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The biorelevant simulation of gastric emptying and its impact on model drug dissolution and absorption kinetics
Journal Pre-proofs Research paper The biorelevant simulation of gastric emptying and its impact on model drug dissolution and absorption kinetics Helena Vrbanac, Jurij Trontelj, Sandra Berglez, Boštjan Petek, Jerneja Opara, Rebeka Jereb, Dejan Krajcar, Igor Legen PII: DOI: Reference:
S0939-6411(20)30035-7 https://doi.org/10.1016/j.ejpb.2020.02.002 EJPB 13228
To appear in:
European Journal of Pharmaceutics and Biopharmaceutics
Received Date: Revised Date: Accepted Date:
16 July 2019 7 January 2020 4 February 2020
Please cite this article as: H. Vrbanac, J. Trontelj, S. Berglez, B. Petek, J. Opara, R. Jereb, D. Krajcar, I. Legen, The biorelevant simulation of gastric emptying and its impact on model drug dissolution and absorption kinetics, European Journal of Pharmaceutics and Biopharmaceutics (2020), doi: https://doi.org/10.1016/j.ejpb. 2020.02.002
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Title:
The biorelevant simulation of gastric emptying and its impact on model drug dissolution and absorption kinetics
Authors:
Helena Vrbanac1, Jurij Trontelj1*, Sandra Berglez2, Boštjan Petek2, Jerneja Opara2, Rebeka Jereb2, Dejan Krajcar2, Igor Legen2
* Corresponding Author: Assist. Prof. Jurij Trontelj, Ph D, Mag. Pharm. e-mail: [email protected] tel. 00386 1 476 95 00 Aškerčeva 7, 1000 Ljubljana, Slovenia
Author affiliations:
1 The
Chair for Biopharmaceutics and Pharmacokinetics, University of Ljubljana, Faculty of Pharmacy, Aškerčeva 7, 1000 Ljubljana, Slovenia
2 Lek
Pharmaceuticals, Inc., a Sandoz company, Verovškova ulica 57, 1000 Ljubljana, Slovenia
The biorelevant simulation of gastric emptying and its impact on model drug dissolution and absorption kinetics
Abstract
The highly variable physiological conditions within the gastrointestinal tract can cause variable drug release and absorption from the orally administrated dosage forms. The emptying of the gastric content is one of the most critical physiological processes, dictating the amount of the active ingredient available for absorption into the systemic circulation. In this study, we prepared two water gastric emptying regimes on advanced gastric simulator 1
(AGS) with programmable “pyloric” valve. Gastric emptying regimes were designed in such a way to capture the main findings of the MRI (magnetic resonance imaging) in vivo studies, conducted under fasted conditions according to the EMA and FDA guidelines for bioavailability and bioequivalence studies. Four immediate release formulations containing a model drug of BCS class III were tested. Comparative dissolution tests were also performed with the USP2 apparatus. In vitro release profiles were compared to the in vivo data in order to evaluate the importance of gastric emptying for subsequent absorption of the active substance from the tested formulations. Our bio-relevant in vitro dissolution model showed good discriminatory power for all of the tested formulations. Moreover, a better relation to in vivo data was achieved with AGS with respect to the tested conventional dissolution method. Key words: stomach; gastric emptying, biorelevant dissolution testing, immediate release; Magenstrasse Introduction One of the challenges in the field of drug development is to find a correlation between in vitro drug release of various formulations and in vivo occurring plasma levels. Biorelevant dissolution testing has been shown as a very useful tool for in vivo product performance prediction. When a suitable in vitro-in vivo correlation (IVIVC) is demonstrated, the number of bioavailability or bioequivalence studies needed is reduced and the process of a new formulation development leads to a product with improved safety and quality [1–3]. Oral drug administration remains the preferred administration route with the highest patient compliance among drug delivery systems [1]. The passage of the ingested dosage form through the stomach and exposure to stomach conditions is necessary in order for the drug to be later absorbed in the small intestine and/or colon [4]. The active pharmaceutical ingredient (API) from the conventional solid dosage form must first dissolve before it can be absorbed to systemic blood circulation. The majority of drugs are absorbed in the small intestine. The rate and extent to which the drug is dissolved and transferred to the small intestine is thus important for the resulting mean plasma concentration. The rate and extent of the API absorption are therefore highly dependent on the volume and distribution of fluid in the stomach and its passage to the small intestine [5]. For rapidly dissolving and highly permeable drugs (BCS class I compounds), gastric emptying represents a rate limiting step for drug absorption in the small intestine [5–7]. In the 2
case of drugs exhibiting high solubility and low membrane permeability (BCS class III compounds), the membrane transport is often limited to a specific region in the small intestine (absorption window). One of the causes for uneven permeability in the GIT (gastrointestinal tract) is the heterogeneous distribution of efflux and influx transporters along the GIT. Gastric residence time and dissolution rate in the stomach have been also described as important factors affecting in vivo pharmacokinetics of such substances [8, 9]. Retention time in the stomach varies depending on the volume and composition of food/fluid intake. Non-caloric liquids, such as water are emptied relatively quickly from the stomach, with gastro-duodenal pressure gradient being the driving force [10–14]. However, solid food components must first be processed into smaller particles with a diameter of 1-2 mm, small enough to pass through the pyloric sphincter. Greater, undigested particles are eliminated in phase III of the migrating motor complex (MMC) of the fasted stomach ("housekeeping wave") [10, 11]. In the postprandial stomach, fast emptying of ingested liquids, known as “the Magenstrasse” (the stomach road) has been observed. Liquids, administered together with food finding a shortcut through the fed stomach can have an important impact on the therapeutic onset of coadministered drugs [14]. On the other hand, high inter- and intraindividual variability of fasted human stomach emptying has been demonstrated as well [15]. The motor activity of the fasted stomach is carried out in cycles (MMC), characterized by a fluctuation of the muscle contraction intensity [16]. Different intensity of motility patterns can have an influence on disintegration time and distribution of drug and formulation particles within the various stomach regions. Random dosing with respect to different phases of the MMC is thus an important factor that can lead to increased variability in systemic drug exposure [17]. According to FDA guidelines [18] for bioavailability and bioequivalence studies (under fasting as well as under fed conditions), it is recommended for the tested formulation is to be administrated together with 240 mL of water at room temperature, on the other hand, EMA suggests administration with a constant volume of at least 150 mL [19]. The distribution of such a volume of water in the stomach immediately after ingestion, the rate of its discharge and occurrence in the small intestine is therefore the main research topic of several studies. Gastric emptying has been monitored with magnetic resonance imaging (MRI), scintigraphy, non-absorbable marker technique, ultrasonography, breath test, intraluminal sampling, with wireless motility capsule (WMC) and other techniques, as presented in the extensive review 3
article by Hens et al. [20]. Among all, scintigraphy is considered as the golden standard in gastric emptying monitoring, however measurements can be highly variable due to the influence of many factors. In 2007, guidelines have been applied to standardize experiments (standardized meals, subject posture, frequency and duration of imaging, etc.) [21]. MRI is a multiplanar technique used to image organ fluids and volumes. In addition to studying the gastric emptying, this technique is also very useful for monitoring the fluid distribution in small intestine, the movement of marked dosage form along the GIT or the bowel movement [22]. Ultrasound is a non-invasive method for gastric emptying monitoring, without radiation exposure. A drawback of this method is the need of expertise knowledge in interpreting sonograms [23]. The passage of individual parts of the GIT can also be monitored with a wireless motility capsule (WMC), which contains the sensors for detecting the pH, temperature and pressure exerted on the capsule wall. The WMC can thus be very useful for studying the motor functions and retention times of individual GIT compartments under fed or fasted conditions. However, using only the WMC the exact profile of liquid gastric emptying cannot be determined [24–26]. Based on the in vivo measurements, several in silico and in vitro models simulating gastric emptying have been proposed; Talattof et al. [27] have developed physiologically based pulsed-packet mathematical model resembling the conditions of a normal subject in a typical fasted BE study. Brümen et al. have performed mathematical modelling of individual gastric emptying profiles of pellets in the fed state [28]. Kurentas et al. [6, 29] have developed in vitro biorelevant gastrointestinal transfer system (BioGIT) to reproduce the concentrations of active ingredients in the fasted upper small intestine. Tsume et al. [30] developed the Gastrointestinal Simulator (GIS) mimicking the physiological conditions of human GIT in fasted state for the dissolution prediction of orally administrated drugs with pH dependent solubility. In this study we explore the possibility of simulating the fasted gastric emptying on the newly developed advanced gastric simulator (AGS). AGS is an innovative device, which has proved to be a successful model in achieving biorelevant conditions [31, 32]. The aim of our research was to test the applicability of the proposed biorelevant gastric dissolution methodology in establishing a better IVIVC/IVIVR (in vitro – in vivo correlation/ in vitro-in vivo relationship) compared to conventional methods for formulations with release and absorption susceptible to the process of gastric emptying.
