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Oral drug absorption is a complex process depending on many factors, including the physicochemical properties of the drug, formulation characteristics and their interplay with gastrointestinal physiology and biology. Physiological-based pharmacokinetic (PBPK) models integrate all available information on gastro-intestinal system with drug and formulation data to predict oral drug absorption. The latter together with in vitro-in vivo extrapolation and other preclinical data on drug disposition can be used to predict plasma concentration-time profiles in silico. Despite recent successes of PBPK in many areas of drug development, an improvement in their utility for evaluating oral absorption is much needed. Current status of predictive performance, within the confinement of commonly available in vitro data on drugs and formulations alongside systems information, were tested using 3 PBPK software packages (GI-Sim (ver.4.1), Simcyp® Simulator (ver.15.0.86.0), and GastroPlus™ (ver.9.0.00xx)). This was part of the Innovative Medicines Initiative (IMI) Oral Biopharmaceutics Tools (OrBiTo) project. Fifty eight active pharmaceutical ingredients (APIs) were qualified from the OrBiTo database to be part of the investigation based on a priori set criteria on availability of minimum necessary information to allow modelling exercise. The set entailed over 200 human clinical studies with over 700 study arms. These were simulated using input parameters which had been harmonised by a panel of experts across different software packages prior to conduct of any simulation. Overall prediction performance and software packages comparison were evaluated based on performance indicators (Fold error (FE), Average fold error (AFE) and absolute average fold error (AAFE)) of pharmacokinetic (PK) parameters. On average, PK parameters (Area Under the Concentration-time curve (AUC 0-tlast ), Maximal concentration (C max ), half-life (t 1/2 )) were predicted with AFE values between 1.11 and 1.97. Variability in FEs of these PK parameters was relatively high with AAFE values ranging from 2.08 to 2.74. Around half of the simulations were within the 2-fold error for AUC 0-tlast and around 90% of the simulations were within 10-fold error for AUC 0-tlast . Oral bioavailability (F oral ) predictions, which were limited to 19 APIs having intravenous (i.v.) human data, showed AFE and AAFE of values 1.37 and 1.75 respectively. Across different APIs, AFE of AUC 0-tlast predictions were between 0.22 and 22.76 with 70% of the APIs showing an AFE > 1. When compared across different formulations and routes of administration, AUC 0-tlast for oral controlled release and i.v. administration were better predicted than that for oral immediate release formulations. Average predictive performance did not clearly differ between software packages but some APIs showed a high level of variability in predictive performance across different software packages. This variability could be related to several factors such as compound specific properties, the quality and availability of information, and errors in scaling from in vitro and preclinical in vivo data to human in vivo behaviour which will be explored further. Results were compared with previous similar exercise when the input data selection was carried by the modeller rather than a panel of experts on each in vitro test. Overall, average predictive performance was increased as reflected in smaller AAFE value of 2.8 as compared to AAFE value of 3.8 in case of previous exercise., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Purpose: Fixed-combination drug products (FCDPs) for patients with type 2 diabetes mellitus (T2DM) may show efficacy comparable to their individual components (ICs) while improving adherence to treatment. This study evaluated the bioequivalence and safety of 2 dapagliflozin/saxagliptin/metformin extended-release (XR) FCDPs relative to their ICs: saxagliptin and dapagliflozin/metformin XR., Methods: This randomized, open-label, single-dose, single-center crossover study was conducted in 84 healthy subjects aged 18-55 years. The primary objective was to evaluate the fed-state bioequivalence of a dapagliflozin 5-mg/saxagliptin 2.5-mg/metformin 1000-mg XR FCDP and a dapagliflozin 10-mg/saxagliptin 5-mg/metformin 1000-mg XR FCDP relative to the ICs. Secondary objectives included the evaluation of the effect of food on the pharmacokinetic (PK) parameters of saxagliptin, dapagliflozin, and metformin in both FCDPs and characterization of the PK parameters of the active metabolite of saxagliptin, 5-hydroxy saxagliptin, in healthy subjects. PK parameters (AUC 0-∞ , AUC 0-t , and C max ) were used to assess the bioequivalence of the 2 FCDPs with their ICs. The C max and AUC 0-t of the study drugs were compared between female and male subjects to assess sex differences in exposure. Safety and tolerability of both FCDPs and ICs were also assessed with adverse events, vital signs (systolic and diastolic blood pressures and pulse rate), 12-lead ECG, physical examinations, and laboratory assessments., Findings: Both dapagliflozin/saxagliptin/metformin XR FCDPs were bioequivalent to their ICs. For the dapagliflozin 5-mg/saxagliptin 2.5-mg/metformin 1000-mg XR FCDP, the 90% CI for the geometric mean ratio of dapagliflozin C max was slightly above the 80%-125% bioequivalence limit, which is unlikely to be clinically relevant. Food delayed the absorption of the study drugs in both FCDPs, which is unlikely to have a clinically relevant impact on efficacy. In both cohorts, exposure was higher in female subjects compared with male subjects, potentially due to the lower body weight of the female subjects. The safety profile and tolerability of the FCDPs were similar to those of their ICs, and no deaths or serious adverse events were reported., Implications: These data support the use of the dapagliflozin/saxagliptin/metformin XR FCDP in patients with T2DM. ClinicalTrials.gov identifier: NCT03169959., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Protein stability is often a limiting factor in the development of commercial proteins and biopharmaceuticals, as well as for biochemical and structural studies. Unfortunately, identifying stabilizing mutations is not trivial since most are neutral or deleterious. Here we describe a high-throughput colony-based stability screen, which is a direct and biophysical read-out of intrinsic protein stability in contrast to traditional indirect activity-based methods. By combining the method with a random mutagenesis procedure, we successfully identify thermostable variants from 10 diverse and challenging proteins, including several biotechnologically important proteins such as a single-chain antibody, a commercial enzyme and an FDA-approved protein drug. We also show that thermostabilization of a protein drug using our approach translates into dramatic improvements in long-term stability. As the method is generic and activity independent, it can easily be applied to a wide range of proteins.

