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The Caco-2 cell line derived from human colon carcinoma is commonly used to assess the permeability of compounds in in vitro conditions. Due to the significant increase in permeability studies using the Caco-2 cell line in recent years, the need to standardize this biological model seems necessary. The pharmaceutical requirements define only the acceptance criteria for the validation of the Caco-2 cell line and do not specify the protocol for its implementation. Therefore, the aim of this study is to review the conditions for permeability studies across the Caco-2 monolayer reported in the available literature concerning validation guidelines. We summarized the main aspects affecting the validation process of the Caco-2 cell line, including the culture conditions, cytotoxicity, cell differentiation process, and monolayer transport conditions, and the main conclusions may be useful in developing individual methods for preparing the cell line for validation purposes and further permeability research.

Perindopril arginine (PA) as an angiotensin-converting enzyme (ACE) inhibitor is widely used in cardiovascular diseases, especially in systemic hypertension and heart failure. Although the pharmacokinetics of PA are well documented, there is no available detailed data on its permeation in in vitro conditions. The present study aimed to assess the transport of PA across both biological membranes and artificial biomimetic ones. For the determination of PA transport, the Caco-2 cell line was selected as a reliable in vitro model of gastrointestinal biological barriers. Additionally, a novel 96-well plate with phospholipid membrane PermeaPad was used to evaluate the transport of PA by passive diffusion. We confirmed that PA is relatively poorly permeable across the Caco-2 monolayer. The permeability results obtained from the non-cell-based model demonstrated higher transport of PA as compared to that of Caco-2. Thus, PA transport across the biological membranes might be suggested to be regulated by the membrane transporters.

The Biopharmaceutics Classification System (BCS) was conceived to classify drug substances by their in vitro aqueous solubility and permeability properties. The essential activity of naftidrofuryl oxalate (NF) has been described as the inhibition of the serotonin receptors (5-HT 2 ), resulting in vasodilation and decreasing blood pressure. Since the early 1980s, NF has been used to treat several venous and cerebral diseases. There is no data available on the BCS classification of NF. However, based on its physical-chemical properties, NF might be considered to belong to the 1st or the 3rd BCS class. The present study aimed to provide data concerning the solubility and permeability of NF through Caco-2 monolayers and propose its preliminary classification into BCS. We showed that NF is a highly soluble and permeable drug substance; thus, it might be suggested to belong to BCS class I. Additionally, a high dissolution rate of the encapsulated NF based on Praxilene ® 100 mg formulation was revealed. Hence, it might be considered as an immediate-release (IR).

Ethylene plays a crucial role in various biological processes and therefore its biosynthesis is strictly regulated by multiple mechanisms. Posttranslational regulation, which is pivotal in controlling ethylene biosynthesis, impacts 1-aminocyclopropane 1-carboxylate synthase (ACS) protein stability via the complex interplay of specific factors. Here, we show that the Arabidopsis thaliana protein phosphatase type 2C, ABI1, a negative regulator of abscisic acid signaling, is involved in the regulation of ethylene biosynthesis under oxidative stress conditions. We found that ABI1 interacts with ACS6 and dephosphorylates its C-terminal fragment, a target of the stress-responsive mitogen-activated protein kinase, MPK6. In addition, ABI1 controls MPK6 activity directly and by this means also affects the ACS6 phosphorylation level. Consistently with this, ozone-induced ethylene production was significantly higher in an ABI1 knockout strain (abi1td) than in wild-type plants. Importantly, an increase in stress-induced ethylene production in the abi1td mutant was compensated by a higher ascorbate redox state and elevated antioxidant activities. Overall, the results of this study provide evidence that ABI1 restricts ethylene synthesis by affecting the activity of ACS6. The ABI1 contribution to stress phenotype underpins its role in the interplay between the abscisic acid (ABA) and ethylene signaling pathways., (© The Author 2014. Published by the Molecular Plant Shanghai Editorial Office in association with Oxford University Press on behalf of CSPB and IPPE, SIBS, CAS.)