDA is tested predominantly via the paddle method, with the basket method being employed less frequently, and the reciprocating cylinder method has not been used for dissolution quality control testing¹⁰. Mesalazine enteric-coated tablets are listed in the imported registration standard of China, and research on the dissolution of homemade mesalazine enteric-coated tablets by Li Fuying and Dong Wendi et al. was conducted by the paddle method¹¹. To determine the similarities and differences between the reciprocating cylinder results in pH 1.2 and pH 6.8 media and those obtained by the paddle method and to highlight the advantages of the reciprocating cylinder method, the in vitro dissolution results from the two methods in both media were compared.

Studying the methods to test the in vitro dissolution of mesalazine enteric-coated tablets is important. In recent years, several studies have reported the in vitro dissolution of mesalazine enteric-coated tablets, but these studies have predominantly used the paddle method. Additionally, mechanistic development and the relationship between dissolution and release kinetics after reaching the target site in vitro are rarely reported.

In this study, pharmacokinetic and in vitro dissolution data of mesalazine enteric-coated tablets (both homemade and reference tablets) by the reciprocating cylinder method were collected to provide more comprehensive results. The designed pH-dependent mechanism of drug release and zero-order kinetics after reaching the target site are discussed. The findings of this study provide a more comprehensive introduction to the use of a reciprocating cylinder to test in vitro dissolution and model studies of mesalazine enteric-coated tablet dissolution.

Methods

Materials

The dissolution test was conducted using a UV-2600i spectrophotometer (Suzhou Shimadzu Technology Co., Ltd.), and the dissolution apparatus was sourced from BIO-DIS Vkanel Technology. The mesalazine reference standard was purchased from the US Pharmacopeia, and the mesalazine enteric-coated tablets (500 mg each) were manufactured by Elite Pharmatech Co., Ltd. (Shanghai; batch numbers: 20210525-3, 21082303-3, 21082305-2, 21091801-2, 20220228-1, and 20220927-1). The reference mesalazine enteric-coated tablets (500 mg each), SALOFALK^®, were manufactured by German GmbH (batch number L20084A). Concentrated hydrochloric acid, sodium hydroxide, and monobasic potassium phosphate were purchased from Merck.

Development of the in vitro dissolution test method

to Considering both mesalazine enteric-coated tablet absorption in the human digestive tract and the general residence time of drugs in the human body¹², the gastric transit time is approximately 1 to 2 h (depending on whether the individual is fasting). Gastric emptying time is approximately 40 min, with drug transfer in the small intestine taking approximately 3 ± 1 h, and the drug remains in the colon for approximately 1 h.

The pH of the human stomach ranges from 0.9 to 1.5, while the pH in the small intestine ranges from 6.0 to 6.8 and that in the colon is approximately 6.5 to 7.5. Therefore, the drug is designed as an oral colon-targeted drug delivery system (OCDDS)¹³. Although there is a risk of early drug release in the stomach and small intestine, the enteric coating serves as a protective layer to ensure that the drug does not dissolve in gastric juice or the small intestine and is only released in the colon with first-order kinetics.

In this study, 0.1 mol/L HCl was used to simulate the dissolution of the enteric-coated tablet in gastric juice. Media with a pH ranging from 4.5 to 6.0 was used to simulate the dissolution of the product from the stomach to the small intestine, whereas a media ranging from pH 6.8 to 7.2 was used to simulate the colon environment for product release.

According to the study by Frank Karkossa and Sandra Klein on the in vitro biopredictive comparison of oral topical mesalazine using a new dissolution model, which was employed to evaluate drug release and transport time in the digestive tract of individual subjects¹⁴, the sampling times for the in vitro dissolution method were set as shown in Table 1.

Table 1 Sampling times in media with different pH values to evaluate in vitro dissolution.

Dissolution apparatus and related parameters

The parameters, sampling volume, and medium volume of the reciprocating cylinder apparatus are shown in Table 2. Dissolution testing was conducted using the paddle method, and the testing details are presented in Tables 3 and 4. The parameters for the second stage of dissolution using the paddle method are shown in Table 4.

Table 2 Dissolution apparatus and reciprocating cylinder parameters.

Table 3 Paddle method parameters – acid stage.

Table 4 Paddle method parameters – buffer stage.