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Materials Tablets For the purpose of this study, immediate release tablets with a high drug load (more than 200 mg) were prepared with fluid-bed granulation process, followed by tablet compression. The model drug is a weak base, with a dissociation constant of approximately 4.5. The compound is an aromatic, heteromonocyclic compound, highly soluble (4 mg/mL) over a pH range of 1.0─8.0. Substance shows high permeability, however, due to transporters dominated absorption it is classified as BCS class III compound, with bioavailability in adults between 80 and 85%. It is a nucleoside reverse transcriptase inhibitor, so it is used as an antiretroviral drug. The weight of tablets was approximately 600-800 mg Formulation D was acquired from the market and was used as a reference in the present study to which the in vitro and in vivo performance of the formulations A, B and C were compared. The formulations A and B differ in technological process of preparation, while the formulation C differs from B in extragranularly added excipient. These formulations also differ in disintegration time; the formulation A has the shortest disintegration time of [2min 42s–2min 48s; n=6] (minimal to maximal disintegration time interval), followed by formulation B [3min 54s–4min 38s; n=6] and C [4min 20s–4min 44s; n=6]. Methods Advanced gastric simulator (AGS) – apparatus description
AGS is an innovative dissolution apparatus simulating motility of the human stomach. The apparatus’ main features are a flexible silicone vessel, eight constriction mechanism, “pyloric” valve, scale, thermal chamber enabling 37 ± 0.5°C and a custom made software. The shape and the size of the container resemble closely those of the human stomach. The thin walls of the vessel are collapsed when the container is empty, but when the vessel is filled with dissolution medium the flexibility of the walls allows it to stretch. Both ends of the vessel are opened; the upper end of the vessel is wider (up to 10 cm in width), resembling the cardia, while the lower end of the vessel narrows to a tubular opening (up to 1 cm narrow), resembling the pyloric sphincter. The vessel allows a volume up to 1 L to be filled into the container. The silicone rubber (DragonSkin FX, Smooth-On Inc., United States) used for formation of the vessel is soft and exhibits the required chemical inertness. The inner lining on the vessel surface is structured with folds spreading in longitudinal direction to enable
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repeatability of the vessel deformation. The change in shape and volume of the vessel is caused by closing and opening of the constriction mechanism around the vessel (Fig. 1). [Insert Figure 1] Figure 1: Left: Advanced gastric simulator (AGS) with main elements: (1) “pyloric” valve, (2) constriction mechanisms, (3) step motors and (4) flexible vessel. Red arrows mark direction of the contraction movement. Right: Schematic presentation of the apparatus set-up.
The structure of each constriction mechanism is derived from optical iris diaphragms (57-586, Edmund optics INC., United States), driven by electric step motor (ST2818, Nanotec) and a worm gear. Synchronised opening and closing of each constriction mechanisms can be individually programmed through a custom made software, allowing a combination of different motion parameters in a programme sequence, mimicking realistic gastric motion regimes. The change in the aperture diameter (75−25 mm) starts with closing of the first constriction mechanism, set closest to the upper part of the stomach and follows with closing of subsequent constriction mechanisms toward the pyloric part of the stomach, with a pre-set delay between each constriction mechanism (Fig. 1). This simulates the contraction waves of the stomach wall. Consequently, the fluid dynamics in the vessel is changed and the pressure from the containers’ wall is transferred to its interior content. The majority of constriction mechanisms is positioned in the antropyloric part of the stomach, where the strong mixing and grinding of ingested food take place [10]. Another important element of the apparatus, enabling the simulation of gastric emptying, is an artificial “pyloric” valve. It consists of two steel pins (PFCLEA-D8-L55-M4), one positioned stationary relative to the stomach container and the other – movable – attached to a liner slide (SE2BZ10-115, Misumi, United States). The pins are tightening the tubular outlet of the flexible vessel and are acting as a pyloric sphincter. The valve aperture is adjusted with an individual electric step motor and custom made software, enabling independent regulation from the constriction mechanisms’ activity. Desired flow rate (mg/min) of the media through the tubular outlet is set in the software and is controlled with the weight of collected sample on the balance (XS6002S, Mettler-Toledo International Inc., United States) outside the thermal chamber (SP-55 EASY, Kambič d.o.o., Slovenia). The information of the current weight of collected sample on the balance is a trigger for software to automatically adjust the flow rate with opening/closing of the pins and thus reaching the desired flow rate (Fig. 2). More detailed structure and function of individual parts of the apparatus can be found in a recently published article [33]. 6
[Insert Figure 2] Figure 2: Real time print screen of the pylorus software displaying the course of desired and actual mass flow through the “pyloric” valve of the advanced gastric simulator.
Dissolution testing with USP2 apparatus Dissolution tests were performed with USP2 apparatus (AT dissolution apparatus, Xtend™, Sotax, Switzerland) at rotation speed of 75 rpm and 900 mL of 0.01M HCl with ions. Dissolution media was prepared by adding 10 mL of 1M HCl Titrisol® concentrate (ampoule, which contains a precisely defined quantity of substance) to 900 mL of deionised water, dissolving 4 g of NaCl, 1.20 g of KCl and 0.084 g of CaCl2x2H2O and then filling the volume of the flask to 1000 mL. The media temperature was set to 37 ± 0.5°C. After 5, 10, 15, 20, 30, 45 and 60 minutes samples of 1.7 mL were automatically collected with an automatic sampler (CP Xtend™ pump, Sotax, Switzerland) and filtered through the Full Flow 1 m filters (P/N FIL001-EW) to 2.0 mL vials. Analysis of dissolution samples The collected samples were analysed with a HPLC system with UV detection (Waters, USA). The chromatographic column Xbridge C18 (Waters, USA) with dimensions of 70x4.6 mm was thermostated at 60°C. Mobile phase was a mixture of ammonium acetate buffer pH 4.2 and methanol (ultra HPLC gradient grade, J. T. Baker, USA) in 80:20 volume ratio. Vials with sample solution were maintained at 25°C. Total run time of the analysis was 2.2 minutes. The simulation of gastric emptying The experiments of gastric emptying on advanced gastric simulator (AGS) can be performed in two ways: with a constant flow rate throughout the entire experiment duration or with changing the flow of container’s content and thus creating different gastric emptying regimes. The aim of this study was to prepare the programs of different gastric emptying rates on AGS, capturing the basic principles of liquid gastric emptying in the fasted state, as observed in vivo. The regimes of gastric emptying were prepared based on the MRI studies performed on healthy volunteers (Mudie et. al and Grimm et al.). Gastric flow rate in fasted state depends on the phase of the MMC; therefore testing different flow rates in the AGS is very relevant. The research group of Mudie et al. has observed gastric emptying in the study of 12 healthy 7
volunteers and had presented it as the average gastric emptying profile. However, Grimm et al. had observed high intraindividual day-to day variations in residual gastric volumes and water emptying in healthy volunteers. Therefore, we prepared two extreme examples of gastric emptying in order to explore the possible influences of varying the flow rate on the in vitro drug dissolution; the “ags_GE_fast” with t1/2 of 5.2 minutes and the “ags_GE_slow”, with t1/2 of 10.7 minutes (Table 1). For the constriction mechanisms activity, a program with a high mechanical impact, resembling the MMC phase III was chosen. 35 mL of 0.01M HCl with ions, preheated to 37±0.5 °C was first added to the flexible vessel, simulating the average resting gastric volume in the fasted state. Formulation was then placed in the container and 240 mL of room temperature water was slowly purred over, while simultaneously starting the program for “pyloric” valve and constriction mechanisms around the flexible vessel. At predetermined time points (2.5, 5, 10, 15, 20, 25 and 30 minutes) samples with respective volumes (76, 60, 70, 20,10, 5, 5mL) were collected at the container’s outlet (Table 1). Samples were further filtered through the LLG-Syringe nonsterile filter (PVDF, 0.22-μm) to 2.0-mL vials and further diluted 30-times with a 20% methanol solution. The residual volume, which was left in the container after the last sampling time, represented the final, infinite dissolution point. This volume was quantitatively transferred to the beaker and stirred for 15 minutes and then further filtered and diluted according to the same procedure as already described for previous sample points. The diluted samples were analysed by a validated reverse phase HPLC method with UV detection. Table 1: Parameters of prepared gastric emptying patterns.
