Biorelevant dissolution models to assess precipitation of weak base drug

The impact of precipitation can affect the amount of drug absorbed, thereby affecting the amount of drug in the systemic body. The precipitation process is preceded by a supersaturation phase, caused by decreased drug solubility in the gastrointestinal tract. This precipitation occurs for weak base drugs with low solubility. When the drug entering the small intestine, the solubility of weak base drugs decrease, then occurs supersaturation, which leads to precipitation, so that drug precipitation is one of a challenge for the pharmaceutical industry in drug development. Precipitation testing of water-soluble weak base drugs can be carried out by the pH shift method to describe the gastrointestinal pH gradient from gastric to small intestine. This pH change can cause supersaturation and then trigger precipitation, especially for weak base drugs. The methods that can used to assess precipitation drug is modi(cid:976)ication of the USP dissolution which are two compartment and multi compartment model. The choice of dissolution medium play an important role in the test results. The use of bio relevant medium can produce closer in vitro and in vivo correlations than the use of buffers. Generally, the medium used to simulate the weakly condition in the small intestine is FaSSIF (Fasted State Simulated Intestinal Fluid) or FeSSIF (Fed State Stimulate Intestinal Fluid) medium. The medium used to simulate the acidic condition in the stomach is FaSSGF (Fasted state simulated gastric (cid:976)luid) or FeSSGF (Fed state simulated gastric (cid:976)luid) medium.

The impact of precipitation can affect the amount of drug absorbed, thereby affecting the amount of drug in the systemic body. The precipitation process is preceded by a supersaturation phase, caused by decreased drug solubility in the gastrointestinal tract. This precipitation occurs for weak base drugs with low solubility. When the drug entering the small intestine, the solubility of weak base drugs decrease, then occurs supersaturation, which leads to precipitation, so that drug precipitation is one of a challenge for the pharmaceutical industry in drug development. Precipitation testing of water-soluble weak base drugs can be carried out by the pH shift method to describe the gastrointestinal pH gradient from gastric to small intestine. This pH change can cause supersaturation and then trigger precipitation, especially for weak base drugs. The methods that can used to assess precipitation drug is modi ication of the USP dissolution which are two compartment and multi compartment model. The choice of dissolution medium play an important role in the test results. The use of bio relevant medium can produce closer in vitro and in vivo correlations than the use of buffers. Generally, the medium used to simulate the weakly condition in the small intestine is FaSSIF (Fasted State Simulated Intestinal Fluid) or FeSSIF (Fed State Stimulate Intestinal Fluid) medium. The medium used to simulate the acidic condition in the stomach is FaSSGF (Fasted state simulated gastric luid) or FeSSGF (Fed state simulated gastric luid) medium.

INTRODUCTION
Gastrointestinal pH plays an important role in the solubility of ionized drugs. The solubility of drugs with weak bases and weak acids depends on the gastrointestinal pH. Weak acid drugs easier dissolved in base pH, while weak base drugs easier dissolved in acidic pH. In weak base drugs, although the solubility is high at the pH of gastric acid, only a few drugs are absorbed in the stomach because the drug is mostly absorbed in the small intestine due to a wider intestinal surface area. This plays an important role in the amount of drug absorbed so that it affects the bio availability of the drug (Kostewicz et al., 2004). If the weak base drugs with poor solubility go into intestine, their solubility will decrease drastically and then undergo supersaturation. Supersaturation can potentially lead to precipitation (Kambayashi and Dressman, 2019). Drug precipitation is the result of nucleation and crystal growth as a thermodynamically undesirable phase when supersaturated. The separation process usually referred to as crystallization is divided into 2 stages, including the nucleation stage and the crystal growth stage. In the irst stage, the molecules of the solute particles accumulate then con igurate to small nuclei bodies in the solution. Aggregation of nuclei bodies causes their size to be larger than the critical size, then initiation of crystal growth (Kumar and Sureshkumar, 2020;Singh et al., 2019).
Drug precipitation is one of the dominant factors causing decreased drug bio availability. It is necessary to carry out tests to predict drug precipitation in the gastrointestinal tract. Generally, testing method of drug precipitation is carried out using dissolution methods (Dai, 2010;Rubbens et al., 2016). When designing in vitro tests for supersaturation evaluation, it is necessary to attention about the various test conditions because they can play a signi icant role in the results of supersaturation testing. Media that are usually used such as buffers do not represent the pH of the stomach or intestines because they are not biorelevant with the gastrointestinal composition, such as osmolarity, ionic strength, buffer capacity, viscosity, bile and pancreatic secretion, and facial tension. Media that biorelevant with stomach is FaSSGF (Fasted state simulated gastric luid) or FeSSGF (Fed state simulated gastric luid) media, while biorelevant wiht intestinal is FaSSIF (Fasted State Simulated Intestinal Fluid) or FeSSIF (Fed State Stimulate Intestinal Fluid) media (Klein, 2010).