Detection conditions

Mesalazine has different characteristic absorptions in various media, and the ratio of the absorbance of the blank (excipient) to that of the control solution sample at a selected specific wavelength should not exceed 1%¹⁵, as shown in Table 2. In 0.1 N hydrochloric acid, the characteristic absorption wavelength of mesalazine is 303 nm, and in media at pH 4.0 to 6.8, the characteristic absorption wavelength is 332 nm.

Liquid chromatography–ultraviolet (UV) spectrophotometric analysis¹⁶ revealed that the amounts of dissolved samples from the same batch were not significantly different. Thus, UV spectrophotometry was used to rapidly determine the dissolution of mesalazine enteric-coated tablets; the analysis parameters are shown in Table 5.

Table 5 UV analysis parameters.

The absorbance peak at 303 nm was used for the determination of sample dissolution in hydrochloric acid media, while the peak at 332 nm was used for the determination of dissolution in pH 4.5, 5.5, 6.0, and 6.8 media.

A 1 cm thick cuvette was used for determining dissolution in hydrochloric acid and pH 4.5, 5.5 and 6.0 media, and a 0.1 cm thick cuvette was used for determining dissolution in pH 6.8 media.

Experimental procedure

Solution Preparation

The 0.1 N HCl standard solution was prepared by dissolving 20 mg of mesalazine in 100 ml of medium in a measuring flask and thorough mixing. One millilitre of this solution was pipetted into a 20 ml volumetric flask and diluted to scale with medium.

The reference standard solutions in pH 4.5, 5.5 and 6.0 media were prepared in the same manner.

The pH 6.8 medium reference solution was prepared by dissolving 22 mg of test compound in 50 ml of medium in a volumetric flask with thorough mixing.

The dissolution medium was prepared in accordance with the Buffer Solutions section of the USP BUFFER SOLUTIONS guide¹⁷.

Note

The dissolution samples in pH 6.8 medium were subjected to a 10-fold dilution prior to analysis.

Method validation

According to the ICH Q2(R1) guidelines¹⁸ for analytical method validation, the specificity, linearity, precision, accuracy, and solution stability of the method for determining mesalazine enteric-coated tablet dissolution using the reciprocating cylinder method were comprehensively investigated so that the method could be used for sample analysis. Testing was conducted as follows.

A sample was collected at each predetermined sampling time and filtered through a 0.45 μm filter. The first 2 ml of filtrate was discarded and the remaining filtrate was analysed.

Method specificity

The medium, blank excipients (placebo), control solution, and sample solution were subjected to specificity testing.

Method linearity and range

On the basis of the quality by design (QbD) concept, the active ingredient in mesalazine enteric-coated tablets should not be released in 0.1 N hydrochloric acid or pH 4.5, 5.5, or 6.0 media. The upper and lower measured absorption value limits of the dissolution solutions should be fall within a suitable range for the instrument. In this experiment, the lowest dissolution concentration was set to 25% of 1% total drug release in the control solution (the specific no release standard was controlled by setting a release limit of 1%). The low and high concentrations of both the reference standard and sample were 0.005 mg/ml and 0.03 mg/ml, respectively.

By design, in vitro release in pH 6.8 medium should not exceed 20% after 15 min; therefore, the low dissolution concentration was set to 20% of the total drug release, and the concentration of the reference standard and sample was 0.44 mg/ml. Thus, 20% of the low dissolution concentration was used as the lower limit of linearity (0.09 mg/ml), and 1.5 times the high dissolution concentration of 0.44 mg/ml (equal to 0.66 mg/ml) was considered the upper limit of linearity.

Linear regression of the absorption values was performed at these concentrations. The intercept, correlation coefficient, and sum of squares of the variance in the linear equation were subsequently calculated.

Method precision and accuracy

The intrabatch repeatability of the method to evaluate the same batch of products was determined by the reciprocating cylinder method and applied to investigate the intrabatch precision of the homemade batch.

The samples were prepared with three concentrations of mesalazine, and three samples were prepared at each concentration.

Solution stability

Sample dissolution stability was determined in pH 6.8 medium only because the sample did not dissolve in 0.1 N hydrochloric acid or phthalate buffer at pH 4.5, 5.5 or 6.0.