Despite the importance of Mg(2+) for numerous cellular activities, the mechanisms underlying its import and homeostasis are poorly understood. The CorA family is ubiquitous and is primarily responsible for Mg(2+) transport. However, the key questions-such as, the ion selectivity, the transport pathway, and the gating mechanism-have remained unanswered for this protein family. We present a 3.2 Å resolution structure of the archaeal CorA from Methanocaldococcus jannaschii, which is a unique complete structure of a CorA protein and reveals the organization of the selectivity filter, which is composed of the signature motif of this family. The structure reveals that polar residues facing the channel coordinate a partially hydrated Mg(2+) during the transport. Based on these findings, we propose a unique gating mechanism involving a helical turn upon the binding of Mg(2+) to the regulatory intracellular binding sites, and thus converting a polar ion passage into a narrow hydrophobic pore. Because the amino acids involved in the uptake, transport, and gating are all conserved within the entire CorA family, we believe this mechanism is general for the whole family including the eukaryotic homologs.

Epstein-Barr virus (EBV) belongs to the gamma subfamily of herpes viruses, among the most common pathogenic viruses in humans worldwide. The viral ribonucleotide reductase small subunit (RNR R2) is involved in the biosynthesis of nucleotides, the DNA precursors necessary for viral replication, and is an important drug target for EBV. RNR R2 generates a stable tyrosyl radical required for enzymatic turnover. Here, the electronic and magnetic properties of the tyrosyl radical in EBV R2 have been determined by X-band and high-field/high-frequency electron paramagnetic resonance (EPR) spectroscopy recorded at cryogenic temperatures. The radical exhibits an unusually low g₁-tensor component at 2.0080, indicative of a positive charge in the vicinity of the radical. Consistent with these EPR results a relatively high C-O stretching frequency associated with the phenoxyl radical (at 1508 cm⁻¹) is observed with resonance Raman spectroscopy. In contrast to mouse R2, EBV R2 does not show a deuterium shift in the resonance Raman spectra. Thus, the presence of a water molecule as a hydrogen bond donor moiety could not be identified unequivocally. Theoretical simulations showed that a water molecule placed at a distance of 2.6 Å from the tyrosyl-oxygen does not result in a detectable deuterium shift in the calculated Raman spectra. UV/VIS light spectroscopic studies with metal chelators and tyrosyl radical scavengers are consistent with a more accessible dimetal binding/radical site and a lower affinity for Fe²⁺ in EBV R2 than in Escherichia coli R2. Comparison with previous studies of RNR R2s from mouse, bacteria, and herpes viruses, demonstrates that finely tuned electronic properties of the radical exist within the same RNR R2 Ia class.