Supersaturation of Weak Base Drugs
Supersaturation is a thermodynamically unstable state in the presence of an increase in the concentration of free drug in solution above the saturation solubility value (Kaur et al., 2018). Supersaturation can be occur by co-solvents in liquid preparations, meta stable form in solid form such as salt or amorphous form, and decreased solubility of weak bases caused by a shift in pH when the drug from the stomach enters the small intestine (Carlert et al., 2014). Several techniques can be used to create supersaturation conditions, there are removing solvents, dissolving the meta stable solid phase, changing temperatures, changing pH, and adding solvents to reduce solubility (Warren et al., 2010). Supersaturation that occurs in the gastrointestinal tract can be explained in 2 cases. The irst case is supersaturation occurs in weak drugs which ionize in an acidic environment, then when it enters the small intestine occurring the pH shift to higher pH so that the degree of ionization and equilibrium solubility have decreased.
The second case is supersaturation occurs by lossing of drug solubilization capacity (Warren et al., 2010). The presence of food can contribute to increase signi icantly in solubility as long as the drug is in the gastrointestinal tract. The concentration of bile components containing bile salts and lecithin will increase when food is present. Solubility is increased through micellar solubilization or increased wetness of the intestinal contents (Kostewicz et al., 2004). Weak base drug interactions with drugs that inhibit gastric acid secretion, such as Proton Pump Inhibitor (PPI) drugs, H2 antagonists, and antacids can decrease the solubility of weak base drugs (Kambayashi and Dressman, 2019).
An increase in gastric acid pH can decrease drug solubility, the strength of force absorption, and intestinal supersaturation (Rubbens et al., 2016). Super-saturated drug preparations are effective for increasing the absorption of oral drugs with poor solubility. However, the supersaturation phase is thermodynamically unstable so that the drug has the potential to undergo precipitation as a result of changes in physiological conditions (Kataoka et al., 2019). A higher degree of supersaturation has a greater risk of precipitation. The degree of supersaturation is de ined as the ratio between the activity of the drug dissolved in the supersaturated solution compared to the activity of the drug in the saturated solution of the thermodynamically stable form (Blaabjerg et al., 2018).
Several strategies can be used to inhibit precipitation by selecting excipients that inhibit precipitation, such as dissolved polymers (for example: HPMC, PVA, PVP, Eudragit) surfactants (for example: Tween, SDS, Span) or cyclodextrin derivatives (Taupitz et al., 2013). Saturated drug formulas such as salt form or amorphous solid dispersions are suitable for amorphous or polymorphic precipitated drugs because they increase solubility so that absorption is also increased. For drugs that are crystalline precipitated from the super saturation phase, a controlled release formulation is an appropriate strategy to increase drug absorption by keeping the small intestine concentration below the nucleation critical concentration. The use of watersoluble polymers can form a super saturation phase which can also inhibit precipitation (Kataoka et al.,

2019
). An understanding of the small bowel super saturation and precipitation of weak base drugs is important for predicting the pharmacokinetic proile of the drug (Kaur et al., 2020).