Solubility in different media

Five portions of mesalazine were weighed and added to 0.1 N hydrochloric acid, pH 4.5 or 5.5 phthalate buffer, or pH 6.0 or 6.8 phosphate buffer. The samples were subsequently placed in a constant temperature water bath (37 ± 0.5 °C) on a thermostatic shaker with constant shaking. Samples were taken at 2 h, 4 h, 6 h, 8 h, and 24 h and centrifuged, and the supernatant was collected for testing. Mesalazine was added until the measured concentration no longer increased (NMT0.2%/h). The external standard method was used to calculate the saturated solubility of mesalazine in each type of media.

Experimental results

In vitro dissolution

Mesalazine is classified in BCS category 4, indicating its low solubility and permeability. The mesalazine raw material is poorly soluble in 0.1 N hydrochloric acid, and its solubility in the pH range of 6.8–7.2 is significantly higher than that in the pH range of 4.0–6.0. Therefore, this drug was designed for colonic release, as the colonic environment is more compatible with its solubility. Mesalazine enteric-coated tablets were designed for local drug release at colon lesion sites; therefore, it is insoluble in the stomach (does not dissolve after meals) and is not released at the proximal or distal ends of the small intestine. Notably, the drug should be released at a constant rate in the colon to ensure its effectiveness and full release at the target site. Consequently, the product is primarily coated with an enteric coating that is typically formulated with acrylic resins such as S-100 or L-100. The thickness of the coating affects the dissolution rate of the product, thereby determining whether it can reach the colon.

The paddle test method is recommended by the United States Pharmacopoeia to determine dissolution, and alternative paddle methods exist. Owing to the design of the product, the reciprocating cylinder method has certain advantages for dissolution testing, including the easy transfer of samples from one medium to another and the ability to set different time intervals in different test tubes. Therefore, USP ˂711˃ Apparatus 3¹⁹ (hereinafter abbreviated as USP App3) was selected to explore the in vitro dissolution of mesalazine and simulate the diverse environments of different human digestive tract regions, as shown in Fig. 1 below.

Fig. 1

In vitro dissolution testing of mesalazine enteric-coated tablets using the reciprocating cylinder apparatus.

Determination of the detection wavelength

The mesalazine solutions in different media were analysed from 200 ~ 400 nm to obtain the UV spectral absorption data, as shown in Fig. 2.

Fig. 2

UV scan spectra of mesalazine solutions in different media.

The characteristic absorption wavelength of mesalazine in 0.1 N hydrochloric acid is 303 nm, whereas that in pH 4.5, 5.5, 6.0, and 6.8 media is 332 nm. Additionally, the absorbance values of blank excipient solutions in different media (prepared by accurately weighing the placebo excipients for dissolution in 100 ml of media) were measured at these characteristic wavelengths, and the results are presented in Table 6.

Table 6 Ratios of the absorbance of the excipients to the absorbance of the control solution at the characteristic absorption wavelength.

The absorbance of the placebo solution remained less than 1% of the absorbance of the test solution under all conditions tested.

Investigation of reciprocating frequency

The volume of each type of medium in the reciprocating cylinder was 250 ml to simulate human gastric and intestinal juices. The number of reciprocations simulates peristalsis of the human GI tract and is generally 10–15 times per minute. The effect of the number of reciprocations (10 and 15) on dissolution was investigated with three batches of the homemade formulation and the reference preparation, and the results are shown in Table 7.

Table 7 Dissolution of the homemade formulation with different number of reciprocations (reciprocating frequencies).

Table 7 shows that the use of 10 or 15 reciprocations did not significantly affect the dissolution results, and the similarity factors (f2 values)²⁰ of the dissolution curves constructed at different speeds were greater than 80, indicating that the number of reciprocations had a small effect on the results. The number of reciprocations is selected for further experiments was 10 times/min.

Note

The similarity factor (f2)​ for the above comparative data was calculated from the dissolution results at 5 sampling time points: 2 h in pH 1.2 medium, 1 h in pH 6.0 medium, 15 min in pH 6.8 medium, 30 min in pH 6.8 medium, and 45 min in pH 6.8 medium.

The similarity factor (f2)​ values in the following sections were calculated in the same manner.