[Insert Table 1] Pharmacokinetic study protocol The pharmacokinetic (PK) study for comparing formulations A, B, C and D was a single dose, randomized, four-period, four-sequence crossover comparative study under fasting conditions. 20 healthy volunteers were included in the study after obtaining written informed consent and 18 subjects completed all study periods. The study was conducted by a thirdparty contract research organization (CRO). There was a 5-day washout period between single-doses administered with 240 ml of water at room temperature. A total of 25 blood samples were collected up to 60h post-dose from each individual in each period. For formulations A and B, 19 volunteers were included (two volunteers haven’t finished period 4
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of the study and there were no concentrations available) in PK and statistical analysis, while for formulations C and D all 20 volunteers were included. The study was approved by the National Health Authority and Independent Ethics Committee. Concentrations of the API in plasma samples were determined by a highperformance liquid chromatography using MS-MS detection (Biological matrix: K2-EDTA plasma, validated quantification range: 10-10000 ng/ml, internal standard: stable isotope of drug), validated according to the FDA guidelines for bioanalytical method validation. Results and discussion Apparatus performance According to the available literature data, the gastric emptying rate is a highly variable process, both within the same subject as well as between different individuals [15]. Mudie et al. [22] for instance studied gastric emptying of 240 mL water administered in fasted state using the MRI technique. At rest, an average of 35 mL of fluid (basal conditions, residual gastric volume) was measured in the stomach. Immediately after water ingestion, the volume of the stomach fluid rose to an average of 242 mL in the form of a continuous fluid pocket. The observed gastric emptying was exponential, with the initial gastric volume decreasing by half in 13 minutes (t1/2), and re-establishment of the basal conditions 45 minutes after the water ingestion. Experiments in the AGS were designed in the way to capture some of the parameters, closely resembling the ones found in human stomach during the fasted state PK studies. Our study was designed to capture the reported differences of gastric emptying. For this purpose, two programs with different gastric emptying rates were prepared, one with fast (ags_GE_fast) and the other with slow gastric emptying profile (ags_GE_slow). Discharging of the stomach contents through the “pyloric” valve is controlled by a custom computer software with incorporated PID controller (proportional–integral–derivative controller). The software continuously calculates the difference between the desired weight and the measured value on the balance and applies the necessary correction to the opening of the “pyloric” valve. Emptying of the container’s content was not set as a continuous process of slow liquid dripping with a constant flow value, but rather as a series of short steps involving passage of small fluid volumes with little or no fluid emptying (Fig. 2). The programmed emptying profile was based on observations in MRI studies as shown in Figure 3, where the Figure 3a is included with permission from Grimm et al., 2018: Interindividual and intraindividual 9
variability of fasted state gastric fluid volume and gastric emptying of water. Eur J Pharm Biopharm, 127, 309-317, DOI:10.1016/j.ejpb.2018.03.002; Copyright (2018) Elsevier, and is available under the 4586410356437 license number. [Insert Figure 3] Figure 3: a) Intraindividual variability in gastric emptying of 240 mL room temperature water in fasted state, as observed in MRI study. Reprinted from Eur J Pharm Biopharm, 127, Grimm et al, Interindividual and intraindividual variability of fasted state gastric fluid volume and gastric emptying of water/314/2018, with permission from Elsevier. b) Gastric emptying curves of two tested programs; ags_GE_fast and ags_GE_slow: theoretical gastric emptying is marked with a dashed line and experimentally observed with a solid line.
The flow value was changed throughout the experiment duration at predetermined sampling points (Table 1). Apparatus performance was estimated by comparing the theoretical fraction weight at each sampling time point with experimentally collected mass on the balance. All experiments were performed in at least 3 parallels, and in the case of higher variability up to 5 parallels were performed. The collected mass fractions did not differ by more than 5% from the theoretical ones, which indicates precise software and hardware control of the pyloric flow (Fig. 3). The final average percentage of the released active substance was higher than 95% for all formulations. Comparison of drug release using the AGS and USP2 apparatus In Fig. 4 the immediate release of API obtained with three dissolution methods can be observed. The entire dose has been released in less than 10 minutes of the experiment conducted with USP2 apparatus, showing no major differences between the tested formulations. Furthermore, dissolution of formulation A and D was almost identical (release profiles in Fig. 4 are overlapping). On the other hand, the release profiles obtained in AGS differ from those, obtained in USP2 apparatus. In 30 minutes of experiment, approximately 60% of the dose has emptied from the stomach. In this case, the samples were collected cumulatively at the outlet of the flexible vessel – the dissolved drug was further away from the tablet’s initial dissolving location, leading to the apparent slower drug release rate in AGS compared to the tests in USP2 apparatus. In order to resemble the realistic gastric volume during the fasted PK studies, the volume of dissolution media in experiments, conducted with AGS was more than 3 times lower than the volume used in USP2 apparatus. The volume of the dissolution media at the beginning of the test was high enough to establish the sink conditions. However, to illustrate relevant 10
biological conditions the volume of the media was reduced to the value of residual gastric volume (approximately 35 mL) observed in vivo. The solubility could therefore be a limiting factor. Nevertheless, together with residual gastric content, the amount of drug released was 95 100% for all four formulations, meaning that the volume was still large enough to allow the entire dose to dissolve. The main physical determinant affecting the dissolution rate in the case of USP2 apparatus is dissolution medium hydrodynamics [34], resulting from the paddle rotation, while in AGS, the tablets were subjected to presumably higher shear forces and direct mechanical contact applied by changing the volume and the shape of the flexible vessel. Variability was higher for experiments conducted in AGS, most likely due to the random movement of the tablet in the flexible vessel and the combination of hydrodynamics and direct physical contact effects on tablet. Differences in drug release kinetics between the tested formulations were more prominent in AGS, suggesting this dissolution method has a better discriminatory power compared to the conventional one. Admittedly, in the USP2 experiments, the conditions could be further modified to possibly show the differences in formulations, for example the paddle rotation speed could be reduced as well as the volume of the media. However, the authors believe that the AGS model offers better experimental platform to more closely mimic the in vivo gastric volume and mechanical contact conditions. Release profiles in AGS with two different gastric emptying regimes differed as well. Release rate was slightly slower in the case of the ags_GE_fast, compared to the ags_GE_slow programme (Fig. 4). The observed difference could be due to the greater volume of dissolution media emptying from the stomach in shorter period of time with the fast programme, leaving less time for the substance to entirely dissolve. Considering the fact that the volume of fluid present in a flexible vessel is rapidly decreasing, it could be assumed that the hydrodynamics is less intense towards the end of the test. The distribution of formulations according to the release rate was maintained when changing the gastric emptying rate, except for the formulation A, where the release rate was faster in the case of ags_GE_fast programme, presumably due to simultaneous discharge of disintegrated particles of the dosage form along with the leaving dissolution medium. This was observed as a strong opacity of the collected sample fractions and correlates to the shortest disintegration time interval among tested formulations. [Insert Figure 4] 11
Figure 4: Release profiles of the tested formulations with different dissolution methodologies: slow and fast gastric emptying (upper diagrams) and dissolution in USP2 apparatus (lower diagram).
Evaluation of in vivo relevance A PK study was performed to evaluate the tested formulations’ behaviour in vivo. The PK parameters, such as the absorption rate (Cmax) and the extent of absorption (AUC) of formulations A, B and C were compared to the reference formulation D. The PK parameters for each tested formulation are presented in Table 2 and the mean plasma concentration profiles in Fig. 5. In PK study, formulations A, B and C showed lower rate of absorption compared to formulation D, with Cmax geometric mean ratios A/D, B/D and C/D at 83.0%, 82.3% and 86.9%, respectively, and smaller extents of absorption with AUC geometric mean ratios for A/D, B/D and C/D at 94.1%, 94.8% and 97.8%, respectively. From the average plasma profiles, it can be observed that the tmax for tested formulations is short, meaning that the rate of drug absorption into the systemic circulation is fast. Despite the considerable variability among all of the tested formulations, it can also be clearly observed that tmax of the reference formulation D is shorter, indicating a faster absorption rate compared to the other formulations. Elimination of the drug is slower, with a half-time of 9 hours. Table 2: Pharmacokinetic parameters of formulations A, B (n=19), C and D (n=20).
[Insert Table 2] [Insert Figure 5] Figure 5: Average plasma profiles of the tested formulations.