Drug Precipitation
Drug precipitation is formed from nucleation and crystal growth as a thermodynamically undesirable supersaturated phase. The separation process usually referred to crystallization, is divided into 2 stages, which are the nucleation stage and the crystal growth stage. The irst stage is the molecules of the solute particles accumulate and form small nuclei bodies in the solution. Aggregation of the nuclei body causes its size to be larger than the critical size, then initiation of crystal growth occurs. The second stage is crystal growth, the molecules are periodically organized to form a crystal skeleton (Kumar and Sureshkumar, 2020;Singh et al., 2019). The two stages occur simultaneously which are in luenced by physical conditions. The rate and mechanism of crystallization are determined by the solubility of the solute, degree of supersaturation, speed of supersaturation, diffusivity, temperature, and reactivity of the nucleation surface (Warren et al., 2010). Precipitation can be caused by a drastic change in pH, dilution of the drug preparation in body luids, or digestion of the solubilized excipient in the formulation. The molecular structure of active substance has tendency to crystallize of the drug. The crowded drug structures with many rotating bonds are usually slow to crystallize, whereas rigid structures are usually quick to crystallize (Raina et al., 2015). The precipitates form as a crystalline or amorphous material. The precipitation mechanism in crystalline and amorphous materials is generally the same, the difference is only in the natural packaging of the solid material that is formed (Warren et al., 2010). Drug precipitation can reduce the drug concentration in the aqueous phase thereby reducing the amount of drug that will be absorbed into the systemic circulation. The reduced amount of absorption will decrease the AUC of the drug as well as the ef icacy of the drug (Dai, 2010). Kinetics of precipitation have a various result depending on the physiological conditions and physical and chemical properties of the drug.
Physiological conditions, such as gastric acid pH, time of residence in the stomach, gastric emptying time, pancreatic secretion, bile secretion, gastrointestinal transfer, hydrodynamics, presence or absence of food in the gastrointestinal tract, while the chemical-physical properties of drugs, such as pKa, solubility, molecular structure, velocity dissolution, and disintegration (Bevernage et al., 2013;Hens et al., 2016). Drug precipitation from supersaturated solutions can be accelerated by increasing degree of supersaturation, increasing solubility, decreasing temperature, decreasing solution viscosity, and decreasing interface stress. If the rate of precipitation is slow during the drug absorption process, there is no precipitation (Warren et al., 2010).

Biorelevant Medium
The choice of dissolution media plays an important role in the test results. Media that are usually used such as buffers do not represent the pH of the stomach or intestines because they are not biorele-vant with the gastrointestinal composition, such as osmolarity, ionic strength, buffer capacity, viscosity, bile and pancreatic secretion, and facial tension. The use of this biorelevant medium resulted in closer in vitro and in vivo correlations than the use of buffers (Klein, 2010).

SIF (Simulated Intestinal Fluid) Media
Generally, the media used for simulating base conditions in the small intestine is FaSSIF (Fasted State Simulated Intestinal Fluid) or FeSSIF (Fed State Stimulate Intestinal Fluid) media Table 1. FaSSIF was developed to stimulate fasting conditions in the proximal small intestine. This medium contains leticin as a representation of bile salts and phospholipids which facilitate to wet solids as well as the solubilization of lipophilic drugs into the mixed micelle. Sodium taurocholate is chosen as the representation of bile salts because cholic acid is one of the bile salts in human bile. The phosphate buffer serves to avoid instability in the pH value. NaCl functions to achieve conditions close to iso-osmolar (Dressman and Reppas, 2000;Klein, 2010).
The presence of food causes hydrodynamic changes and intralumenal volume. After the entry of solids, the pH of the chyme decreased while the buffer capacity and osmolarity increase. The dissolution medium that re lects the condition of the small intestine when eating is FeSSIF. The bile salt and leticin content in FeSSIF media were higher than in FaSSIF media. The acetate buffer functions to adjust the desired pH to keep the pH value low while the buffer capacity and osmolarity remain high. The content of sodium taurocholate and leticin is higher in FeSSIF than in FaSSIF as a representation of the response of bile to food intake (Dressman and Reppas, 2000;Klein, 2010). FeSSIF does not contain lipolysis products so together with bile it can increase solubility of poor soluble drugs. Data in humans shows that the pH in the upper small intestine decreases slowly after food is entered so that a review is needed to obtain better predictions of in vivo data (Jantratid et al., 2008).

