Department of Pharmaceutics, Centre for Pharmaceutical sciences, University college of engineering, science and technology JNTUH, Kukatpally, Hyderabad, 500085
More than 40% of new chemical entities exhibit low aqueous solubility, which leads to poor bioavailability due to poor water solubility. Several approaches have been employed to improve the oral bioavailability of those drugs. Among them, oral lipid – based drug delivery systems have shown immense potential in improving the poor and inconsistent drug absorption of many poorly water-soluble drugs. The examples of those systems are liposomes, niosomes, self-emulsifying drug delivery systems, etc. These lipid-based formulations undergo in the gastrointestinal tract, which influences the absorption of the drug molecules. So, in vitro lipolysis models have been developed to study the fate of lipid-based formulations in the gastrointestinal tract. This review focuses on different in vitro lipolysis models, challenges and limitations.
Often, 40% newly discovered drug compounds elicit poor water solubility, which results in low oral bioavailability2. Now-a-days lipid-based formulations are used to increase the oral bioavailability of poorly soluble drugs. The lipid-based formulations can be formulated as oily solutions, emulsions, self-emulsifying drug delivery systems, microemulsions and vesicular systems like liposomes and niosomes8,43,44,51. These types of formulations are made up of pure or a mixture of triglycerides or diglycerides, surfactants and co-solvents. The mechanism beyond lipid-based formulations is increasing solubility and dissolution of the lipophilic drug by stimulating pancreatic as well as biliary secretions, increasing the GI residence time and by stimulating lymphatic transport1. The processes of lipid digestion as well as absorption in the body are the factors used to determine the oral bioavailability of lipid-based drug delivery systems3. Many events take place after intake of lipid-based formulations as they move from the mouth, stomach, small intestine, and large intestine3,4. Some of the lipid-based formulations undergo the normal digestion process, which is considered the most important release of the drug from the formulation8. Due to this, simple dissolution/dispersion tests are not suitable for invitro characterization of lipid-based formations5. So, in vitro digestion models, often called as in vitro lipolysis models, are used, which establish similar in vivo lipid digestion conditions mainly in the stomach and small intestine5,8. The crucial and most important steps in lipid digestion occur in the proximal part of the small intestine; the majority of the in vitro lipolysis models focus on the intestinal digestion3,5,7. The in vivo lipid digestion process is carried out by pancreatic enzymes, named like co-lipase, which can be simulated in vitro by using in vitro lipolysis models. After ingestion of a lipid-based formulation, intestinal lipases hydrolyse the triglycerides present in the lipid-based formulation into free fatty acids and monoglycerides; by this, the dissolved drug enters the small intestine. The bile salts and phospholipids are the major components in the medium of in vitro lipolysis models, which are mostly taken in the ratio of 4:1. Porcine pancreatin is used in the in vitro lipolysis because of having similar pancreatic enzyme composition to that of humans3,50.
2. IN VIVO LIPID DIGESTION
It is very important to understand normal lipid digestion that occurs in humans in order to develop an accurate in vitro lipolysis model. After administration of lipid drugs, they undergo several events as they pass from the mouth to the large intestine. These events may influence drug absorption.
FIG 1: Schematic diagram of physiological conditions in different regions of the human
The lipid droplets in the food and pharmaceutical formulations vary in their physical states and structural properties11,23. The lipid droplets in the food are usually surrounded by emulsifying agents, which are proteins, polysaccharides, surfactants and phospholipids25. The fate and absorption of lipid droplets depend on many of these factors26,27,28. When the lipid formulations are administered, the structure and properties of lipid droplets change when they mix with saliva. Liquids pass through the mouth very quickly, but solids require more time to swallow. The stomach is a bag-shaped organ where ingested food is stored, mixed, and a small amount is digested11. The length of time taken by the ingested material to leave the stomach and enter the intestine affects digestion as well as absorption. When food enters the stomach, then it mixes with the gastric juice and some enzymes. The pH of the stomach, which is acidic, turns into basic temporarily and after some time returns to its original pH. The gastric lipase present in the stomach breaks the triglycerides into fatty acids and glycerol11,17,22.
The small intestine is the region where most of the fat digestion and absorption takes place. Bile salts and phospholipids further break down the fat droplets into smaller ones. Pancreatic lipase is one of the important enzymes involved in the digestion of fats. It works by breaking down the triglycerides into fatty acids and monoglycerides. Co-lipase is a co-factor that helps in the working of lipase. The main functions of the large intestine are absorption of water and salts, and reabsorption of bile salts and elimination of faeces. Usually, fats are digested before they reach the colon, but some of them reach the colon if they are surrounded by protective coatings or fibres. The colon consists of bacteria that breaks downs the undigested food. These bacteria are capable of releasing the trapped lipids in dietary fibres by fermenting the dietary fibres.