In order to compare the in vitro observed release profiles of the tested formulations to the observed mean plasma concentrations, in vivo absorption profiles were calculated based on Loo-Riegelmann two-compartment deconvolution method with the GastroPlusTM software. Assuming the absorption of the released drug is mostly dependent on the gastric emptying rate, dissolution experiments were compared to the first 30 minutes of the absorption profiles. As it can be observed in Figure 6, the dissolution profiles obtained with tests in AGS imitate better the in vivo absorption compared to the tests performed in USP2 apparatus. However, a lag time of approximately 5 minutes was observed in the case of in vivo absorption, therefore lag time correction was applied also to the dissolution profiles obtained in AGS. According to several research articles [35, 36], this type of correction in the process of setting up in vitro in 12
vivo correlation is recommended to be avoided in some cases. Nevertheless, the lag time correction in this study was considered to be properly justified since the in vivo observed time lag for the studied drug absorption is explained by the documented existence of an absorption window within a specific region of the small intestine. Dissolution conditions in AGS with slow gastric emptying programme (ags_GE_slow) showed the best fit to the in vivo absorption data, allocating the formulations appropriately according to the absorption profiles, from the fastest to the slowest formulation (D
Based on the present study, it may be concluded that AGS as a biorelevant model showed a better discriminatory power in revealing the differences in the drug release of the tested immediate release formulations. Furthermore, a better relation to the in vivo results compared to the conventional USP2 apparatus was also observed. The presented examples showed a significant effect of the modelled gastric emptying rate programme on the release kinetics of the tested BCS III class drug, which is also reflected. These kinds of experiments are therefore highly valuable when greater discriminatory power is needed. One such example are the immediate release dosage forms where the release rate is often so fast that it is very difficult to detect any differences between formulations using only conventional dissolution methodologies Despite the good correlation with in vivo results in the present study, it needs to be emphasized that the method should be tested on individual cases since different formulations and compounds may respond differently to in vitro and in vivo conditions. To achieve good discriminatory power, a wide array of options is available with the AGS: variation of constriction frequency and intensity, the flow through the lumen, the dissolution medium volume and composition and the possibility to monitor the dissolution for longer periods of time. Furthermore, the anatomical similarity of the model's volume and shape and the control over its mechanical and flow functions offers achieving the physiologically relevant in vitro conditions which may fruitfully support the drug development process.
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Conclusion In this paper we present a dynamic biorelevant dissolution model – advanced gastric simulator, capable of simulating realistic gastric contractions movement and gastric emptying. The model showed the ability to precisely control the opening and closing of the “pyloric” valve. Specific movements of the constriction mechanism and the course of the containers’ outlet flow can be set for individual formulation testing. This study shows that biorelevant dissolution methods can be used as a powerful tool to predict the in vivo formulation performance and to minimize the human studies in a new formulation development process. The AGS showed better discriminating capability between all four of the tested formulations compared to the standard dissolution method with USP2 apparatus. These results will undoubtedly lead to further model optimisation and additional investigation of the novel apparatus applicability in cases where gastric emptying rate is considered to be crucial.
Declaration of competing interest The authors declared no conflicts of interest related to this article. The authors from University of Ljubljana, Faculty of Pharmacy were supported by the Slovenian Research Agency (ARRS) [P1-0189].
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[20]. B. Hens, M. Corsetti, R. Spiller, L. Marciani, T. Vanuytsel, J. Tack, et al, Exploring gastrointestinal variables affecting drug and formulation behavior: Methodologies, challenges and opportunities, Int. J. Pharm. 519 (2017) 79–97. https://doi.org/10.1016/j.ijpharm.2016.11.063 [21]. T.–L. Abell, M. Camilleri, K. Donohoe, W.–L. Hasler WL, H.–C. Lin, A.–H. Maurer et al, Consensus recommendations for gastric emptying scintigraphy: A joint report of the american neurogastroenterology and motility society and the society of nuclear medicine, J. Nucl. Med. Technol. 36 (2008) 44–54. https://doi.org/10.2967/jnmt.107.048116 [22]. D.−M. Mudie, K. Murray, C.−L. Hoad, S.−E. Pritchard, M.−C. Garnett, G.−L. Amidon, et al, Quantification of gastrointestinal liquid volumes and distribution following a 240 mL dose of water in the fasted state, Mol. Pharm. 11 (2014) 3039–3047. https://doi.org/10.1021/mp500210c [23]. Z. Liu, Y. Li, J. Guo, J. Li, W. Ren, S. Tang et al, Evaluation of gastric emptying by transabdominal ultrasound after oral administration of semisolid cellulose-based gastric ultrasound contrast agents, Ultrasound. Med. Biol. 44 (2018) 2183–2188. https://doi.org/10.1016/j.ultrasmedbio.2018.04.019 [24]. M. Koziolek, F. Schneider, M. Grimm, C. Modeβ, A. Seekamp, T. Roustom et al, Intragastric pH and pressure profiles after intake of the high-caloric, high-fat meal as used for food effect studies, J. Control. Release. 220 (2015) 71–78. https://doi.org/10.1016/j.jconrel.2015.10.022 [25]. F.−K. Friedenberg, S. Maqbool, H.−P. Parkman, Wireless capsule motility: Comparison of the SmartPill_GI monitoring system with scintigraphy for measuring whole gut transit, Dig. Dis. Sci. 54 (2009) 2167–2174. https://doi.org/10.1007/s10620-009-0899-9 [26]. F. Schneider, M. Grimm, M. Koziolek, C. Modeß, A. Dokter, T. Roustom et al, Resolving the physiological conditions in bioavailability and bioequivalence studies: Comparison of fasted and fed state, Eur. J. Pharm. Biopharm. 108 (2016) 214–219. https://doi.org/10.1016/j.ejpb.2016.09.009
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[27]. A. Talattof, G.−L. Amidon, Pulse packet stochastic model for gastric emptying in the fasted state: A physiological approach, Mol. Pharm. 15 (2018) 2107–2115. https://doi.org/10.1021/acs.molpharmaceut.7b01077 [28]. B. Bürmen, I. Locatelli, A. Bürmen, M. Bogataj, A. Mrhar, Mathematical modeling of individual gastric emptying of pellets in the fed state, J. Drug. Deliv. Sci. Technol. 24 (2014) 418–424. https://doi.org/10.1016/S1773-2247(14)50083-4 [29]. A. Kourentas, M. Vertzoni, N. Stavrinoudakis, A. Symillidis, J. Brouwers, P. Augustijns, et al, An in vitro biorelevant gastrointestinal transfer (BioGIT) system for forecasting concentrations in the fasted upper small intestine: Design, implementation, and evaluation, Eur. J. Pharm. Sci. 20 (2016) 106–114. https://doi.org/10.1016/j.ejps.2015.11.012 [30]. Y. Tsume, S. Takeuchi, K. Matsui, G.−E. Amidon, G.−L. Amidon, In vitro dissolution methodology, mini-Gastrointestinal Simulator (mGIS), predicts better in vivo dissolution of a weak base drug, dasatinib, Eur. J. Pharm. Sci. 76 (2015) 203–212. https://doi.org/10.1016/j.ejps.2015.05.013 [31]. I. Legen, A. Bevc, S. Berglez, J. Diaci, L. Kuscer, Apparatus for simulating the function of human stomach and/or human intestine, U.S. Patent 20160351079 (2018). [32]. M. Hribar, O. Jakasanovski, J. Trontelj, I. Grabnar, I. Legen, Determining the pressuregenerating capacity of the classical and alternative in vitro dissolution methods using a wireless motility capsule, J. Pharm. Innov. 13 (2018) 226–236. https://doi.org/10.1007/s12247-018-9317-1 [33]. M. Hribar, J. Trontelj, S. Berglez, A. Bevc, L. Kuščer, J. Diaci, I. Legen, Design of an innovative advanced gastric simulator, Dissol. Technol. 26 (2019) 20-29. https://doi.org/10.14227/DT260219P20 [34]. V. Todaro, T. Persoons, G. Grove, A.−M. Healy, D.−M. D’Arcy, Characterization and simulation of hydrodynamics in the paddle, basket and flow-through dissoluiotn testing apparatuses – a review, Dissolut. Technol. 24 (2017) 24–36. https://doi.org/10.14227/DT240317P24
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[35]. J.−M. Cardot, B.−M. Davit. In vitro-in vivo correlations: Tricks and traps, The AAPS. J. 14 (2012) 491–499. https://doi.org/10.1208/s12248-012-9359-0 [36]. J. Emami, In vitro - in vivo correlation: from theory to applications, J. Pharm. Sci. 9 (2006) 169–189.
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Figure 1: Left: Advanced gastric simulator (AGS) with main elements: (1) “pyloric” valve, (2) constriction mechanisms, (3) step motors and (4) flexible vessel. Red arrows mark direction of the contraction movement. Right: Schematic presentation of the apparatus set-up.