SGF (Simulated Gastric Fluid) Media
The media used to simulate the gastric atmosphere is FaSSGF (Fasted state simulated gastric luid) or FeSSGF (Fed state simulated gastric luid) media Table 2. FaSSGF contains pepsin and a small amount of bile salts and leticin. This medium is more suitable for physiological conditions because the decrease in facial tension in the media is caused by the presence of pepsin like physiological conditions (Jantratid et al., 2008). The use of FaSSGF is highly recommended for in vitro dissolution testing.
However, this volume does not represent the volume of stomach acid in the fasted state. The volume in the stomach is estimated to be 200-300 ml, but it is dif icult to obtain reproducible results on dissolution tests using the standard USP apparatus with too little volume. Therefore, it is advisable to use a "mini paddle" for volumes less than 300 ml (Klein, 2010).
The condition of the stomach containing food is very dependent on the composition of the food being digested. The ideal medium that represents the condition of the stomach at a mealtime should be similar to the nutritional and physical chemical properties of food. There are two alternative media that approach the condition of the stomach when eating, namely milk containing 3.5% fat and Ensure Plus. Both media have physical and chemical properties similar to the food standards recommended by the American HHS-FDA for the study of the effect of food on the bioavailability-bioequivalence test (Jantratid et al., 2008). Ensure Plus is closer to the chemical properties of FDA breakfast standards, whereas milk is more suitable for simulating a low-fat breakfast. However, both media still have drawbacks related to pH or pepsin (Klein, 2010).

Modi ication of Dissolusion Methods
Precipitation testing on water-soluble weak base drugs can be carried out by the pH shift method to describe the gastrointestinal pH gradient from gastric pH to small intestine pH. This process of pH changes that can cause super saturation and trigger precipitation, especially for immediate reaction weak base drugs. The pH shift method can test the supersaturation potential of ionized drugs. The shift in pH can decrease the solubility of ionized drugs according to the acidic or base properties of the drug. Acidic drugs are more soluble at base pH, while base drugs will dissolve more in acidic pH. Compared to the solvent square method, this method is more biorelevant for weak base drugs that are transferred from the stomach to the small intestine. Then there is supersaturation due to decreased solubility of weak base drugs. The pH shifting can be performed with one compartment or multiple compartments (Bevernage et al., 2013). Modi ications of the USP dissolution method have been carried out to obtain results that are close to physiological conditions. This modi ication can be a two-compartment or a multi-compartment model.