3. INVITRO DIGESTION MODELS
For LBDDS, in vitro release studies are not accurate to build an in vitro in vivo correlation [IVIVC]. The main drawback of in vitro studies is unable to simulate the in vivo lipid digestion process. In vitro lipolysis models are able to simulate in vivo conditions of lipid digestion. In order to get an accurate and strong IVIVC correlation, it is very difficult to simulate human GI tract conditions like PH, enzymes, etc. No single currently developed model can simulate complex Gi conditions 4,31.
Different types of models are used, some based on focusing on one particular region of the GIT, others use sequential steps to accurately mimic the digestion process 44. These models are classified as:
3.1. SINGLE STEP MODELS
In this model, only one particular region of the GIT is simulated, such as the mouth, stomach, small intestine or colon.
EXAMPLE: PH-stat model
3.2. MULTI-STEP MODELS
In this model, two or more regions of the GIT are simulated. It is developed to mimic the complete human GIT 45,46.
FIG 2: Schematic representation multi step in vitro lipolysis model
4. PH-STAT MODEL
PRINCIPLE
This method is mainly used for evaluating the self-microemulsifying drug delivery systems [SMEDDS]. It is an analytical model mainly used for pharmaceutical and food research for the invitro characterization of lipid digestion by using conditions similar to the small intestine52. It is mainly based on measurements of the quantity of free fatty acids which are released from lipids, especially triacylglycerols, after addition of lipase at neutral PH. The sample containing which contains lipids, is placed within the temperature-controlled reaction chamber that contains simulated small intestinal fluid [SSIF]. The SSIF should contain lipase, co-lipase, bile salts, phospholipids and mineral ions, which are important digestive components involved in lipid digestion. The alkali, like NaoH is added in order to neutralise the free fatty acids produced by the digestion of lipids and thereby the initial PH can be maintained [PH:7]4,11,12,16. Then the PH change versus time was recorded. The pH-stat lipolysis model is one of the important bases for the development of other in vitro lipolysis methods15.
The % of total free fatty acids [F] as a function of time[t] are measured in this model by using the following equation:
∅=∅max (1-(1+3kMt÷2d0ρ0) −2)
Here, ∅ max provides a measure of the total extent of digestion (i.e., the maximum percentage of the total FFA present that is released at the end of the reaction), k provides a measure of the rate of digestion (i.e., moles of FFA released per unit droplet surface area per unit time), d0 is the initial droplet diameter, ρ0 is an oil droplet density, M is the molar mass of the oil. A pH stat profile can then be characterised in terms of just two parameters: ∅max and k, which can be determined by finding the values that give the best fit between the experimental data and the mathematical model4,11.
4.1. COMPOSITION OF SIMULATED SMALL INTESTINAL FLUID
The effectiveness of the lipid digestion process mainly depends on the composition of the SSIF.
4.1.1. LIPASE AND OTHER ENZYMES
Lipase is one of the major compounds used in in vitro lipolysis models. It is an important component because it simulates lipid digestion that usually occurs in the small intestine. The type and concentration of pancreatic lipase that is used in the PH stat model are very important. Various types of lipases are used till now, like pancreatin, pancreatic lipase and non-pancreatic lipases4,11,19.
4.1.2. CALCIUM AND OTHER MINERALS
Calcium is used in the PH stat model and considered an important component because it determines the rate and extent of lipid digestion4,11,21,39. Calcium determines the rate of lipid digestion by different physiological mechanisms, like calcium ions acting as the co-factor for the stimulation of lipase activity, removing the free fatty acids from the surfaces of lipid droplets4,30.
4.1.3. BILE
Bile simulates the in vivo lipid digestion process that takes place in the small intestine29. Usually, bile extract or a mixture of bile acids is used. Bile extract contains different components like phospholipids, bile acids and minerals4,11,16. So, mostly bile extract is used rather than selecting a different mixture of bile acids. As bile extract is an insoluble component, it should be filtered to carry out the analysis accurately.
4.1.4.PH
The pH of the small intestine varies depending upon several factors, like the type of food ingested, the volume of the food and the quantity of the food taken. The lipid droplets, when they are present in the stomach, are in an acidic pH. As they pass through the intestine, the pH increases4,11,18. The results of the in vitro digestion mainly depend upon the pH. The pH may affect the activity of lipase, the enzyme that plays a major role in the digestion of lipids and have effect on the ionisation of free fatty acids, which are produced after lipid digestion.
