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Compared with rodents and many other animal species, the human cytochrome P450 (P450) Cyp2c gene cluster varies significantly in the multiplicity of functional genes and in the substrate specificity of its enzymes. As a consequence, the use of wild-type animal models to predict the role of human CYP2C enzymes in drug metabolism and drug-drug interactions is limited. Within the human CYP2C cluster CYP2C9 is of particular importance, because it is one of the most abundant P450 enzymes in human liver, and it is involved in the metabolism of a wide variety of important drugs and environmental chemicals. To investigate the in vivo functions of cytochrome P450 Cyp2c genes and to establish a model for studying the functions of CYP2C9 in vivo, we have generated a mouse model with a deletion of the murine Cyp2c gene cluster and a corresponding humanized model expressing CYP2C9 specifically in the liver. Despite the high number of functional genes in the mouse Cyp2c cluster and the reported roles of some of these proteins in different biological processes, mice deleted for Cyp2c genes were viable and fertile but showed certain phenotypic alterations in the liver. The expression of CYP2C9 in the liver also resulted in viable animals active in the metabolism and disposition of a number of CYP2C9 substrates. These mouse lines provide a powerful tool for studying the role of Cyp2c genes and of CYP2C9 in particular in drug disposition and as a factor in drug-drug interaction.
Generation and Characterization of a Novel Multidrug Resistance Protein 2 Humanized Mouse Line
(2012)
The multidrug resistance protein (MRP) 2 is predominantly expressed in liver, intestine, and kidney, where it plays an important role in the excretion of a range of drugs and their metabolites or endogenous compounds into bile, feces, and urine. Mrp knockout [Mrp2(−/−)] mice have been used recently to study the role of MRP2 in drug disposition. Here, we describe the first generation and initial characterization of a mouse line humanized for MRP2 (huMRP2), which is nulled for the mouse Mrp2 gene and expresses the human transporter in the organs and cell types where MRP2 is normally expressed. Analysis of the mRNA expression for selected cytochrome P450 and transporter genes revealed no major changes in huMRP2 mice compared with wild-type controls. We show that human MRP2 is able to compensate functionally for the loss of the mouse transporter as demonstrated by comparable bilirubin levels in the humanized mice and wild-type controls, in contrast to the hyperbilirubinemia phenotype that is observed in MRP2(−/−) mice. The huMRP2 mouse provides a model to study the role of the human transporter in drug disposition and in assessing the in vivo consequences of inhibiting this transporter by compounds interacting with human MRP2.
In humans, 75% of all drugs are metabolized by the cytochrome P450-dependent monooxygenase system. Enzymes encoded by the CYP2C, CYP2D, and CYP3A gene clusters account for ∼80% of this activity. There are profound species differences in the multiplicity of cytochrome P450 enzymes, and the use of mouse models to predict pathways of drug metabolism is further complicated by overlapping substrate specificity between enzymes from different gene families. To establish the role of the hepatic and extrahepatic P450 system in drug and foreign chemical disposition, drug efficacy, and toxicity, we created a unique mouse model in which 30 cytochrome P450 genes from the Cyp2c, Cyp2d, and Cyp3a gene clusters have been deleted. Remarkably, despite a wide range of putative important endogenous functions, Cyp2c/2d/3a KO mice were viable and fertile, demonstrating that these genes have evolved primarily as detoxification enzymes. Although there was no overt phenotype, detailed examination showed Cyp2c/2d/3a KO mice had a smaller body size (15%) and larger livers (20%). Changes in hepatic morphology and a decreased blood glucose (30%) were also noted. A five-drug cocktail of cytochrome P450 isozyme probe substrates were used to evaluate changes in drug pharmacokinetics; marked changes were observed in either the pharmacokinetics or metabolites formed from Cyp2c, Cyp2d, and Cyp3a substrates, whereas the metabolism of the Cyp1a substrate caffeine was unchanged. Thus, Cyp2c/2d/3a KO mice provide a powerful model to study the in vivo role of the P450 system in drug metabolism and efficacy, as well as in chemical toxicity.