Determination of cuvette thickness

to The control solution was prepared according to the sample specifications, and the absorbance values of both the sample and the control solution were monitored to minimize the need for secondary dilution during intermediate operations, as the absorbance of the final solution should remain within the range of 0.3 to 1.2. The amount of reference substance weighed meets the minimum weight requirements, and appropriate cuvettes of suitable thickness were selected. Additionally, the comparability of test results across different media was confirmed to reduce systematic errors caused by the cuvettes. Finally, drug dissolution in 0.1 N hydrochloric acid and pH 4.5, 5.5, and 6.0 media was determined by measuring the absorbance of each solution using 1 cm cuvettes, whereas dissolution in pH 6.8 media was assessed using a 0.1 cm cuvette.

Method validation

Specificity

The absorbances of the blank medium, blank excipients, and reference solution in each type of media were determined. Additionally, the ratios of the absorbances of the blank medium and blank excipients to that of the reference solution were calculated, as presented in Table 8, according to the following formula:

Formula¹⁵: Interference value = (Ap/As) × Cs × (V/L) × 100.

where:

• Ap is the absorbance of the blank excipients;

• As is the absorbance of the reference solution;

• Cs is the absorbance of the corresponding reference solution;

• V is the media volume (ml); and.

• L is the label claim (mg).

Table 8 shows that the interference effect of each blank medium on the measurement results was not greater than 1%, indicating that the blank medium did not interfere.

Table 8 Blank medium interference.

Table 9 Placebo solution interference.

As shown in Table 9, the placebo interference is less than 1%; thus, the blank excipients do not interfere with the measurement results.

Linearity and range

In accordance with Sect. 2.2.3.4, the linearity of the control samples was evaluated at concentrations ranging from 25 to 150% (and at concentrations ranging from 20 to 150% in pH 6.8 medium) by plotting the control sample concentration on the x-axis and the absorption value on the y-axis. Linear regression was performed to determine the linear equations, correlation coefficients, intercepts, and sum of squares of variance for the standard solutions in different media. The results are presented in Table 10.

Table 10 Results of the examination of the linear relationships between concentrations in different media and absorption Values.

In pH 1.2, 4.5, 5.5, and 6.0 media, the linear concentration range is 5 to 30 µg/ml. In contrast, the linear range in pH 6.8 media was 90 to 660 µg/ml. The linear relationship between concentration and absorbance is strong within these ranges, with correlation coefficient (r) values exceeding 0.999.

Precision and accuracy

The precision of the method was validated using batch 20220228-1 (n = 6) following the instrumental methods outlined in Tables 1 and 2. The within-batch plate-to-plate precision was assessed by calculating the absolute differences to determine if the method’s precision met the specified requirements for different instruments and analysts on different days with a difference no greater than 2%. These results are presented in Tables 11 and 12.

Table 11 Method precision (dissolution instrument A).

Table 12 Method precision (dissolution instrument B).

The accuracy was assessed by determining the recovery rate, which confirmed that the method meets the established requirements. These results are shown in Table 13.

Table 13 Method accuracy in pH 6.8 buffer via the reciprocating cylinder method.

The formulation was dissolved in pH 6.8 media, and the average recovery rates of nine samples at four concentrations (5%, 50%, 100% and 120%) were 105.8%, 102.8%, 100.9%, and 101.2%, respectively, all of which fell within the acceptable range of 90–110%. The relative standard deviation (RSD) of the recovery rates for the nine samples was 1.4%, which is less than the acceptability criterion of no more than 2% (excluding the 5% concentration), which meets the predetermined requirements.

Stability of the dissolution solution

The samples of the pH 6.8 dissolution solution taken at 30 and 45 min were stored for 2, 4, 6, 8, 12, and 24 h, and the absolute difference between each initial value and the value after storage was not greater than the acceptance limit of 2%. These results are shown in Table 14.

Table 14 Stability in the pH 6.8 medium dissolution solution.

As shown in Table 14, the dissolution solutions sampled at 30 min and 45 min exhibited minimal changes after being stored for 24 h, demonstrating good stability.

Results

The in vitro dissolution method established for determining the dissolution of mesalazine enteric-coated tablets using the reciprocating cylinder method was validated, and it specificity, linearity, precision, accuracy, and solution stability met the established standards, confirming the method’s applicability.