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Figure 2: Real time print screen of the pylorus software displaying the course of desired and actual mass flow through the “pyloric” valve of the advanced gastric simulator.
Table 1: Parameters of prepared gastric emptying patterns. 21
ags_GE_fast
ags_GE_slow
lag time time estimated volume of volume collected desired flow estimated volume of volume collected desired flow correction* [min] media in bag [mL] [mL] [g/min] media in bag [mL] [mL] [g/min] [min] -5 -5 0 0 0 275 2,5 7,5 200 75 5 10 140 60 10 15 70 70 15 20 50 20 20 25 40 10 25 30 35 5 30 35 30 5 *arbitrarily delayed in vitro release time by 5 min
0 275 240 205 145 100 70 50 30
30 24 14 4 2 1 1
22
35 35 60 45 30 20 20
14 14 12 9 6 4 4
Figure 3: a) Intraindividual variability in gastric emptying of 240 mL room temperature water in fasted state, as observed in MRI study. Reprinted from Eur J Pharm Biopharm, 127, Grimm et al, Interindividual and intraindividual variability of fasted state gastric fluid volume and gastric emptying of water/314/2018, with permission from Elsevier. b) Gastric emptying curves of two tested programs; ags_GE_fast and ags_GE_slow: theoretical gastric emptying is marked with a dashed line and experimentally observed with a solid line.
23
Figure 4: Release profiles of the tested formulations with different dissolution methodologies: slow and fast gastric emptying (upper diagrams) and dissolution in USP2 apparatus (lower diagram).
24
Table 2: Pharmacokinetic parameters of formulations A, B (n=19), C and D (n=20).
PK parameters formulation
A
B
C
D
parameter
arithmetic mean ± SD
Median [range]
geometric mean ratio
Cmax [ng/mL]
2210 ± 430
−
83.02
AUCt [h*ng/mL]
12610 ± 2171
−
tmax [h]
−
1.33 [0.83−4.50]
−
Cmax [ng/mL]
2200 ± 536
−
82.34
AUCt [h*ng/mL]
12760 ± 2580
−
tmax [h]
−
2.50 [0.83−5.00]
−
Cmax [ng/mL]
2320 ± 574
−
86.88
AUCt [h*ng/mL]
13110 ± 2093
−
tmax [h]
−
1.67 [0.67−5.00]
Cmax [ng/mL]
2690 ± 833
−
−
−
AUCt [h*ng/mL]
13410 ± 2184
−
−
−
tmax [h]
−
1.00 [0.67−2.03]
−
−
25
formulation A vs D
formulation B vs D
formulation C vs D
94.12
94.84
97.80 −
Figure 5: Average plasma profiles of the tested formulations.
26
Figure 6: Comparison of in vitro dissolution methods with 30 minutes of in vivo observed absorption profile for each tested formulation.
27
28
S0939-6411(20)30035-7 https://doi.org/10.1016/j.ejpb.2020.02.002 EJPB 13228
To appear in:
European Journal of Pharmaceutics and Biopharmaceutics
Received Date: Revised Date: Accepted Date:
16 July 2019 7 January 2020 4 February 2020
Please cite this article as: H. Vrbanac, J. Trontelj, S. Berglez, B. Petek, J. Opara, R. Jereb, D. Krajcar, I. Legen, The biorelevant simulation of gastric emptying and its impact on model drug dissolution and absorption kinetics, European Journal of Pharmaceutics and Biopharmaceutics (2020), doi: https://doi.org/10.1016/j.ejpb. 2020.02.002
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Title:
The biorelevant simulation of gastric emptying and its impact on model drug dissolution and absorption kinetics
Authors:
Helena Vrbanac1, Jurij Trontelj1*, Sandra Berglez2, Boštjan Petek2, Jerneja Opara2, Rebeka Jereb2, Dejan Krajcar2, Igor Legen2
* Corresponding Author: Assist. Prof. Jurij Trontelj, Ph D, Mag. Pharm. e-mail: [email protected] tel. 00386 1 476 95 00 Aškerčeva 7, 1000 Ljubljana, Slovenia
Author affiliations:
1 The
Chair for Biopharmaceutics and Pharmacokinetics, University of Ljubljana, Faculty of Pharmacy, Aškerčeva 7, 1000 Ljubljana, Slovenia
2 Lek
Pharmaceuticals, Inc., a Sandoz company, Verovškova ulica 57, 1000 Ljubljana, Slovenia
The biorelevant simulation of gastric emptying and its impact on model drug dissolution and absorption kinetics
Abstract
The highly variable physiological conditions within the gastrointestinal tract can cause variable drug release and absorption from the orally administrated dosage forms. The emptying of the gastric content is one of the most critical physiological processes, dictating the amount of the active ingredient available for absorption into the systemic circulation. In this study, we prepared two water gastric emptying regimes on advanced gastric simulator 1
(AGS) with programmable “pyloric” valve. Gastric emptying regimes were designed in such a way to capture the main findings of the MRI (magnetic resonance imaging) in vivo studies, conducted under fasted conditions according to the EMA and FDA guidelines for bioavailability and bioequivalence studies. Four immediate release formulations containing a model drug of BCS class III were tested. Comparative dissolution tests were also performed with the USP2 apparatus. In vitro release profiles were compared to the in vivo data in order to evaluate the importance of gastric emptying for subsequent absorption of the active substance from the tested formulations. Our bio-relevant in vitro dissolution model showed good discriminatory power for all of the tested formulations. Moreover, a better relation to in vivo data was achieved with AGS with respect to the tested conventional dissolution method. Key words: stomach; gastric emptying, biorelevant dissolution testing, immediate release; Magenstrasse Introduction One of the challenges in the field of drug development is to find a correlation between in vitro drug release of various formulations and in vivo occurring plasma levels. Biorelevant dissolution testing has been shown as a very useful tool for in vivo product performance prediction. When a suitable in vitro-in vivo correlation (IVIVC) is demonstrated, the number of bioavailability or bioequivalence studies needed is reduced and the process of a new formulation development leads to a product with improved safety and quality [1–3]. Oral drug administration remains the preferred administration route with the highest patient compliance among drug delivery systems [1]. The passage of the ingested dosage form through the stomach and exposure to stomach conditions is necessary in order for the drug to be later absorbed in the small intestine and/or colon [4]. The active pharmaceutical ingredient (API) from the conventional solid dosage form must first dissolve before it can be absorbed to systemic blood circulation. The majority of drugs are absorbed in the small intestine. The rate and extent to which the drug is dissolved and transferred to the small intestine is thus important for the resulting mean plasma concentration. The rate and extent of the API absorption are therefore highly dependent on the volume and distribution of fluid in the stomach and its passage to the small intestine [5]. For rapidly dissolving and highly permeable drugs (BCS class I compounds), gastric emptying represents a rate limiting step for drug absorption in the small intestine [5–7]. In the 2
case of drugs exhibiting high solubility and low membrane permeability (BCS class III compounds), the membrane transport is often limited to a specific region in the small intestine (absorption window). One of the causes for uneven permeability in the GIT (gastrointestinal tract) is the heterogeneous distribution of efflux and influx transporters along the GIT. Gastric residence time and dissolution rate in the stomach have been also described as important factors affecting in vivo pharmacokinetics of such substances [8, 9]. Retention time in the stomach varies depending on the volume and composition of food/fluid intake. Non-caloric liquids, such as water are emptied relatively quickly from the stomach, with gastro-duodenal pressure gradient being the driving force [10–14]. However, solid food components must first be processed into smaller particles with a diameter of 1-2 mm, small enough to pass through the pyloric sphincter. Greater, undigested particles are eliminated in phase III of the migrating motor complex (MMC) of the fasted stomach ("housekeeping wave") [10, 11]. In the postprandial stomach, fast emptying of ingested liquids, known as “the Magenstrasse” (the stomach road) has been observed. Liquids, administered together with food finding a shortcut through the fed stomach can have an important impact on the therapeutic onset of coadministered drugs [14]. On the other hand, high inter- and intraindividual variability of fasted human stomach emptying has been demonstrated as well [15]. The motor activity of the fasted stomach is carried out in cycles (MMC), characterized by a fluctuation of the muscle contraction intensity [16]. Different intensity of motility patterns can have an influence on disintegration time and distribution of drug and formulation particles within the various stomach regions. Random dosing with respect to different phases of the MMC is thus an important factor that can lead to increased variability in systemic drug exposure [17]. According to FDA guidelines [18] for bioavailability and bioequivalence studies (under fasting as well as under fed conditions), it is recommended for the tested formulation is to be administrated together with 240 mL of water at room temperature, on the other hand, EMA suggests administration with a constant volume of at least 150 mL [19]. The distribution of such a volume of water in the stomach immediately after ingestion, the rate of its discharge and occurrence in the small intestine is therefore the main research topic of several studies. Gastric emptying has been monitored with magnetic resonance imaging (MRI), scintigraphy, non-absorbable marker technique, ultrasonography, breath test, intraluminal sampling, with wireless motility capsule (WMC) and other techniques, as presented in the extensive review 3