Organic anion transporting polypeptide (Oatp) 1a/1b knockout and OATP1B1 and -1B3 humanized mouse models are promising tools for studying the roles of these transporters in drug disposition. Detailed characterization of these models will help to better understand their utility for predicting clinical outcomes. To advance this approach, we carried out a comprehensive analysis of these mouse lines by evaluating the compensatory changes in mRNA expression, quantifying the amounts of OATP1B1 and -1B3 protein by liquid chromatography–tandem mass spectrometry, and studying the active uptake in isolated hepatocytes and the pharmacokinetics of some prototypical substrates including statins. Major outcomes from these studies were 1) mostly moderate compensatory changes in only a few genes involved in drug metabolism and disposition, 2) a robust hepatic expression of OATP1B1 and -1B3 proteins in the respective humanized mouse models, and 3) functional activities of the human transporters in hepatocytes isolated from the humanized models with several substrates tested in vitro and with pravastatin in vivo. However, the expression of OATP1B1 and -1B3 in the humanized models did not significantly alter liver or plasma concentrations of rosuvastatin and pitavastatin compared with Oatp1a/1b knockout controls under the conditions used in our studies. Hence, although the humanized OATP1B1 and -1B3 mice showed in vitro and/or in vivo functional activity with some statins, further characterization of these models is required to define their potential use and limitations in the prediction of drug disposition and drug-drug interactions in humans.
The constitutive androstane receptor (CAR) and the pregnane X receptor (PXR) are closely related nuclear receptors involved in drug metabolism and play important roles in the mechanism of phenobarbital (PB)-induced rodent nongenotoxic hepatocarcinogenesis. Here, we have used a humanized CAR/PXR mouse model to examine potential species differences in receptor-dependent mechanisms underlying liver tissue molecular responses to PB. Early and late transcriptomic responses to sustained PB exposure were investigated in liver tissue from double knock-out CAR and PXR (CARᴷᴼ-PXRᴷᴼ), double humanized CAR and PXR (CARʰ-PXRʰ), and wild-type C57BL/6 mice. Wild-type and CARʰ-PXRʰ mouse livers exhibited temporally and quantitatively similar transcriptional responses during 91 days of PB exposure including the sustained induction of the xenobiotic response gene Cyp2b10, the Wnt signaling inhibitor Wisp1, and noncoding RNA biomarkers from the Dlk1-Dio3 locus. Transient induction of DNA replication (Hells, Mcm6, and Esco2) and mitotic genes (Ccnb2, Cdc20, and Cdk1) and the proliferation-related nuclear antigen Mki67 were observed with peak expression occurring between 1 and 7 days PB exposure. All these transcriptional responses were absent in CARᴷᴼ-PXRᴷᴼ mouse livers and largely reversible in wild-type and CARʰ-PXRʰ mouse livers following 91 days of PB exposure and a subsequent 4-week recovery period. Furthermore, PB-mediated upregulation of the noncoding RNA Meg3, which has recently been associated with cellular pluripotency, exhibited a similar dose response and perivenous hepatocyte-specific localization in both wild-type and CARʰ-PXRʰ mice. Thus, mouse livers coexpressing human CAR and PXR support both the xenobiotic metabolizing and the proliferative transcriptional responses following exposure to PB.