Solubility studies

The “USP1092 Guidance on Dissolution Method Development and Validation” recommends that solubility testing can be conducted at the same temperature as dissolution testing (37 °C), as this serves as strong evidence for evaluating the concentration conditions under Sink condition. Therefore, based on the equilibrium solubility testing method outlined in USP < 1236>²¹, Mesalazine solubility in various media was evaluated at 37 °C, and the results are presented in Table 15 and plotted in Fig. 3.

Table 15 Solubility of mesalazine in different media.

Fig. 3

Solubility of mesalazine in different media.

The plot in Fig. 3 shows that the mesalazine raw material is highly soluble in 0.1 N hydrochloric acid and more soluble in pH 6.8 medium than in the other buffer solutions. Therefore, among the methods in the USP for mesalazine ER capsules, the paddle method for determining dissolution in 0.1 N hydrochloric acid and pH 6.8 medium is preferred.

Mechanism of product release

Enteric-coated tablets are dissolved and absorbed in the GI tract is as follows. First, upon oral administration, the enteric-coated tablet enters the stomach, where the coating does not dissolve, and drug release does not occur. The tablet then progresses to the small intestine, where the enteric coating remains intact, preventing the release of the active ingredient. After passing through the caecum, it enters the large intestine. Shortly after entering the large intestine, the enteric coating gradually dissolves, leading to minimal release of mesalazine that is controlled at less than 5%. Since the colon is located in the middle and lower parts of the large intestine, concentrated release occurs in the colonic region. This entire process is illustrated in Fig. 4.

Fig. 4

Schematic of in vitro dissolution simulating the in vivo release of mesalazine enteric-coated tablets.

Discussion

Consistent dissolution of the homemade batch and reference Preparation in vitro

The reciprocating cylinder method was used to test the in vitro dissolution of both the reference preparation (SALOFALK^®, Germany) and the homemade preparation (500 mg) (n = 12) to determine if their dissolution profiles were consistent on the basis of the f2 value. The dissolution test results are summarized in Table 16.

Table 16 Dissolution of the generic mesalazine enteric-coated formulation and reference drug determined by USP App3.

Fig. 5

Dissolution of generic mesalazine enteric-coated formulation and the reference drug in media simulating gastrointestinal pH ranges determined by USP App3.

As shown in Fig. 5, after simulating the pH gradient and retention times in various parts of the GI tract, neither the homemade nor reference preparation released the active ingredient in the stomach at pH 1.2, in the duodenum and proximal small intestine at pH 4.5, or in the distal small intestine at pH 5.5 to 6.0. The drug coating begins to break down and mesalazine is released in the colon following zero-order kinetics to ensure that the drug concentration reaches a therapeutic level.

Using the reciprocating cylinder method, the in vitro dissolution of the homemade preparation was completely consistent with that of the reference preparation, with a similarity factor (f2) of greater than 80, which is within the acceptable range of 50–100. These findings indicate that the in vitro dissolution profiles of three batches of homemade samples are similar to those of the reference preparation.

The similarity factor (f2) is commonly used in the pharmaceutical industry to compare the dissolution profiles of test and reference formulations, with values greater than 50 generally indicating similarity^22,23. The reference drug batch was lot L20084A.

Correlation between in vitro dissolution via the reciprocating cylinder method and in vivo drug release

The in vitro–in vivo correlation (IVIVC) is as a critical quality control measure²⁴. Dissolution methods established on the basis of the IVIVC model can accurately predict in vivo drug bioavailability and serve as surrogates for bioequivalence. As shown in Fig. 5, homemade batch samples 21082305-2, 21091801-2, and 20220927-1, along with the reference preparation L20084A, exhibited complete drug release (greater than 85%) in vitro within 5.75 h. The mesalazine enteric-coated tablets (SALFORK^® 500 mg) reference information²⁵ states that the drug is absorbed primarily in the proximal part of the intestine with little absorption in the distal region, indicating that it is released in pH 6.8 media, which is similar to the pH of intestinal fluid. The reference information specified that the drug should be released 3–4 h after administration.

Additionally, the reference information mentions a pharmacokinetic study that used combined scintigraphy scans in patients, showing that the product reaches the ileocecal region 3–4 h after fasting and the ascending colon after approximately 4–5 h. The transit time throughout the entire colon is approximately 17 h. These results are consistent with the findings of C. Bott and M. W. Rudolph²⁶ in their study titled “In vivo evaluation of a novel multi-unit colonic drug delivery system based on pH and time.”

