Role of in vitro two-dimensional (2D) and three-dimensional (3D) cell culture systems for ADME-Tox screening in drug discovery and development: a comprehensive review.

Drug discovery and development have become a very time-consuming and expensive process. Preclinical animal models have become the gold standard for studying drug pharmacokinetic and toxicity parameters. However, the involvement of a huge number of animal subjects and inter-species pathophysiological variations between animals and humans has provoked a lot of debate, particularly because of ethical concerns. Although many efforts are being established by biotech and pharmaceutical companies for screening new chemical entities in vitro before preclinical trials, failures during clinical trials are still involved. Currently, a large number of two- dimensional (2D) in vitro assays have been developed and are being developed by researchers for the screening of compounds. Although these assays are helpful in screening a huge library of compounds and have shown perception, there is a significant lack in predicting human Absorption, Distribution, Metabolism, Excretion and Toxicology (ADME-Tox). As a result, these assays cannot completely replace animal models. The recent inventions in three-dimensional (3D) cell culture-based assays like organoids and micro-physiological systems have shown great potential alternative tools for predicting the compound pharmacokinetic and pharmacodynamic fate in humans. In this comprehensive review, we have summarized some of the most commonly used 2D in vitro assays and emphasized the achievements in next-generation 3D cell culture-based systems for predicting the compound ADME-Tox.

Developing C2-Aroyl Indoles as Novel Inhibitors of IDO1 and Understanding Their Mechanism of Inhibition via Mass Spectroscopy, QM/MM Calculations and Molecular Dynamics Simulation.

Indoleamine-2,3-dioxygenase (IDO1) and tryptophan dioxygenases are two heme based metalloenzymes that catalyze the tryptophan oxidation reaction by inserting molecular dioxygen to cleave the pyrrole ring. The mechanism of such ring cleavage reaction is of carcinogenic importance as the malignant tumors recruit this mechanism for immune invasion. In the presence study, we have synthesized a Novel C2 aroyl indoles inhibitor, 8d, which shows significant inhibition of 180 nM at IC50 scale. The binding and conformational changes that transpire after inhibitor binding were thoroughly studied by molecular docking and MD simulations. The subsequent QM/MM (Quantum Mechanical/Molecular Mechanical) calculations were used to proposed the mechanism of inhibition. The QM/MM calculations show that the reaction proceeds via multistep processes where the dioxygen insertion to the substrate 8a is the rate determining process. Theoretical mechanism is further supported by mass spectroscopy, and drug metabolism/pharmacokinetics study (DMPK) and metabolic stability of compound 8d was investigated in rat and human liver microsomes.

Optimization of Nicotinamides as Potent and Selective IRAK4 Inhibitors with Efficacy in a Murine Model of Psoriasis.

IRAK4 is an attractive therapeutic target for the treatment of inflammatory conditions. Structure guided optimization of a nicotinamide series of inhibitors has been expanded to explore the IRAK4 front pocket. This has resulted in the identification of compounds such as 12 with improved potency and selectivity. Additionally 12 demonstrated activity in a pharmacokinetics/pharmacodynamics (PK/PD) model. Further optimization efforts led to the identification of the highly kinome selective 21, which demonstrated a robust PD effect and efficacy in a TLR7 driven model of murine psoriasis.

Discovery of novel pyrido-pyrrolidine hybrid compounds as alpha-glucosidase inhibitors and alternative agent for control of type 1 diabetes.

A new library of pyrido-pyrrolidine hybrid compounds were designed, developed and screened for their antidiabetic property with α-glucosidase. The design is based on preliminary screening of key fragments identified from literature reported α-glucosidase inhibitors and antidiabetic compounds. The most active fragments were stitched to provide a pyrido-pyrrolidine hybrid molecule as a new motif. A library of these compounds were synthesized and screened against a series of α-glycosidases. Subsequently, compound 3k was the most efficacious analog with IC50 of 0.56 µM. Photoluminescence study and circular dichroism experiments indicated that compound 3k modulates the primary and secondary structure of the enzyme. It successfully brings down the fasting blood glucose level for streptozotocin (STZ, 70 mg/kg, Intraperitoneal) induced type I diabetic male Sprague-Dawley rats (250-320 g). At lower concentration, compound 3k slightly stimulates proliferation of BRIN-BD11 (α-glucose responsive beta cells from rat pancreas islets that secretes insulin) cells.

Altered pharmacokinetics and pharmacodynamics of repaglinide by ritonavir in rats with healthy, diabetic and impaired hepatic function.