Cytochrome b5 Is a Major Determinant of Human Cytochrome P450 CYP2D6 and CYP3A4 Activity In Vivo s
(2015)
Opioid Analgesia in P450 Gene Cluster Knockout Mice: A Search for Analgesia-Relevant Isoforms
(2015)
Breast cancer resistance protein (BCRP) is expressed in various tissues, such as the gut, liver, kidney and blood brain barrier (BBB), where it mediates the unidirectional transport of substrates to the apical/luminal side of polarized cells. Thereby BCRP acts as an efflux pump, mediating the elimination or restricting the entry of endogenous compounds or xenobiotics into tissues and it plays important roles in drug disposition, efficacy and safety. Bcrp knockout mice (Bcrp−/−) have been used widely to study the role of this transporter in limiting intestinal absorption and brain penetration of substrate compounds. Here we describe the first generation and characterization of a mouse line humanized for BCRP (hBCRP), in which the mouse coding sequence from the start to stop codon was replaced with the corresponding human genomic region, such that the human transporter is expressed under control of the murine Bcrp promoter. We demonstrate robust human and loss of mouse BCRP/Bcrp mRNA and protein expression in the hBCRP mice and the absence of major compensatory changes in the expression of other genes involved in drug metabolism and disposition. Pharmacokinetic and brain distribution studies with several BCRP probe substrates confirmed the functional activity of the human transporter in these mice. Furthermore, we provide practical examples for the use of hBCRP mice to study drug-drug interactions (DDIs). The hBCRP mouse is a promising model to study the in vivo role of human BCRP in limiting absorption and BBB penetration of substrate compounds and to investigate clinically relevant DDIs involving BCRP.
NVS123 is a poorly water-soluble protease 56 inhibitor in clinical development. Data from in vitro hepatocyte studies suggested that NVS123 is mainly metabolized by CYP3A4. As a consequence of limited solubility, NVS123 therapeutic plasma exposures could not be achieved even with high doses and optimized formulations. One approach to overcome NVS123 developability issues was to increase plasma exposure by coadministrating it with an inhibitor of CYP3A4 such as ritonavir. A clinical boost effect was predicted by using physiologically based pharmacokinetic (PBPK) modeling. However, initial boost predictions lacked sufficient confidence because a key parameter, fraction of drug metabolized by CYP3A4 (ƒₘCYP3A4), could not be estimated with accuracy on account of disconnects between in vitro and in vivo preclinical data. To accurately estimate ƒₘCYP3A4 in human, an in vivo boost effect study was conducted using CYP3A4-humanized mouse model which showed a 33- to 56-fold exposure boost effect. Using a top-down approach, human ƒₘCYP3A4 for NVS123 was estimated to be very high and included in the human PBPK modeling to support subsequent clinical study design. The combined use of the in vivo boost study in CYP3A4-humanized mouse model mice along with PBPK modeling accurately predicted the clinical outcome and identified a significant NVS123 exposure boost (∼42-fold increase) with ritonavir.
The Pharmacokinetics and Metabolism of Lumiracoxib in Chimeric Humanized and Murinized FRG Mice
(2017)
The pharmacokinetics and metabolism of diclofenac in chimeric humanized and murinized FRG mice
(2018)
The pharmacokinetics of diclofenac were investigated following single oral doses of 10 mg/kg to chimeric liver humanized and murinized FRG and C57BL/6 mice. In addition, the metabolism and excretion were investigated in chimeric liver humanized and murinized FRG mice. Diclofenac reached maximum blood concentrations of 2.43 ± 0.9 µg/mL (n = 3) at 0.25 h post-dose with an AUCinf of 3.67 µg h/mL and an effective half-life of 0.86 h (n = 2). In the murinized animals, maximum blood concentrations were determined as 3.86 ± 2.31 µg/mL at 0.25 h post-dose with an AUCinf of 4.94 ± 2.93 µg h/mL and a half-life of 0.52 ± 0.03 h (n = 3). In C57BL/6J mice, mean peak blood concentrations of 2.31 ± 0.53 µg/mL were seen 0.25 h post-dose with a mean AUCinf of 2.10 ± 0.49 µg h/mL and a half-life of 0.51 ± 0.49 h (n = 3). Analysis of blood indicated only trace quantities of drug-related material in chimeric humanized and murinized FRG mice. Metabolic profiling of urine, bile and faecal extracts revealed a complex pattern of metabolites for both humanized and murinized animals with, in addition to unchanged parent drug, a variety of hydroxylated and conjugated metabolites detected. The profiles in humanized mice were different to those of both murinized and wild-type animals, e.g., a higher proportion of the dose was detected in the form of acyl glucuronide metabolites and much reduced amounts as taurine conjugates. Comparison of the metabolic profiles obtained from the present study with previously published data from C57BL/6J mice and humans revealed a greater, though not complete, match between chimeric humanized mice and humans, such that the liver humanized FRG model may represent a model for assessing the biotransformation of such compounds in humans.