Despite the few studies with USP III (reciprocating cylinder) and IV (flow-through cell) apparatuses, these systems have been highly recommended for IVIVC approaches, mainly in the development of ER products. For modified-release (MR) products²⁷, such as enteric-coated mesalazine, in vitro and in vivo corrective studies have also been conducted.

Because the reciprocating cylinder method can better simulate the pH gradient of the human GI tract and control sampling from different media at different times, this method is more suitable for pH-dependent designs or combined pH-dependent and time-controlled formulations for the treatment of colitis and Crohn’s disease. Sandra Klein, Markus W. Rudolph, and Brigitte Skalsky²⁸, among others, studied the mechanism of caffeine absorption throughout the GI tract by employing the reciprocating cylinder method and a physiologically based pH gradient. The in vitro drug release was subsequently compared with that of the drug in vivo, revealing a certain IVIVC.

Cord J. Andreas, Ying-Chen Chen, Constantinos Markopoulos, et al. studied drug release from MR mesalazine products to predict their effects. They reported that USP apparatus III (reciprocating cylinder method) generally tended to exhibit in faster dissolution rates and forecasted more pronounced food effects for Salofalk^® 250 mg than did USP apparatus IV (flow-through cell). The biorelevant dissolution gradients were also able to reflect the in vivo behaviour of the formulations²⁹.

Su-hua Zhang, Yao Li and Shan-shan Wei studied the effects of different food patterns on the pharmacokinetics of mesalazine enteric-coated tablets in the same cohort of healthy Chinese volunteers³⁰. Under fasting conditions, the peak plasma concentration was reached within 7 h after administration, which is essentially consistent with the approximately 6.4 h to reach the peak time determined by the reciprocating cylinder method in the in vitro dissolution test. The curve of the blood concentration over time is shown in Fig. 6. The in vitro dissolution of mesalazine enteric-coated tablets determined by the reciprocating cylinder method may be correlated with the in vivo release of the drug.

Fig. 6

Su-Hua Zhang, Yao Li and Shan-Shan Wei studied the mean N-AC-5-ASA plasma concentrations over time for enteric-coated mesalazine in high-fat (purple line) and fasted states (black line). (This figure is sourced from the research article by Su Hua zhang, Yao Li, Shan-Shan Wei, et al., which explores the impact of dietary regimen and fasting on the bioavailability of mesalazine enteric-coated tablets.).

Method comparison

The paddle method was used to test the dissolution of the homemade and reference preparations in 0.1 N HCl and pH 6.8 media (n = 6), and the results are given in Table 17 and plotted in Fig. 7.

Table 17 Dissolution of the homemade and reference preparations determined by the USP II paddle method. The test was conducted for two h in pH 6.8 media.

Fig. 7

Dissolution curves of the homemade and reference preparations determined using the paddle method.

The dissolution of the homemade and reference preparations was examined via both the paddle and the reciprocating cylinder methods for 90 min in pH 6.8 media. These results were plotted on a single graph for comparison (Fig. 8).

Fig. 8

Comparison of the dissolution curves.

In pH 6.8 media, less than 1% dissolution occurred via both the reciprocating cylinder and the paddle method from 0 to 15 min, with complete release achieved by 90 min. Notably, the reciprocating cylinder method results in a slightly faster dissolution rate than the paddle method does between 20 and 80 min. This difference may be attributed to the simulation of the human digestive tract with the reciprocating cylinder method, where the enteric coating of the tablets may thin upon exposure to the duodenum and jejunum segments; in contrast, the paddle method lacks these intermediate pH segments (pH 4.5, 5.5, and 6.0). Additionally, the test applying the paddle method, a small number of homemade and reference mesalazine enteric-coated tablets were found at the bottom of the cup, a phenomenon not observed with the reciprocating cylinder method. This accumulation likely contributes to the slower dissolution observed with the paddle method. Frank Karkossa and Sandra Klein noted that dissolution was slightly faster with the reciprocating cylinder method than with the paddle method in their study. Notably, L20084A is the batch number of the reference preparation.