Two compartment model
The two compartment method is designed to simulate the condition of the stomach and small intestine. This method uses USP II apparatus based on pH shift with 2 compartments, which are the donor com-   (Kostewicz et al., 2004). The drug solution in the donor compartment is transferred to the acceptor compartment, then the drug precipitates at the acceptor phase are evaluated. The speed of the paddle on the vessel represents the motility of the small intestine upon drug precipitation. In this system, the precipitation is evaluated without considering the absorption of the drug. For drugs that have rapid absorption, precipitations can have overestimated result (Dai, 2010). The drug in the donor compartment is dissolved in a gastric biorelevant medium such as FeSSGF or FaSSGF. This is then transferred to an acceptor compartment containing biorelevant medium with the small intestine such as FeSSIF or FaSSIF.
Transfer of solution from the donor compartment to the acceptor can use a peristaltic pump. Transfer rate represents the low rate from the stomach (Dai, 2010). The other way is pouring the solution of drug directly from the donor compartment to the acceptor, or vice versa. Kambayashi, 2016 and 2019 (Kambayashi et al., 2016;Kambayashi and Dressman, 2019) tested dipyridamole precipitation in FaSSIF-V2 with a 2-stage dumping setup Figure 1a. The ketoconazole dissolution test (200 mg Nizoral Tablets) was carried out by Ruff, 2017(Ruff et al., 2017 in FaSSGF-V2 and FaSSIF-V2 using the mini vessel method and USP II Apparatus with a peristaltic pump transfer system Figure 1b. Berlin, 2014(Berlin et al., 2014 conducted a dissolution test of cinnarizine tablets using 30 mg Cinnarizine tablets, 20 mg Arlevert (Cinnarizine / Dimenhidrinat) and 25 mg Stugeron (Cinnarizine) transfer systems with a peristaltic pump transfer system in FaSSGF and FaSSIF-V2 media Figure 1c.

Multi compartment model
The multi-compartment method is designed as closely as possible to the gastrointestinal tract, there are 4 compartments, which are the gastric, small intestine, absorption/sink, and reservoir compartment Figure 2. The multi-compartment model is more advantageous than the single compartment. The multi-compartment method not only can evaluate drug precipitation directly, but also can estimate the precipitation potential to diagnose whether the precipitation contributes to bio availability. This method also obtains results that are closer to in vivo simulations and more ef icient than conventional dissolution methods (Dai, 2010). The dissolution test of a formula that has the potential to be supersaturated and precipitated can use the USP I or II apparatus method. The dissolution method of USP I or II has been widely used for the study of dissolution or precipitation of oral drugs under different parameter conditions, such as dissolution medium, dissolution volume, stirring rate, and hydrodynamics (Dai, 2010). There are many advantages of using the USP apparatus, namely that it provides robust results, is easy to operate, and is widely available (Klein, 2010).
It should be noted that several factors contribute to dissolution and absorption. These factors include: physical and chemical properties of drugs, bio pharmaceuticals, and physiological conditions. In order to develop a model that has a good correlation between in vitro and in vivo, it is important to consider these factors. Physical and chemical factors affect the dissolution of drugs including solubility, environmental pH, salt form, and particle size. Bio pharmaceutical factors are represented from the permeability of the drug, the non-ionized form of the drug is easier to permeate than the ionized form. Partition coef icient value (Log P) which has good permeability is between 0 and 3. Physiological conditions that need to be considered is the pH in the gastrointestinal tract. A pH gradient from a low pH in the stomach (1 to 2) to a high pH in the intestinal (5 to 8) can change the solubility, dissolution, stability, and permeability of the drug entering. Apart from pH, other physiological factors are gastric emptying time and existence of food (Lu et al., 2011).

Case Study
Dipyridamole Kambayashi, 2016(Kambayashi et al., 2016 tested dipyridamole precipitation in FaSSIF-V2 with a 2stage dumping setup. The dipyridamole precipitation pro ile was faster at higher dipyridamole concentrations. The result shows that the predicted total and dissolved drug concentration curves have similar results to the in vivo pro ile so that the test method could be used to predict drug precipitation in the small intestine, particularly for early development. Kambayashi, 2019 (Kambayashi andDressman, 2019) conducted another dissolution test of dipyridamol (Persantine tablets) used the same method. Persantine tablet dissolved in FaSSGF about 80% for 30 minutes, whereas in FaSSIF-V2 only 9% dissolved after 1 hour.
The dipyridamol precipitation test (2x Persantine 25 mg tablets) in various acidic pH was carried out by Gu, 2005(Gu et al., 2005 used a multi compartment method with a peristaltic pump transfer system. The dissolution rate of dipyridamole is higher at pH 1.2 than at pH 2.0. Dipyridamol was easier to dissolve at a more acidic stomach pH. Dipyridamol was dissolved at both pH for 25 minutes. The rate of drug transfer from the stomach compartment to the intestine was slower at pH 2.0, whereas the amount of drug transferred to the absorption compartment at pH 1.2 and 2.0 was almost the same. An experiment was also conducted on stomach acid with a pH of 5.9 using famotidine to increase the pH of stomach acid. The test results of the multi compartment method showed exceeds the estimate result than in vivo test, but was still closer than the conventional dissolution method.