article by Hens et al. [20]. Among all, scintigraphy is considered as the golden standard in gastric emptying monitoring, however measurements can be highly variable due to the influence of many factors. In 2007, guidelines have been applied to standardize experiments (standardized meals, subject posture, frequency and duration of imaging, etc.) [21]. MRI is a multiplanar technique used to image organ fluids and volumes. In addition to studying the gastric emptying, this technique is also very useful for monitoring the fluid distribution in small intestine, the movement of marked dosage form along the GIT or the bowel movement [22]. Ultrasound is a non-invasive method for gastric emptying monitoring, without radiation exposure. A drawback of this method is the need of expertise knowledge in interpreting sonograms [23]. The passage of individual parts of the GIT can also be monitored with a wireless motility capsule (WMC), which contains the sensors for detecting the pH, temperature and pressure exerted on the capsule wall. The WMC can thus be very useful for studying the motor functions and retention times of individual GIT compartments under fed or fasted conditions. However, using only the WMC the exact profile of liquid gastric emptying cannot be determined [24–26]. Based on the in vivo measurements, several in silico and in vitro models simulating gastric emptying have been proposed; Talattof et al. [27] have developed physiologically based pulsed-packet mathematical model resembling the conditions of a normal subject in a typical fasted BE study. Brümen et al. have performed mathematical modelling of individual gastric emptying profiles of pellets in the fed state [28]. Kurentas et al. [6, 29] have developed in vitro biorelevant gastrointestinal transfer system (BioGIT) to reproduce the concentrations of active ingredients in the fasted upper small intestine. Tsume et al. [30] developed the Gastrointestinal Simulator (GIS) mimicking the physiological conditions of human GIT in fasted state for the dissolution prediction of orally administrated drugs with pH dependent solubility. In this study we explore the possibility of simulating the fasted gastric emptying on the newly developed advanced gastric simulator (AGS). AGS is an innovative device, which has proved to be a successful model in achieving biorelevant conditions [31, 32]. The aim of our research was to test the applicability of the proposed biorelevant gastric dissolution methodology in establishing a better IVIVC/IVIVR (in vitro – in vivo correlation/ in vitro-in vivo relationship) compared to conventional methods for formulations with release and absorption susceptible to the process of gastric emptying.
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Materials Tablets For the purpose of this study, immediate release tablets with a high drug load (more than 200 mg) were prepared with fluid-bed granulation process, followed by tablet compression. The model drug is a weak base, with a dissociation constant of approximately 4.5. The compound is an aromatic, heteromonocyclic compound, highly soluble (4 mg/mL) over a pH range of 1.0─8.0. Substance shows high permeability, however, due to transporters dominated absorption it is classified as BCS class III compound, with bioavailability in adults between 80 and 85%. It is a nucleoside reverse transcriptase inhibitor, so it is used as an antiretroviral drug. The weight of tablets was approximately 600-800 mg Formulation D was acquired from the market and was used as a reference in the present study to which the in vitro and in vivo performance of the formulations A, B and C were compared. The formulations A and B differ in technological process of preparation, while the formulation C differs from B in extragranularly added excipient. These formulations also differ in disintegration time; the formulation A has the shortest disintegration time of [2min 42s–2min 48s; n=6] (minimal to maximal disintegration time interval), followed by formulation B [3min 54s–4min 38s; n=6] and C [4min 20s–4min 44s; n=6]. Methods Advanced gastric simulator (AGS) – apparatus description
AGS is an innovative dissolution apparatus simulating motility of the human stomach. The apparatus’ main features are a flexible silicone vessel, eight constriction mechanism, “pyloric” valve, scale, thermal chamber enabling 37 ± 0.5°C and a custom made software. The shape and the size of the container resemble closely those of the human stomach. The thin walls of the vessel are collapsed when the container is empty, but when the vessel is filled with dissolution medium the flexibility of the walls allows it to stretch. Both ends of the vessel are opened; the upper end of the vessel is wider (up to 10 cm in width), resembling the cardia, while the lower end of the vessel narrows to a tubular opening (up to 1 cm narrow), resembling the pyloric sphincter. The vessel allows a volume up to 1 L to be filled into the container. The silicone rubber (DragonSkin FX, Smooth-On Inc., United States) used for formation of the vessel is soft and exhibits the required chemical inertness. The inner lining on the vessel surface is structured with folds spreading in longitudinal direction to enable
5
repeatability of the vessel deformation. The change in shape and volume of the vessel is caused by closing and opening of the constriction mechanism around the vessel (Fig. 1). [Insert Figure 1] Figure 1: Left: Advanced gastric simulator (AGS) with main elements: (1) “pyloric” valve, (2) constriction mechanisms, (3) step motors and (4) flexible vessel. Red arrows mark direction of the contraction movement. Right: Schematic presentation of the apparatus set-up.
The structure of each constriction mechanism is derived from optical iris diaphragms (57-586, Edmund optics INC., United States), driven by electric step motor (ST2818, Nanotec) and a worm gear. Synchronised opening and closing of each constriction mechanisms can be individually programmed through a custom made software, allowing a combination of different motion parameters in a programme sequence, mimicking realistic gastric motion regimes. The change in the aperture diameter (75−25 mm) starts with closing of the first constriction mechanism, set closest to the upper part of the stomach and follows with closing of subsequent constriction mechanisms toward the pyloric part of the stomach, with a pre-set delay between each constriction mechanism (Fig. 1). This simulates the contraction waves of the stomach wall. Consequently, the fluid dynamics in the vessel is changed and the pressure from the containers’ wall is transferred to its interior content. The majority of constriction mechanisms is positioned in the antropyloric part of the stomach, where the strong mixing and grinding of ingested food take place [10]. Another important element of the apparatus, enabling the simulation of gastric emptying, is an artificial “pyloric” valve. It consists of two steel pins (PFCLEA-D8-L55-M4), one positioned stationary relative to the stomach container and the other – movable – attached to a liner slide (SE2BZ10-115, Misumi, United States). The pins are tightening the tubular outlet of the flexible vessel and are acting as a pyloric sphincter. The valve aperture is adjusted with an individual electric step motor and custom made software, enabling independent regulation from the constriction mechanisms’ activity. Desired flow rate (mg/min) of the media through the tubular outlet is set in the software and is controlled with the weight of collected sample on the balance (XS6002S, Mettler-Toledo International Inc., United States) outside the thermal chamber (SP-55 EASY, Kambič d.o.o., Slovenia). The information of the current weight of collected sample on the balance is a trigger for software to automatically adjust the flow rate with opening/closing of the pins and thus reaching the desired flow rate (Fig. 2). More detailed structure and function of individual parts of the apparatus can be found in a recently published article [33]. 6
[Insert Figure 2] Figure 2: Real time print screen of the pylorus software displaying the course of desired and actual mass flow through the “pyloric” valve of the advanced gastric simulator.