Method discrimination

Coating weight gain is a key process parameter (CPP)³¹ for enteric-coated drug formulations and directly influences the key quality attributes (CQA) of the product. Investigating coating weight gain under various process conditions is crucial to ensure that the active ingredient is released at the target site in accordance with the design. The dissolution method must accurately and sensitively reflect changes in the process on the basis of adjustments to key parameters. The dissolution of the tablets with varying coating weights determined by the reciprocating cylinder method are shown in Table 18.

Table 18 Dissolution of tablets with different film thicknesses.

The dissolution curves constructed using the data from the reciprocating cylinder method are shown in Fig. 9.

Fig. 9

In vitro dissolution of generic enteric-coated tablets with different coating thicknesses.

There is a strong correlation between film thickness and dissolution, which effectively reflect the adjustments in the process parameters. As the film thickness increases, the dissolution rate decreases. The dissolution curves constructed from the reciprocating cylinder method data demonstrate suitable discrimination.

Drug release kinetics

Deriving a mathematical model to predict drug release is challenging. A strong mathematical model can reduce the number of required experiments owing to its predictive ability, and these estimates save time and money by helping to propose more efficient experiments³². However, does in vitro release align with the zero-order kinetic model, Higuchi release kinetics, the Hixson–Crowell model or the Korsmeyer–Peppas model? The drug release kinetics of three consecutive batches of both homemade and reference preparations were investigated, and their fits to several commonly used drug release models, including the Higuchi model, zero-order kinetics, the Hixson–Crowell model and Korsmeyer–Peppas model, were compared^33,34 to identify the kinetic model of drug release from this formulation. The dissolution data of the tested preparations are presented in Table 19, and the simulated curves are shown in Figs. 10, 11, 12 and 13.

Table 19 Dissolution of three batches of mesalazine enteric-coated and reference formulations at pH 6.8.

Table 20 Fits of the dissolution data to different kinetic models, including the equation and r value.

Table 21 Fits of the dissolution data to different kinetic models, including the equation and r value.

Tables 19, 20 and 21 show that the correlation coefficient (r) for the zero-order kinetics model is greater than 0.95, and the Hixson‒Crowell model gives the smallest mean squared error (MSE). However, in terms of the correlation of dissolution over time, the Hixson‒Crowell model is slightly inferior to the zero-order release model. When considering both the correlation coefficient and the MSE³⁵, the homemade enteric-coated mesalamine tablets and the reference preparation match the zero-order dissolution model. The zero-order kinetic model more accurately characterized drug release in pH 6.8 media.

The Higuchi plot, which shows the square root of the dissolution percentage versus time for the three batches of homemade and reference preparations, is presented in Fig. 10.

Linear regression of the zero-order kinetic equation of the percent drug release over time in pH 6.8 media is presented in Fig. 11.

Linear regression of the cube root of the dissolution percentage versus time for the mesalazine enteric-coated tablet samples is shown in Fig. 12, and linear regression of the natural logarithm of the dissolution percentage versus the natural logarithm of the sampling time is presented in Fig. 13.

Fig. 10

Percent drug release versus the square root of time (Higuchi model).

Fig. 11

Linear regression of the percent drug release over time in pH 6.8 media (zero-order kinetics).

Fig. 12

Linear regression of the cube root of the dissolution percentage versus time (Hixson‒Crowell model).

Fig. 13

Linear regression of the natural logarithm of the dissolution percentage versus the natural logarithm of the sampling time (Korsmeyer‒Peppas model).

As shown in Fig. 11, both the homemade preparation and reference preparation in pH 6.8 media fit the zero-order kinetics model³⁶.

Notes: Curve B represents sample 21082305-2.

Curve C1 represents sample 21082303-3.

Curve D1 represents sample 20220927-1.

Curve E1 represents the reference preparation L20084A.

Conclusions

The reciprocating cylinder method was used to study the in vitro dissolution of mesalazine enteric-coated tablets. Compared with the commonly used basket and paddle methods, the reciprocating cylinder method can more conveniently and effectively simulate drug release from the samples in media with different pH values that mimic various parts of the GI tract. An advantage of the reciprocating cylinder method is its ability to measure the dissolution of drugs in different media simultaneously, as manual medium replacement and sample transfer operations are not needed. The in vitro dissolution kinetics of mesalazine enteric-coated tablets were also studied and fit the zero-order kinetics model. These models offer new research ideas, and this study provides a new application scenario to study the in vitro release of drugs absorbed in the intestine considering the site of absorption and target.