Slot die coating is applied to deposit thin and homogenous films in roll-to-roll and sheet-to-sheet applications. The critical step in operation is to choose suitable process parameters within the process window. In this work, we investigate an upper limit for stripe coatings. This maximum film thickness is characterized by stripe merging which needs to be avoided in a stable process. It is shown that the upper limit reduces the process window for stripe coatings to a major extent. As a result, stripe coatings at large coating gaps and low viscosities are only possible for relatively thick films. Explaining the upper limit, a theory of balancing the side pressure in the gap region in the cross-web direction has been developed.
A future bio-economy should not only be based on renewable raw materials but also in the raise of carbon yields of existing production routes. Microbial electrochemical technologies are gaining increased attention for this purpose. In this study, the electro-fermentative production of biobutanol with C. acetobutylicum without the use of exogenous mediators is investigated regarding the medium composition and the reactor design. It is shown that the use of an optimized synthetic culture medium allows higher product concentrations, increased biofilm formation, and higher conductivities compared to a synthetic medium supplemented with yeast extract. Moreover, the optimization of the reactor system results in a doubling of the maximum product concentrations for fermentation products. When a working electrode is polarized at −600 mV vs. Ag/AgCl, a shift from butyrate to acetone and butanol production is induced. This leads to an increased final solvent yield of Yᴀᴃᴇ = 0.202 gg⁻¹ (control 0.103 gg⁻¹), which is also reflected in a higher carbon efficiency of 37.6% compared to 23.3% (control) as well as a fourfold decrease in simplified E-factor to 0.43. The results are promising for further development of biobutanol production in bioelectrochemical systems in order to fulfil the principles of Green Chemistry.
Monitoring of organic acids (OA) and volatile fatty acids (VFA) is crucial for the control of anaerobic digestion. In case of unstable process conditions, an accumulation of these intermediates occurs. In the present work, two different enzyme-based biosensor arrays are combined and presented for facile electrochemical determination of several process-relevant analytes. Each biosensor utilizes a platinum sensor chip (14 × 14 mm²) with five individual working electrodes. The OA biosensor enables simultaneous measurement of ethanol, formate, d- and l-lactate, based on a bi-enzymatic detection principle. The second VFA biosensor provides an amperometric platform for quantification of acetate and propionate, mediated by oxidation of hydrogen peroxide. The cross-sensitivity of both biosensors toward potential interferents, typically present in fermentation samples, was investigated. The potential for practical application in complex media was successfully demonstrated in spiked sludge samples collected from three different biogas plants. Thereby, the results obtained by both of the biosensors were in good agreement to the applied reference measurements by photometry and gas chromatography, respectively. The proposed hybrid biosensor system was also used for long-term monitoring of a lab-scale biogas reactor (0.01 m³) for a period of 2 months. In combination with typically monitored parameters, such as gas quality, pH and FOS/TAC (volatile organic acids/total anorganic carbonate), the amperometric measurements of OA and VFA concentration could enhance the understanding of ongoing fermentation processes.
Background
Culture media containing complex compounds like yeast extract or peptone show numerous disadvantages. The chemical composition of the complex compounds is prone to significant variations from batch to batch and quality control is difficult. Therefore, the use of chemically defined media receives more and more attention in commercial fermentations. This concept results in better reproducibility, it simplifies downstream processing of secreted products and enable rapid scale-up. Culturing bacteria with unknown auxotrophies in chemically defined media is challenging and often not possible without an extensive trial-and-error approach. In this study, a respiration activity monitoring system for shake flasks and its recent version for microtiter plates were used to clarify unknown auxotrophic deficiencies in the model organism Bacillus pumilus DSM 18097.