Dissolution testing with USP2 apparatus Dissolution tests were performed with USP2 apparatus (AT dissolution apparatus, Xtend™, Sotax, Switzerland) at rotation speed of 75 rpm and 900 mL of 0.01M HCl with ions. Dissolution media was prepared by adding 10 mL of 1M HCl Titrisol® concentrate (ampoule, which contains a precisely defined quantity of substance) to 900 mL of deionised water, dissolving 4 g of NaCl, 1.20 g of KCl and 0.084 g of CaCl2x2H2O and then filling the volume of the flask to 1000 mL. The media temperature was set to 37 ± 0.5°C. After 5, 10, 15, 20, 30, 45 and 60 minutes samples of 1.7 mL were automatically collected with an automatic sampler (CP Xtend™ pump, Sotax, Switzerland) and filtered through the Full Flow 1 m filters (P/N FIL001-EW) to 2.0 mL vials. Analysis of dissolution samples The collected samples were analysed with a HPLC system with UV detection (Waters, USA). The chromatographic column Xbridge C18 (Waters, USA) with dimensions of 70x4.6 mm was thermostated at 60°C. Mobile phase was a mixture of ammonium acetate buffer pH 4.2 and methanol (ultra HPLC gradient grade, J. T. Baker, USA) in 80:20 volume ratio. Vials with sample solution were maintained at 25°C. Total run time of the analysis was 2.2 minutes. The simulation of gastric emptying The experiments of gastric emptying on advanced gastric simulator (AGS) can be performed in two ways: with a constant flow rate throughout the entire experiment duration or with changing the flow of container’s content and thus creating different gastric emptying regimes. The aim of this study was to prepare the programs of different gastric emptying rates on AGS, capturing the basic principles of liquid gastric emptying in the fasted state, as observed in vivo. The regimes of gastric emptying were prepared based on the MRI studies performed on healthy volunteers (Mudie et. al and Grimm et al.). Gastric flow rate in fasted state depends on the phase of the MMC; therefore testing different flow rates in the AGS is very relevant. The research group of Mudie et al. has observed gastric emptying in the study of 12 healthy 7
volunteers and had presented it as the average gastric emptying profile. However, Grimm et al. had observed high intraindividual day-to day variations in residual gastric volumes and water emptying in healthy volunteers. Therefore, we prepared two extreme examples of gastric emptying in order to explore the possible influences of varying the flow rate on the in vitro drug dissolution; the “ags_GE_fast” with t1/2 of 5.2 minutes and the “ags_GE_slow”, with t1/2 of 10.7 minutes (Table 1). For the constriction mechanisms activity, a program with a high mechanical impact, resembling the MMC phase III was chosen. 35 mL of 0.01M HCl with ions, preheated to 37±0.5 °C was first added to the flexible vessel, simulating the average resting gastric volume in the fasted state. Formulation was then placed in the container and 240 mL of room temperature water was slowly purred over, while simultaneously starting the program for “pyloric” valve and constriction mechanisms around the flexible vessel. At predetermined time points (2.5, 5, 10, 15, 20, 25 and 30 minutes) samples with respective volumes (76, 60, 70, 20,10, 5, 5mL) were collected at the container’s outlet (Table 1). Samples were further filtered through the LLG-Syringe nonsterile filter (PVDF, 0.22-μm) to 2.0-mL vials and further diluted 30-times with a 20% methanol solution. The residual volume, which was left in the container after the last sampling time, represented the final, infinite dissolution point. This volume was quantitatively transferred to the beaker and stirred for 15 minutes and then further filtered and diluted according to the same procedure as already described for previous sample points. The diluted samples were analysed by a validated reverse phase HPLC method with UV detection. Table 1: Parameters of prepared gastric emptying patterns.
[Insert Table 1] Pharmacokinetic study protocol The pharmacokinetic (PK) study for comparing formulations A, B, C and D was a single dose, randomized, four-period, four-sequence crossover comparative study under fasting conditions. 20 healthy volunteers were included in the study after obtaining written informed consent and 18 subjects completed all study periods. The study was conducted by a thirdparty contract research organization (CRO). There was a 5-day washout period between single-doses administered with 240 ml of water at room temperature. A total of 25 blood samples were collected up to 60h post-dose from each individual in each period. For formulations A and B, 19 volunteers were included (two volunteers haven’t finished period 4
8
of the study and there were no concentrations available) in PK and statistical analysis, while for formulations C and D all 20 volunteers were included. The study was approved by the National Health Authority and Independent Ethics Committee. Concentrations of the API in plasma samples were determined by a highperformance liquid chromatography using MS-MS detection (Biological matrix: K2-EDTA plasma, validated quantification range: 10-10000 ng/ml, internal standard: stable isotope of drug), validated according to the FDA guidelines for bioanalytical method validation. Results and discussion Apparatus performance According to the available literature data, the gastric emptying rate is a highly variable process, both within the same subject as well as between different individuals [15]. Mudie et al. [22] for instance studied gastric emptying of 240 mL water administered in fasted state using the MRI technique. At rest, an average of 35 mL of fluid (basal conditions, residual gastric volume) was measured in the stomach. Immediately after water ingestion, the volume of the stomach fluid rose to an average of 242 mL in the form of a continuous fluid pocket. The observed gastric emptying was exponential, with the initial gastric volume decreasing by half in 13 minutes (t1/2), and re-establishment of the basal conditions 45 minutes after the water ingestion. Experiments in the AGS were designed in the way to capture some of the parameters, closely resembling the ones found in human stomach during the fasted state PK studies. Our study was designed to capture the reported differences of gastric emptying. For this purpose, two programs with different gastric emptying rates were prepared, one with fast (ags_GE_fast) and the other with slow gastric emptying profile (ags_GE_slow). Discharging of the stomach contents through the “pyloric” valve is controlled by a custom computer software with incorporated PID controller (proportional–integral–derivative controller). The software continuously calculates the difference between the desired weight and the measured value on the balance and applies the necessary correction to the opening of the “pyloric” valve. Emptying of the container’s content was not set as a continuous process of slow liquid dripping with a constant flow value, but rather as a series of short steps involving passage of small fluid volumes with little or no fluid emptying (Fig. 2). The programmed emptying profile was based on observations in MRI studies as shown in Figure 3, where the Figure 3a is included with permission from Grimm et al., 2018: Interindividual and intraindividual 9
variability of fasted state gastric fluid volume and gastric emptying of water. Eur J Pharm Biopharm, 127, 309-317, DOI:10.1016/j.ejpb.2018.03.002; Copyright (2018) Elsevier, and is available under the 4586410356437 license number. [Insert Figure 3] Figure 3: a) Intraindividual variability in gastric emptying of 240 mL room temperature water in fasted state, as observed in MRI study. Reprinted from Eur J Pharm Biopharm, 127, Grimm et al, Interindividual and intraindividual variability of fasted state gastric fluid volume and gastric emptying of water/314/2018, with permission from Elsevier. b) Gastric emptying curves of two tested programs; ags_GE_fast and ags_GE_slow: theoretical gastric emptying is marked with a dashed line and experimentally observed with a solid line.
The flow value was changed throughout the experiment duration at predetermined sampling points (Table 1). Apparatus performance was estimated by comparing the theoretical fraction weight at each sampling time point with experimentally collected mass on the balance. All experiments were performed in at least 3 parallels, and in the case of higher variability up to 5 parallels were performed. The collected mass fractions did not differ by more than 5% from the theoretical ones, which indicates precise software and hardware control of the pyloric flow (Fig. 3). The final average percentage of the released active substance was higher than 95% for all formulations. Comparison of drug release using the AGS and USP2 apparatus In Fig. 4 the immediate release of API obtained with three dissolution methods can be observed. The entire dose has been released in less than 10 minutes of the experiment conducted with USP2 apparatus, showing no major differences between the tested formulations. Furthermore, dissolution of formulation A and D was almost identical (release profiles in Fig. 4 are overlapping). On the other hand, the release profiles obtained in AGS differ from those, obtained in USP2 apparatus. In 30 minutes of experiment, approximately 60% of the dose has emptied from the stomach. In this case, the samples were collected cumulatively at the outlet of the flexible vessel – the dissolved drug was further away from the tablet’s initial dissolving location, leading to the apparent slower drug release rate in AGS compared to the tests in USP2 apparatus. In order to resemble the realistic gastric volume during the fasted PK studies, the volume of dissolution media in experiments, conducted with AGS was more than 3 times lower than the volume used in USP2 apparatus. The volume of the dissolution media at the beginning of the test was high enough to establish the sink conditions. However, to illustrate relevant 10
biological conditions the volume of the media was reduced to the value of residual gastric volume (approximately 35 mL) observed in vivo. The solubility could therefore be a limiting factor. Nevertheless, together with residual gastric content, the amount of drug released was 95 100% for all four formulations, meaning that the volume was still large enough to allow the entire dose to dissolve. The main physical determinant affecting the dissolution rate in the case of USP2 apparatus is dissolution medium hydrodynamics [34], resulting from the paddle rotation, while in AGS, the tablets were subjected to presumably higher shear forces and direct mechanical contact applied by changing the volume and the shape of the flexible vessel. Variability was higher for experiments conducted in AGS, most likely due to the random movement of the tablet in the flexible vessel and the combination of hydrodynamics and direct physical contact effects on tablet. Differences in drug release kinetics between the tested formulations were more prominent in AGS, suggesting this dissolution method has a better discriminatory power compared to the conventional one. Admittedly, in the USP2 experiments, the conditions could be further modified to possibly show the differences in formulations, for example the paddle rotation speed could be reduced as well as the volume of the media. However, the authors believe that the AGS model offers better experimental platform to more closely mimic the in vivo gastric volume and mechanical contact conditions. Release profiles in AGS with two different gastric emptying regimes differed as well. Release rate was slightly slower in the case of the ags_GE_fast, compared to the ags_GE_slow programme (Fig. 4). The observed difference could be due to the greater volume of dissolution media emptying from the stomach in shorter period of time with the fast programme, leaving less time for the substance to entirely dissolve. Considering the fact that the volume of fluid present in a flexible vessel is rapidly decreasing, it could be assumed that the hydrodynamics is less intense towards the end of the test. The distribution of formulations according to the release rate was maintained when changing the gastric emptying rate, except for the formulation A, where the release rate was faster in the case of ags_GE_fast programme, presumably due to simultaneous discharge of disintegrated particles of the dosage form along with the leaving dissolution medium. This was observed as a strong opacity of the collected sample fractions and correlates to the shortest disintegration time interval among tested formulations. [Insert Figure 4] 11
Figure 4: Release profiles of the tested formulations with different dissolution methodologies: slow and fast gastric emptying (upper diagrams) and dissolution in USP2 apparatus (lower diagram).