4.1.5. IONIC COMPOSITION
Calcium ions may produce long-chain fatty acids, which they react with free fatty acids that are produced after lipid digestion. It may further increase the lipid digestion, but calcium ions also lead to the formation of soap, which may decrease the lipid digestion. Some of the biopolymers, when they react with the calcium forms gel, then the digestive enzymes cannot reach the lipid droplets, therefore the digestion process decreases.
4.1.6. ENZYME ACTIVITY
There are several enzymes that are involved in the digestion, such as lipase that digests lipids, amylases that digest starch, proteases that digest proteins, etc. The ability of these enzymes to reach their specific substrate influences the lipid digestion process. When the lipid droplets are surrounded by a protective coating, fibre matrix or protein coat, the lipase reaches the lipid droplets. Sometimes, the ingested food contains enzyme inhibitors like polyphenols and soybean peptides, which inhibit the activity of the enzymes. Then the lipid absorption decreases.
4.1.7. SURFACE ACTIVE AGENTS
There are several exogenous[surfactants], endogenous[proteins]and internally generated surface-active agents surrounding the lipid droplets in the aqueous phase and compete with the surfactants that are in the water-oil interface, which may vary the interfacial properties and influence the digestion of lipids.
4.1.8. OTHER FACTORS
Lipids are normally ingested in the normal diet along with carbohydrates, proteins, fibres, etc. These components may influence the digestion of lipids and may increase or decrease the rate of lipid digestion4,20,40.
Table 1: Proposed standardised pH-stat method for testing emulsified lipids using the in vitro digestion model under fed state conditions.
|
Experimental parameter |
proposed value |
|
PH Reaction cell volume Temperature Stirring speed [NaOH] in the titration unit Lipid content in the reaction cell NaCl in the reaction cell CaCl2 in the reaction cell Bile Extract in the reaction cell Pancreatin (100–400 units/mg protein) in the reaction cell
|
7 37.5 mL 370C 4 s-1 0.1mM 300 mg 150 mM 10 mM 20 mg/mL 2.4 mg/mL |
5. ONE-COMPARTMENT INTESTINAL DIGESTION MODEL
This model includes the thermos stated vessel, generally maintained at 37?, stirrer, PH electrode an a titrator. LBFs are dispersed in the medium, simulating the in vivo intestinal conditions. The lipid digestion starts after the addition of lipase and colipase and leads to the formation of fatty acids, then the PH starts. The change in the PH is measured using the electrode, and the liberated fatty acids are titrated with sodium hydroxide using the titrator. The extent can be quantified based on the rate of addition of sodium hydroxide. The samples can be taken during the digestion process, and the three phases of lipid digestion can be determined. That is the oil phase containing the lipids that are undigested, micelles consisting of the digested lipid droplets and the pellet phase containing the precipitated drug. Determining the amount of drug in three phases predicts the amount of solubilised drug. The amount of the drug in the micelle phases can be correlated with the pharmacokinetic parameters4,32,15.
FIG 3: Schematic representation of a one-compartment PH-stat model
6. GI DIGESTION MODEL
The one-compartment model is one of the simplest and most widely used methods for evaluating the LBFs. The model is based on the principle that the small intestine is the main site for the digestion of lipids and drug absorption. But the model cannot mimic GI physiology and stomach conditions4,11,34. Lipid digestion in the stomach contributes to 15% of the overall lipid digestion in the
FIG 4: Schematic representation of a two-compartment PH-stat model
GIT. So, GI digestion PH-stat models, either two-step one-compartment or two step two compartments, are developed in order to simulate both stomach and intestinal conditions. In the one-compartment model, the simulated gastric and intestinal digestion was carried out in two steps. First, the LBFs are dispersed in stimulated gastric fluid, and gastric digestion is initiated by adding gastric lipase. After some time, intestinal conditions are mimicked by the addition of a concentrated simulated gastric fluid and pancreatic lipases4,11. During the two sequential steps, automatic titration with sodium hydroxide is carried out, in order to maintain a constant PH simulating the gastric and intestinal PH. Two individual setups are used in the two-compartmental model for stomach and small intestinal conditions. The simulated gastric and intestinal fluids, along with respective lipases, are added to the two reaction vessels and connected with the peristaltic pumps40. During the process of digestion, the medium used in the gastric compartment is pumped to the intestinal compartment to simulate the gastric. Therefore, the two-compartment model is considered too closely simulate the in vivo conditions compared to the one-compartment model 35.