Ketoconazole
The ketoconazole precipitation test (Nizoral 200 mg tablets) was carried out by Ruff, 2017(Ruff et al., 2017 in FaSSGF-V2 and FaSSIF-V2 media used the mini vessel method and USP II Apparatus with a peristaltic pump transfer system. In FaSSGF-V2 medium more than 80% dissolved after 20 minutes and completely dissolved after 45 minutes, whereas in FaSSIF-V2 media only 2% of the drug was dissolved. Ketoconazole at a higher concentration results in a faster precipitation rate. If it compared with dipyridamole, ketoconazole had a higher degree of supersaturation (the ratio of initial solute concentration to saturation solubility) is higher. This was probably due to the pKa value of ketoconazole (pKa 2.9) which was lower than dipyridamole (pKa 5.7-6.4) so that it had a greater potential to change into a non-ionized form and then its solubility decreased at neutral pH (Kambayashi et al., 2016).

Cinnarizine
The precipitation test for cinnarizine tablets used Cinnarizine 30 mg, Arlevert 20 mg (Cinnarizine / Dimenhidrinat) and Stugeron 25 mg (Cinnarizine) tablets was carried out by Berlin, 2014(Berlin et al., 2014 with a peristaltic pump transfer system. In FaSSGF media pH 1.6, Arlevert tablets were dissolved for 15 minutes, while Stugeron tablets were dissolved for 30 minutes. In FaSSIF-V2 media both tablets were slowly dissolved, releasing only 5.6% and still continued to reach the solubility value after 240 minutes. Arlevart tablets released as much as 47% in FeSSGF media after 240 minutes, whereas cinnarizine tablets 30 mg were significantly slower to release only 27% in 240 minutes. In FeSSIF-V2 medium, both Arlevart and Cinnarizine 30 mg tablets had greater dissolution than in FeSSGF media. Cinnarizine tablets have a lower percentage dissolution value than Artlevart. The cinnarizine precipitation test (cinnarizine tablets 25 mg and 50 mg) with various acidic pH was carried out by (Gu et al., 2005) multi compartment method with peristaltic pump transfer system. The dissolution rate of cinnarizine is higher at pH 2.0 than at pH 5.0. Cinnarizine dissolved faster at a more acidic stomach pH. Cinnarizine was completely dissolved at pH 2.0 for 30 minutes. When compared the dissolution pro ile between the multi compartment method and the conventional dissolution test method there were differences. The dissolved concentration at pH 2.0 used the multi compartment method was 4.6x higher, whereas with the conventional dissolution method it was 57x higher. The results of the dissolution used the multi compartment method were closer to the in vivo data. Therefore, the multi compartment method was more accurate to estimate cinnarizine concentrations with different gastric pH. This could be due to the multi-compartment simulation method closer to the actual physiological conditions, where the volume of media in the gastric compartment has decreased into the intestinal compartment, while the volume in conventional dissolution was constant or unchanged.

CONCLUSIONS
The dissolution test of a formula that has the potential to precipitate can use a modi ied USP II apparatus method. The dissolution method has been widely used for the study of dissolution or precipitation of oral drugs under different parameter conditions, such as dissolution media, dissolution volume, stirring rate, and hydrodynamics. There are many advantages of using the USP apparatus, that it provides robust results, is easy to operate, and is widely available. The use of bio relevant media resulted in closer in vitro and in vivo correlations than the use of buffers. The bio relevant medium for the stomach is FaSSGF / FeSSSGF, while the bio relevant medium for the intestine is FeSSIF / FeSSIF.