Recent advances in chemical synthesis, compound identification, and bioinformatics have multiplied new drug candidates, requiring decisions about molecular potency to be made quickly and accurately. The pharmaceutical industry uses a combination of high-throughput screening (HTS) technology based on the pharmacological activity and pharmacokinetic (PK) properties of a candidate drug. A few years ago, PK properties were the main cause of wastage in drug discovery and development. To overcome this problem, many HTS methods have emerged to optimize the screening phase and help select molecules with the best PK characteristics. These methods are cost-effective and efficient, preventing unnecessary expenditures.
The main goal of the pharmaceutical industry is to generate promising molecules based on efficacy, adequate safety, and appropriate PK characteristics, thereby avoiding clinical trial failure. In addition to solubility, most drugs are administered orally, and permeability has become a key issue for gastrointestinal drug absorption. In this way, it is easy to understand the importance of knowledge about the potential permeability of drug candidates. Nowadays, for permeability screening, three important in vitro models are available: the Parallel Artificial Membrane Permeability Assay (PAMPA), the Caco-2 Cell Assay, and the Rat Eversion Test. These methods should not be considered as competitive permeability assays, but as complementary methods used in different stages of drug discovery. They can be used in different steps of drug discovery and development processes, and filter a large number of potential drug candidates.
Modern drug discovery involves screening from a large number of candidate compounds by using high-throughput technology, ADME screening (absorption, distribution, metabolism, and excretion), and then, once a compound that meets all of these requirements is identified, it will begin before clinical trials Drug development process.
Caco-2 cells, an epithelial human colon adenocarcinoma cell line, have been used as a model for intestinal absorption studies in 1989. Since then, the assay has become widely used. In addition, the use of human cells avoids the study of interspecific differences that often occur in animals. Screening monolayers with Caco-2 cells for permeability involves three stages: cell culture, transport experiments, and data analysis.
A research drug should be evaluated in vitro to determine if they are potential substrates for P-glycoproteins (recognized efflux transporters in many tissues and organs, such as the brain, kidney, and intestine). MDR1-MDCK permeability measurement is one of the screening methods for drug absorption. This method uses the MDR1-MDCK cell line, which consists of Madin Darby canine kidney (MDCK) cells and the MDR1 gene, and encodes the efflux protein P-gp. The MDR1-MDCK permeability assay is a valuable tool for identifying and characterizing P-gp substrates and inhibitors. It can help understand the mechanism of drug efflux and highlight early potential problems with drug permeability at an early stage. Compared with Caco-2 cells, MDCK cells can form tightly connected monolayer cells faster, with lower transporter expression and metabolic activity.
The challenges the pharmaceutical industry are facing at every step of the drug discovery and development process are enormous. The large number of compounds produced by combinatorial chemistry and high-throughput medicinal chemistry procedures is the reason for the parallel method of screening for pharmacological efficacy and PK properties. In vitro methods such as PAMPA, Caco-2 and rat intestinal canals are valuable research tools currently used to screen compounds for permeability and absorption. The results obtained from these assays should not be interpreted in isolation, but should be evaluated together, even in combination with other compound characteristics (MW, Sw, log P, pKa, PSA, solubility and stability).
These in vitro models should be combined at different stages of drug discovery as they may provide different permeability information. Although these models are popular and acceptable, it is important to recognize that they have limitations and that there are differences within and between laboratories. Therefore, in order to implement them as high-throughput detection methods in the initial stages of drug discovery and development, it is important to standardize experimental variables and develop uniform and validated methods. Finally, it is more important to recognize that these simple in vitro assays are not sufficient to represent the complex absorption mechanisms in the human intestine and must therefore be combined with in vivo studies in animals and humans. However, in the initial stages of drug discovery and development, they are effective tools to predict the permeability characteristics and intestinal absorption of NCE with minimal resources in a short period of time.