Evaluation of in vivo relevance A PK study was performed to evaluate the tested formulations’ behaviour in vivo. The PK parameters, such as the absorption rate (Cmax) and the extent of absorption (AUC) of formulations A, B and C were compared to the reference formulation D. The PK parameters for each tested formulation are presented in Table 2 and the mean plasma concentration profiles in Fig. 5. In PK study, formulations A, B and C showed lower rate of absorption compared to formulation D, with Cmax geometric mean ratios A/D, B/D and C/D at 83.0%, 82.3% and 86.9%, respectively, and smaller extents of absorption with AUC geometric mean ratios for A/D, B/D and C/D at 94.1%, 94.8% and 97.8%, respectively. From the average plasma profiles, it can be observed that the tmax for tested formulations is short, meaning that the rate of drug absorption into the systemic circulation is fast. Despite the considerable variability among all of the tested formulations, it can also be clearly observed that tmax of the reference formulation D is shorter, indicating a faster absorption rate compared to the other formulations. Elimination of the drug is slower, with a half-time of 9 hours. Table 2: Pharmacokinetic parameters of formulations A, B (n=19), C and D (n=20).
[Insert Table 2] [Insert Figure 5] Figure 5: Average plasma profiles of the tested formulations.
In order to compare the in vitro observed release profiles of the tested formulations to the observed mean plasma concentrations, in vivo absorption profiles were calculated based on Loo-Riegelmann two-compartment deconvolution method with the GastroPlusTM software. Assuming the absorption of the released drug is mostly dependent on the gastric emptying rate, dissolution experiments were compared to the first 30 minutes of the absorption profiles. As it can be observed in Figure 6, the dissolution profiles obtained with tests in AGS imitate better the in vivo absorption compared to the tests performed in USP2 apparatus. However, a lag time of approximately 5 minutes was observed in the case of in vivo absorption, therefore lag time correction was applied also to the dissolution profiles obtained in AGS. According to several research articles [35, 36], this type of correction in the process of setting up in vitro in 12
vivo correlation is recommended to be avoided in some cases. Nevertheless, the lag time correction in this study was considered to be properly justified since the in vivo observed time lag for the studied drug absorption is explained by the documented existence of an absorption window within a specific region of the small intestine. Dissolution conditions in AGS with slow gastric emptying programme (ags_GE_slow) showed the best fit to the in vivo absorption data, allocating the formulations appropriately according to the absorption profiles, from the fastest to the slowest formulation (D
Based on the present study, it may be concluded that AGS as a biorelevant model showed a better discriminatory power in revealing the differences in the drug release of the tested immediate release formulations. Furthermore, a better relation to the in vivo results compared to the conventional USP2 apparatus was also observed. The presented examples showed a significant effect of the modelled gastric emptying rate programme on the release kinetics of the tested BCS III class drug, which is also reflected. These kinds of experiments are therefore highly valuable when greater discriminatory power is needed. One such example are the immediate release dosage forms where the release rate is often so fast that it is very difficult to detect any differences between formulations using only conventional dissolution methodologies Despite the good correlation with in vivo results in the present study, it needs to be emphasized that the method should be tested on individual cases since different formulations and compounds may respond differently to in vitro and in vivo conditions. To achieve good discriminatory power, a wide array of options is available with the AGS: variation of constriction frequency and intensity, the flow through the lumen, the dissolution medium volume and composition and the possibility to monitor the dissolution for longer periods of time. Furthermore, the anatomical similarity of the model's volume and shape and the control over its mechanical and flow functions offers achieving the physiologically relevant in vitro conditions which may fruitfully support the drug development process.
13
Conclusion In this paper we present a dynamic biorelevant dissolution model – advanced gastric simulator, capable of simulating realistic gastric contractions movement and gastric emptying. The model showed the ability to precisely control the opening and closing of the “pyloric” valve. Specific movements of the constriction mechanism and the course of the containers’ outlet flow can be set for individual formulation testing. This study shows that biorelevant dissolution methods can be used as a powerful tool to predict the in vivo formulation performance and to minimize the human studies in a new formulation development process. The AGS showed better discriminating capability between all four of the tested formulations compared to the standard dissolution method with USP2 apparatus. These results will undoubtedly lead to further model optimisation and additional investigation of the novel apparatus applicability in cases where gastric emptying rate is considered to be crucial.
Declaration of competing interest The authors declared no conflicts of interest related to this article. The authors from University of Ljubljana, Faculty of Pharmacy were supported by the Slovenian Research Agency (ARRS) [P1-0189].
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Figure 1: Left: Advanced gastric simulator (AGS) with main elements: (1) “pyloric” valve, (2) constriction mechanisms, (3) step motors and (4) flexible vessel. Red arrows mark direction of the contraction movement. Right: Schematic presentation of the apparatus set-up.
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Figure 2: Real time print screen of the pylorus software displaying the course of desired and actual mass flow through the “pyloric” valve of the advanced gastric simulator.
Table 1: Parameters of prepared gastric emptying patterns. 21
ags_GE_fast
ags_GE_slow
lag time time estimated volume of volume collected desired flow estimated volume of volume collected desired flow correction* [min] media in bag [mL] [mL] [g/min] media in bag [mL] [mL] [g/min] [min] -5 -5 0 0 0 275 2,5 7,5 200 75 5 10 140 60 10 15 70 70 15 20 50 20 20 25 40 10 25 30 35 5 30 35 30 5 *arbitrarily delayed in vitro release time by 5 min
0 275 240 205 145 100 70 50 30
30 24 14 4 2 1 1
22
35 35 60 45 30 20 20
14 14 12 9 6 4 4
Figure 3: a) Intraindividual variability in gastric emptying of 240 mL room temperature water in fasted state, as observed in MRI study. Reprinted from Eur J Pharm Biopharm, 127, Grimm et al, Interindividual and intraindividual variability of fasted state gastric fluid volume and gastric emptying of water/314/2018, with permission from Elsevier. b) Gastric emptying curves of two tested programs; ags_GE_fast and ags_GE_slow: theoretical gastric emptying is marked with a dashed line and experimentally observed with a solid line.
23
Figure 4: Release profiles of the tested formulations with different dissolution methodologies: slow and fast gastric emptying (upper diagrams) and dissolution in USP2 apparatus (lower diagram).
24
Table 2: Pharmacokinetic parameters of formulations A, B (n=19), C and D (n=20).
PK parameters formulation
A
B
C
D
parameter
arithmetic mean ± SD
Median [range]
geometric mean ratio
Cmax [ng/mL]
2210 ± 430
−
83.02
AUCt [h*ng/mL]
12610 ± 2171
−
tmax [h]
−
1.33 [0.83−4.50]
−
Cmax [ng/mL]
2200 ± 536
−
82.34
AUCt [h*ng/mL]
12760 ± 2580
−
tmax [h]
−
2.50 [0.83−5.00]
−
Cmax [ng/mL]
2320 ± 574
−
86.88
AUCt [h*ng/mL]
13110 ± 2093
−
tmax [h]
−
1.67 [0.67−5.00]
Cmax [ng/mL]
2690 ± 833
−
−
−
AUCt [h*ng/mL]
13410 ± 2184
−
−
−
tmax [h]
−
1.00 [0.67−2.03]
−
−
25
formulation A vs D
formulation B vs D
formulation C vs D
94.12
94.84
97.80 −
Figure 5: Average plasma profiles of the tested formulations.
26
Figure 6: Comparison of in vitro dissolution methods with 30 minutes of in vivo observed absorption profile for each tested formulation.
27
28