Development of Simulated Intestinal Fluids - Research Paper Example

Introduction Caco-2 cell culture model is an established in vitro intestinal epithelial model which has been used for studying the absorption of numerous biomolecules and drugs. It is a cell line obtained from human adenocarcinoma cells in the intestinal epithelium. Previous studies have shown that Caco-2 is a valuable model to elucidate mechanisms underlying membrane traffic and polarity in epithelial cells and a very successful model to study the mechanisms of absorption of water soluble drugs. Aim & Summary of the Research Paper The authors in the present study have endeavored to develop an ideal transport medium containing appropriate nutrients, which could support the Caco-2 cell culture model thereby facilitating the transport of traditionally hydrophobic molecules like estradiol and etoposide. The study seems well designed and original. It has built upon previous work on the facilitation of lipohilic drugs solubility on the Caco-2 cell models by incorporation of agents like dimethyl sulfoxide (DMSO), ethanol, cyclodextrins and surfactants. The reason cited by the authors for this study is the fact that a large number of recently discovered drug molecules and pharmaceuticals are lipohilic. Just by diluting the lipohilic molecules by the agents described above disturbs the normal physiological environ of the in vitro model which has to be as identical to the in vivo conditions as possible, and can lead to misleading interpretations because the normal physiological status in the living cells is entirely different and needs to be emulated precisely to provide accurate analysis in such studies. This factor has been given prime importance by the authors while undertaking this study. Previous Work The authors have based the study on past attempts to simulate intestinal fluids artificially by addition of bile salts, phospholipids and/or fatty acids. They have analyzed the previous studies well and the paper elaborates upon the various approaches tried and the success or failures thereof in the introduction itself. They have also taken into consideration the altered normal physiology of the digestive tract in the pre and post prandial states. The presence of different levels of digestive enzymes in these two states has been shown to be an important aspect while developing in vitro models. Simulated Intestinal Fluids (SIFs) with varying composition were studied for their effect in maintaining cellular integrity, the production of Lactate dehydrogenase in Caco-2 cell cultures and by measuring the cellular protein content. The authors have built upon this study based on the idea of development of dissolution fluids in 1998, which were biorelevant to the dissolution of drugs, and mimicked the in vivo conditions. These were the fasted state simulated intestinal fluid (FaSSIF) and fed state simulated intestinal fluid (FeSSIF). Since then, these two have then been modified in various ways to suit the Caco-2 cell line model. The authors have also justified the use of lipolysis products as an addition to the ideal SIF for this model, to mimic the post prandial state. Bile salts are also a normal constituent of the intestinal content in the in vivo conditions, but in in vitro conditions, they had exerted toxicity on the Caco-2 cell cultures. The authors have suggested adding nutrients to the SIF in order to overcome this problem. Critique Keeping all relevant facts in view the study is an approach aimed at providing the ideal in vitro model for study on lipid soluble compounds. The facts have been presented well and the major historical research in this area taken into consideration. The experiments have been planned and executed in consistency with the currently available technology for such a study. The sourcing of the biochemicals, cell lines, etc has been from renowned companies and institutions which are known for their rigorous standards and quality (Sigma-Aldrich, USA, Applichem, Germany, Unikem, Denmark, ATCC, USA, etc.). The instruments used are of high standard leaving little margin for error. (Spectrophotometer from Multiskan, Finland, Liquid Scintillation Analyzer from Canberra Packard, Germany, Hewlett Packard HPLC system, etc.). The paper may however be unintelligible to the general reader who has not been exposed to scientific nomenclature and methodology. To be intelligible to the general reader, it has to shed the scientific nomenclature and translated into layman language with the use of general terms which a normal person encounters in his daily exposure to newspapers, magazines and other media. Other Studies relevant to this model There is active current research in this area and other authors have quoted the multifarious utilities of the Caco-2 cell culture in understanding the mechanisms of drug metabolism in in vitro conditions. Some of the other studies have explored the use of this model to study food iron availability (Garcia et al, 1996), effects of ascorbic acid and polyphenolic compounds on iron bioavailability (Yun et al, 2004), regulation of digestion in cell culture (Zweibaum A. et al, 1991), for the characterization of intestinal absorption of antibiotics (Biganzoli E. et al, 1999) and patterns of peptide transportation in the intestines (Herrera-Ruiz D. et al, 2001). It is thus well apparent that this model is an important tool