Results
Bacillus pumilus DSM 18097 was unable to grow in a mineral medium without the addition of complex compounds. Therefore, a rich chemically defined minimal medium was tested containing basically all vitamins, amino acids and nucleobases, which are essential ingredients of complex components. The strain was successfully cultivated in this medium. By monitoring of the respiration activity, nutrients were supplemented to and omitted from the rich chemically defined medium in a rational way, thus enabling a systematic and fast determination of the auxotrophic deficiencies. Experiments have shown that the investigated strain requires amino acids, especially cysteine or histidine and the vitamin biotin for growth.
Conclusions
The introduced method allows an efficient and rapid identification of unknown auxotrophic deficiencies and can be used to develop a simple chemically defined tailor-made medium. B. pumilus DSM 18097 was chosen as a model organism to demonstrate the method. However, the method is generally suitable for a wide range of microorganisms. By combining a systematic combinatorial approach based on monitoring the respiration activity with cultivation in microtiter plates, high throughput experiments with high information content can be conducted. This approach facilitates media development, strain characterization and cultivation of fastidious microorganisms in chemically defined minimal media while simultaneously reducing the experimental effort.
Synthetic mimics of natural high-performance structural materials have shown great and partly unforeseen opportunities for the design of multifunctional materials. For nacre-mimetic nanocomposites, it has remained extraordinarily challenging to make ductile materials with high stretchability at high fractions of reinforcements, which is however of crucial importance for flexible barrier materials. Here, highly ductile and tough nacre-mimetic nanocomposites are presented, by implementing weak, but many hydrogen bonds in a ternary nacre-mimetic system consisting of two polymers (poly(vinyl amine) and poly(vinyl alcohol)) and natural nanoclay (montmorillonite) to provide efficient energy dissipation and slippage at high nanoclay content (50 wt%). Tailored interactions enable exceptional combinations of ductility (close to 50% strain) and toughness (up to 27.5 MJ m⁻³). Extensive stress whitening, a clear sign of high internal dynamics at high internal cohesion, can be observed during mechanical deformation, and the materials can be folded like paper into origami planes without fracture. Overall, the new levels of ductility and toughness are unprecedented in highly reinforced bioinspired nanocomposites and are of critical importance to future applications, e.g., as barrier materials needed for encapsulation and as a printing substrate for flexible organic electronics.
In the present work an optical sensor in combination with a spectrally resolved detection device for in-line particle-size-monitoring for quality control in beer production is presented. The principle relies on the size and wavelength dependent backscatter of growing particles in fluids. Measured interference structures of backscattered light are compared with calculated theoretical values, based on Mie-Theory, and fitted with a linear least square method to obtain particle size distributions. For this purpose, a broadband light source in combination with a process-CCD-spectrometer (charge ? coupled device spectrometer) and process adapted fiber optics are used. The goal is the development of an easy and flexible measurement device for in-line-monitoring of particle size. The presented device can be directly installed in product fill tubes or vessels, follows CIP- (cleaning in place) and removes the need of sample taking. A proof of concept and preliminary results, measuring protein precipitation, are presented.
Multi-analyte biosensors may offer the opportunity to perform cost-effective and rapid analysis with reduced sample volume, as compared to electrochemical biosensing of each analyte individually. This work describes the development of an enzyme-based biosensor system for multi-parametric determination of four different organic acids. The biosensor array comprises five working electrodes for simultaneous sensing of ethanol, formate, d-lactate, and l-lactate, and an integrated counter electrode. Storage stability of the biosensor was evaluated under different conditions (stored at +4 °C in buffer solution and dry at −21 °C, +4 °C, and room temperature) over a period of 140 days. After repeated and regular application, the individual sensing electrodes exhibited the best stability when stored at −21 °C. Furthermore, measurements in silage samples (maize and sugarcane silage) were conducted with the portable biosensor system. Comparison with a conventional photometric technique demonstrated successful employment for rapid monitoring of complex media.