Obtaining mono-disperse and stable protein is a requirement for successful structural and biochemical investigation of proteins. For membrane proteins, such preparation is one of the major hurdles, which consequently has contributed to the slow progress in studying them. During the past few years, many screening methods have been developed to make studies of membrane proteins more efficient. Despite these advances, many membrane proteins remain challenging to even isolate in a stable and homogeneous form. The bacterial zinc transporter ZntB is such a protein, for which no isolation procedure has been reported. Here, we present a systematic approach to obtain homogeneous and mono-disperse zinc transporter ZntB in quantities sufficient for structural and biochemical studies. Important aspects of this study that can be applied to other membrane proteins are also discussed., (Copyright 2010 Elsevier Inc. All rights reserved.)

The Kaposi's sarcoma-associated herpesvirus protein SOX (shut off and exonuclease) and its Epstein-Barr virus homolog, BGLF5, are active during the early lytic phase and belong to the alkaline nuclease family. Both proteins have been shown to be bifunctional, being responsible for DNA maturation as well as host shutoff at the mRNA level. We present the crystal structure of SOX determined at 1.85 A resolution. By modeling DNA binding, we have identified catalytic residues that explain the preferred 5'-exonuclease activity of the alkaline nucleases. The presence of a crevice suitable for binding duplex DNA supports a role for herpes alkaline nucleases in recombination events preceding packaging of viral DNA. Direct interaction with dsDNA is supported by oligonucleotide binding data. Mutations specifically affecting host shutoff map to a surface region of the N-terminal domain, implying an essential role in protein-protein interactions, and link the RNase activity of the enzyme to mRNA degradation pathways.

Objective: Our objective was to investigate the inducing effect of repeated oral administration of St John's wort on the jejunal transport and presystemic extraction of R- and S-verapamil in humans., Methods: Jejunal single-pass perfusion experiments with 120-mg/L (244 micromol/L) R-/S-verapamil were performed in 8 healthy male volunteers for 100 minutes before and after 14 days of oral treatment with St John's wort (300 mg 3 times a day). The enantiomers of verapamil and the cytochrome P450 (CYP) 3A4-formed metabolite norverapamil in perfusate and plasma were quantified by chiral HPLC with fluorescence and tandem mass spectrometry detection, respectively., Results: St John's wort did not affect the jejunal permeability or the fraction absorbed of either R- or S-verapamil. The values for area under the plasma concentration-time curve (AUC) for R- and S-verapamil decreased by 78% and 80%, respectively (P <.0001). The corresponding decreases in the maximum concentration were 76% and 78%, respectively (P <.0001), whereas the terminal half-life did not change significantly for any of the enantiomers. The AUC for R-verapamil was 6 times higher than that for S-verapamil in the control phase, and St John's wort did not change this ratio. The AUC values for R- and S-norverapamil decreased by 51% (P <.01) and 63% (P <.0001), respectively., Conclusions: Repeated administration of St John's wort significantly decreased the bioavailability of R- and S-verapamil. This effect is caused by induction of first-pass CYP3A4 metabolism, most likely in the gut, because the jejunal permeability and the terminal half-life were unchanged for both enantiomers.