in studying the mechanisms of metabolism and absorption of a wide variety of compounds. Summary The present study is very methodical in approach and a thorough effort has been made to eliminate any errors by rigorous testing of the cell culture media for viability in the presence of SIFs with a wide variety of compositions. All matters pertaining to the osmolality of the solutions, their pH and composition have been ensured to deliver the perfect values in accordance with the naturally occurring environment of the intestines in a living individual. The authors have justified the use of osmolality values maintained in the SIFs used as being within the limits of similar values in in vivo conditions which fall within the range of fasted and fed conditions in the living organism. The composition of different SIFs has been thoroughly tested and each component’s percentage accurately determined. The tabular representation of the facts is precise and comprehensible to the reader. Incorporation of antibiotics to the media is also at a previously approved level which increases the viability of such in vitro solutions. The statistical design used for the study has also been quoted and is an approved method of arriving at a correct analysis (Sigma Stat for Windows ver.3.5, Systat Software, USA) has been used. Advanced technique of transepithelial electrical resistance (TER) measurement has been used and applied correctly to determine the viability of the cell cultures. Cellular protein assays have also been done by approved methods. Similarly the measurement of lactate dehydrogenase has been done by using an approved protocol. This has ensured the actual experiment of the transport of estradiol and etoposide to be run in as near natural conditions as possible outside the body. The authors have studied the transportation of the lipid soluble molecules by playing with the composition of the SIFs and arrived at a definite conclusion due to the multiple experiments conducted keeping the cell viability constant throughout. Variations in the composition have definitely lead to changes in the solubility as well as transport of both estradiol and etoposide, their binding to the micelles, availability in the unbound state, and decrease in their intracellular presence leading to enhanced recovery. Results The experiments have identified the most appropriate SIF which should be used for such in vitro studies. According to the study, Leibovitz’s L-15 nutritional medium containing 5mM TC (Sodium taurocholate) and 1.25mM lyso-PC (Lysophosphatidylcholine) is the most ideal medium for such in vitro studies. Addition of lipolysis products to this medium does not affect the viability of Caco-2 cells and increases the physiological relevance of the SIF as compared to the previous media used for such in vitro models. Further Possibilities The authors of this study have been very particular in citing the literature to date in this area of research and have built the work upon other similar studies. Their emphasis on studying lipid soluble molecules like estradiol provides a stepping stone for similar studies using more such molecules which are being produced by the dozen in current advances in therapeutics. Availability of an in vitro model becomes all the more necessary as animal experimentation is being discouraged worldwide. However such pharmacokinetic studies are more accurate in in vivo models or live animal experimentation because in the living state, the dynamics of the on going normal physiological processes keep changing the environment of the organs on which the therapeutic or other natural molecules are acting. The blood flow and lymphatic drainage also play crucial roles in continuously altering the physiological status in a living organism. Such a condition is almost impossible to mimic in an in vitro model. The authors have given a very balanced view of study in this area. They have tried to add more physiologically relevant components to the already proven media developed for the Caco-2 cell cultures for better in vitro studies. References: 1. Elia Biganzoli1, Luigi A. Cavenaghi2, Roberta Rossi, Maria C. Brunati and Maria L. Nolli Use of a Caco-2 cell culture model for the characterization of intestinal absorption of antibiotics Il Farmaco Volume 54, Issue 9, 30 September 1999, Pages 594-599 2. Herrera-Ruiz D, Wang Q, Gudmundsson OS, Cook TJ, Smith RL, Faria TN, Knipp GT. Spatial Expression Patterns of Peptide Transporters in the Human and Rat Gastrointestinal Tracts, Caco-2 In Vitro Cell Culture Model, and Multiple Human Tissues. AAPS PharmSci. 2001; 3 (1): article 9. DOI: 10.1208/ps030109 3. Maria N. Garcia, Carol Flowers and James D. Cook The Caco-2 Cell Culture System Can be Used as a Model to Study Food Iron Availability Journal of Nutrition Vol. 126 No. 1 January 1996, pp. 251-258 4. Yun, S. M., Habicht, J. P., Miller, D. D., Glahn, R. P. An in vitro digestion/Caco-2 cell culture system accurately predicts the effects of ascorbic acid and polyphenolic compounds on iron bioavailability in humans. Journal of Nutrition, 2004 (Vol. 134) (No. 10) 2717-2721 5. Zweibaum, A., Laburthe, M., Grasset, E. and Louvard, D. (1991). The use of cultured cell lines in studies of intestinal cell differentiation and function. In: Handbook of Physiology (Field, A. and Frizzel, R.A., eds.) Vol 4, pp. 223-255. Amer. Physiol. Soc. Bethesda. Read More