7. COMBINED MODELS AND IVIVC
In vivo lipolysis cannot predict the oral bioavailability of LBFs because models cannot mimic the in vivo conditions accurately. These models cannot maintain the absorption sink conditions. This leads to the supersaturation and drug precipitation. Meanwhile, in vivo absorption results in an immediate and sufficient drop in the luminal drug concentration and prevents precipitation. In vivo conditions, in addition to the absorption issue, the absorbed drugs may also undergo first-pass metabolism. In these conditions, the in vitro models can predict a higher amount of solubilised drug. Therefore, combined lipolysis, permeation and digestion-microsomal metabolism models were developed, in order to obtain a better IVIVC4,37.
8. IN VITRO LIPOLYSIS–PERMEATION MODEL
FIG 5: Schematic diagram of in vitro lipolysis-permeation model
The original setup of this model consists of two separate single compartments. The lipolysis and permeation were performed in a sequential manner. Dispersion and digestion of LBFs were performed in a single compartment by using the PH-stat lipolysis model4,38. At regular intervals, samples were collected and transferred to the second compartment for the permeation studies. The absorptive membrane should mimic the intestinal epithelia, and under the conditions of lipolysis, pancreatic enzymes, surfactants and excipients of LBFs. Permeability through the CaCo2 monolayer is considered to be the gold standard for evaluating the oral drug absorption. Differentiated CaCo2 cells resemble the epithelial layer of the intestine, which helps to predict the drug transport mediated by different pathways. Because of the intolerance of CaCo2 cells to the pancreatic enzymes, immobilised lipase was replaced, and this showed a positive result4
9. CHALLENGES
The human digestive system is a very complex process, and it is difficult to mimic those conditions in vitro. After administration of the LBFs, the lipid molecules undergo various physical and chemical changes, which include the breakdown of the lipid into smaller lipid droplets. So, during the development of any in vitro models, it is very important to consider the GIT physiological conditions47,48.
The in vitro lipolysis models vary according to their complexity and how well they resemble the in vivo conditions. In case of simpler models like one- compartmental models, the focus is only on the rate-limiting step that is drug absorption. But simultaneously, different processes occur, like drug release from a supersaturated system and permeation. The complex in vitro models should take into account all of these and need to simulate all the simultaneously occurring processes. In vitro models are used to predict the in vivo behaviour of the drug as closely as possible. Even though the in vitro models require low labour, rapid and cheap, due to the complexity of the human body, there is no perfect in vitro model to predict the in vivo drug performance. The selection of an in vitro model is based on the knowledge of the human body, the type of drug delivery system and the physicochemical characteristics of the drug molecule14.
10. FUTURE ASPECTS
Previously, most of the in vivo studies were carried out using rats. But there are several differences between gastrointestinal conditions in rats and humans. So, now in vitro lipolysis models, which mimic the physiological conditions of humans, are used to predict in vivo performance of lipid-based formulations2. These models eliminate number of animal studies required49. The current in vitro lipolysis models are known to predict the total amount of drug, but cannot differentiate between free drug and the solubilised drug. Particularly, when it comes to BCS-4, it is difficult evaluate the contribution of lipid-based formulations for the absorption of drug molecules41,42. To resolve this problem, tools like fluorescent probes and Forster resonance energy transfer probes must be developed. The fluorescence probe works by exhibiting different signals or intensities before and after the breakage of the formulation. These types of probes are widely used in quantitative and qualitative analysis, and should be taken forward to analyse the lipid-based formulations. Development of this technique provides valuable information for the design of lipid-based formulations15.
CONCLUSION
Various in vitro lipolysis models are developed in order to estimate the in vivo performance of LBFs.Due to the complexity of physiological conditions of GIT, no single in vitro lipolysis model can simulate the in vivo conditions. This article provides an overview of in vitro lipid digestion process and critical insights about current in vitro lipolysis models, particularly emphasizing on PH-stat model and the influence of SSF. Furthermore, the article includes the major challenges faced during the development of any model. And the last, future perspectives are included to predict the in vivo performance of LBFs accurately.
REFERENCES
Shaik Nushrath Bhanu, Dr. K. Anie Vijetha, Dr. M. Sunitha Reddy, In vitro Lipolysis Models for Evaluation of Lipid-Based Drug Delivery Systems: A Comprehensive Review, Int. J. of Pharm. Sci., 2026, Vol 4, Issue 2, 4481--4492. https://doi.org/10.5281/zenodo.18798755
10.5281/zenodo.18798755