BACKGROUND: Ritonavir is an antiretroviral drug to treat HIV AIDS and inhibits cytochrome P450 3A4. To treat diabetes mellitus in HIV, repaglinide is coadministered with ritonavir in the clinic. Multiple cytochrome P450 (CYP) isoforms are involved in the metabolism of repaglinide like CYP2C8 and CYP 3A4. In order to predict and understand drug-drug interactions of these two drugs, the pharmacokinetics and pharmacodynamics (PK/PD) of repaglinide and ritonavir were studied in normal, diabetic and hepatic impaired rats. The purpose of the study was to assess the influence of ritonavir on the PK/PD of repaglinide in rats with normal, diabetic and impaired hepatic function. METHODS: Human oral therapeutic doses of ritonavir and repaglinide were extrapolated to rats based on the body surface area. Ritonavir (20 mg/kg, p.o.), alone and along with repaglinide (0.5 mg/kg, p.o.), was given to normal, diabetic and hepatic impaired rats, and the PK/PD were studied. RESULTS: The pharmacokinetic parameters like peak plasma concentration (Cmax), area under the plasma concentration time profile (AUC) and elimination half life of repaglinide were significantly (p<0.0001) increased when compared to repaglinide control rats. The repaglinide clearance (CL) was significantly (p<0.0001) decreased in the presence of ritonavir treatment. In the presence of ritonavir, repaglinide hypoglycemic activity was increased significantly (p<0.0005) when compared with repaglinide control group. CONCLUSIONS: The significant difference in the PK/PD changes have been due to the increased plasma exposure and decreased total body clearance of repaglinide, which may be due to the inhibition of the CYP P450 metabolic system and organic anion-transporting polypeptide transporter by ritonavir.

Stereoselective binding of chiral anti-diabetic drug nateglinide to plasma proteins.

The binding of nateglinide (NA) enantiomers with human plasma (HP), human serum albumin (HSA) and bovine serum albumin (BSA) was investigated. The protein binding was studied over a drug concentration range of 5-100 µM at a protein concentration of 600 µM. Unbound drug concentrations were determined by direct chiral liquid chromatography using chiralcel OJ-RH column. At therapeutic drug concentrations, the protein binding of each enantiomer was >98%. The results showed that the binding of NA enantiomers was stereoselective, mutually competitive and non-linear. The binding characteristics were, however, opposite for the two most important plasma binding proteins. Opposite stereo-selectivity was observed between BSA and HSA while stereo-selectivity was identical between HSA and HP. Scatchard analysis was used to illustrate the different binding affinities of NA enantiomers to BSA, HSA and HP. The interaction between enantiomers observed in HP and serum albumins was confirmed as a competitive type interaction at the high affinity site. Scatchard analysis was used to illustrate the different binding affinities of NA enantiomers to BSA, HSA and HP.

Stereoselective plasma protein binding of amlodipine.

The binding of the (R)- and (S)-enantiomers of amlodipine to bovine serum albumin (BSA), human serum albumin (HSA), alpha(1)-acid glycoprotein (AGP), and human plasma (HP) was studied by equilibrium dialysis over the concentration range of 75-200 microM at a protein concentration of 150 microM. Unbound drug concentrations were determined by enantioselective capillary electrophoresis using 50 mM phosphate buffer, pH 2.5, containing 18 mM alpha-cyclodextrin as background electrolyte. Saturation of the protein binding sites was not observed over the concentration range tested. Upon application of racemic amlodipine besylate, (S)-amlodipine was bound to a higher extend by HSA and HP compared with (R)-amlodipine, whereas the opposite binding of the enantiomers was observed for BSA and AGP. Scatchard analysis was used to illustrate the different binding affinities of amlodipine besylate enantiomers to BSA, HSA and AGP.

Development and validation of a stereoselective HPLC method for the determination of the in vitro transport of nateglinide enantiomers in rat intestine.

A simple stereoselective high performance liquid chromatographic method was developed for the determination of the in vitro transport of the enantiomers of nateglinide (N-(trans-4-isopropylcyclohexyl-carbonyl)-phenylalanine) in the rat intestine using a Chiralcel OJ-RH column (150 x 4.0 mm, 5 microm). The effects of the mobile phase composition, pH, the flow rate, and the temperature on the chromatographic separation were investigated. The enantioseparation was achieved at 33 degrees C using a mobile phase containing 100 mM potassium dihydrogen phosphate, pH 2.5, and ACN (32:68 v/v) delivered at a flow rate of 1 mL/min. The analytes were monitored at 210 nm and linearity (r >0.99) was obtained for a concentration range of 0.5-50 microg/mL. The LOD and LOQ were 0.2 and 0.5 microg/mL for the R-enantiomer and 0.2 and 0.8 microg/mL for the S-enantiomer, respectively. Both, the intra- and interday accuracy and precision of the calibration curves were determined. The method was successfully applied to estimate the in vitro passage of the enantiomers and the racemate of nateglinide in duodenum, jejunum, and ileum of rats. Generally, higher concentrations of nateglinide and the S-enantiomer were observed when the racemate was administered compared to administration of the individual enantiomers of nateglinide.