We have evaluated the passive and carrier-mediated intestinal transport and CYP3A4-mediated metabolism of R/S-verapamil with respect to dose dependency and enantioselectivity in modified Caco-2 cells. The present in vitro results were compared to published data from human in vivo and rat in situ jejunal perfusions with R/S-verapamil. Caco-2 cell permeability to enantiomers of verapamil and norverapamil was weakly concentration dependent (2.5-100 microM). While Caco-2 permeability to verapamil was 2.6- to 3.7-fold lower than in the human jejunum, it was 1.4- to 2.3-fold higher than in rats. However, all three models classified R- and S-verapamil as high permeability compounds according to the biopharmaceutical classification system. In accordance with human and rat data, R/S-verapamil was transported to a minor extent by carrier-mediated mechanisms in Caco-2 cells. Neither the passive nor the carrier-mediated permeability was enantioselective in any of the three models. CYP3A4-mediated demethylation to R/S-norverapamil was enantioselective in Caco-2 cells. Apparent V(max) and K(m) values for the conversion of R-verapamil were 3.2 pmol/min/insert and 0.7 microM, respectively, and for S-verapamil, 5.4 pmol/min/insert and 0.6 microM, respectively. The enantioselectivity in the CYP3A4-metabolism observed in Caco-2 cells was in agreement with human data, but not with rat data, indicating that Caco-2 cells better reflect the human small intestine in this regard. However, all three models suggested that intestinal permeability to verapamil is unaffected by CYP3A4-activity. In summary, modified Caco-2 cells and human jejunum were qualitatively related with respect to R-and S-verapamil transport and CYP3A4-metabolism.

The aim of this study was to find a cell culture model of the intestinal epithelium for use in studies of CYP3A4-mediated first-pass metabolism of drugs and also for studies of the interplay between CYP3A4 metabolism and P-glycoprotein efflux. For this purpose, the expression of CYP3A4, CYP3A5, and MDR1 mRNA was studied in three cell lines of the normal human intestinal epithelium and three transformed cell lines of colonic (Caco-2) origin. Surprisingly, only transformed cell lines were induced by 1alpha,25-dihydroxy vitamin D3 (D3) to express high amounts of CYP3A4. In contrast to the original findings for this model, the monolayer integrity was maintained during D3 treatment. Levels of CYP3A mRNA expression in Caco-2 and TC7 cells differed dramatically. The highest levels of CYP3A4 and lowest levels of CYP3A5 mRNA expression were observed in D3 treated Caco-2 cells of high passage numbers, resulting in a CYP3A4/3A5 expression ratio greater than fourfold higher than that seen in TC7 cells. Functional studies, using the CYP3A probe testosterone, showed that CYP3A activity was completely absent only in uninduced Caco-2 cells. After D3 induction, high levels of the metabolite were produced in both cell lines (149.4 +/- 12.3 and 86.5 +/- 3.8 pmol 6beta-OH testosterone/min/mg cellular protein from 75 microM testosterone in Caco-2 and TC7 cells, respectively). The maximum velocity (Vmax) and the apparent Michaelis constant (Km) for the 6beta-hydroxylation of testosterone by CYP3A4 in intact Caco-2 monolayers were similar to those obtained from human intestinal microsomes. Only minor changes in P-glycoprotein activity were observed when the metabolically stable P-glycoprotein substrate celiprolol was used. In conclusion, these results show that the features of the generally available Caco-2 cell line from American Type Culture Collection make it suitable for studies of CYP3A4-mediated first-pass metabolism and also for studies of the interplay between CYP3A4 and drug efflux mechanisms., (Copyright 2001 Wiley-Liss, Inc. and the American Pharmaceutical Association)

In Drosophila, the erect wing (ewg) protein is required for proper development of the central nervous system and the indirect flight muscles. The fly ewg gene encodes a novel DNA-binding domain that is also found in four genes previously identified in sea urchin, chicken, zebrafish, and human. To identify mouse ewg homologs, we designed degenerate primers to the conserved DNA-binding domain. The RT-PCR product obtained from mRNA of the mouse muscle cell line C2C12 was used to screen cDNA libraries; a single gene was identified which encodes a predicted 503 amino acid protein. The mouse ewg homolog, termed Nrf1, was mapped to proximal Chr 6. By RT-PCR and Northern analysis, Nrf1 was expressed in all tissues examined, and Northern analysis on adult tissues revealed a complex banding pattern suggesting extensive alternative splicing. Nrf1 hybridized to mRNA transcripts at approximately 2.2 kb, 4.0 kb, 4.4 kb, and 5.0 kb, with additional tissue-specific transcripts at 1.5 kb in testis, 1.9 kb in lung, and 3.7 kb in skeletal muscle. In situ hybridization on whole-mount E9-10.5 embryos showed a broad pattern of expression, with the highest levels of expression in the central nervous system, somites, first branchial arch, optic vesicle, and otic vesicle.