Defining the Contribution of CYP1A1 and CYP1A2 to Drug Metabolism Using Humanized CYP1A1/1A2 and Cyp1a1/Cyp1a2 Knockout Mice.

Cytochrome P450s CYP1A1 and CYP1A2 can metabolize a broad range of foreign compounds and drugs. However, these enzymes have significantly overlapping substrate specificities. To establish their relative contribution to drug metabolism in vivo, we used a combination of mice humanized for CYP1A1 and CYP1A2 together with mice nulled at the Cyp1a1 and Cyp1a2 gene loci. CYP1A2 was constitutively expressed in the liver, and both proteins were highly inducible by 2,3,7,8-tetrachlorodibenzodioxin (TCDD) in a number of tissues, including the liver, lung, kidney, and small intestine. Using the differential inhibition of the human enzymes by quinidine, we developed a method to distinguish the relative contribution of CYP1A1 or CYP1A2 in the metabolism of drugs and foreign compounds. Both enzymes made a significant contribution to the hepatic metabolism of the probe compounds 7-methoxy and 7-ehthoxyresorufin in microsomal fractions from animals treated with TCDD. This enzyme kinetic approach allows modeling of the CYP1A1, CYP1A2, and non-CYP1A contribution to the metabolism of any substrate at any substrate, inhibitor, or enzyme concentration and, as a consequence, can be integrated into a physiologically based pharmacokinetics model. The validity of the model can then be tested in humanized mice in vivo. SIGNIFICANCE STATEMENT: Human CYP1A1 and CYP1A2 are important in defining the efficacy and toxicity/carcinogenicity of drugs and foreign compounds. In light of differences in substrate specificity and sensitivity to inhibitors, it is of central importance to understand their relative role in foreign compound metabolism. To address this issue, we have generated mice humanized or nulled at the Cyp1a gene locus and, through the use of these mouse lines and selective inhibitors, developed an enzyme kinetic-based model to enable more accurate prediction of the fate of new chemicals in humans and which can be validated in vivo using mice humanized for cytochrome P450-mediated metabolism.

An Extensively Humanized Mouse Model to Predict Pathways of Drug Disposition and Drug/Drug Interactions, and to Facilitate Design of Clinical Trials.

Species differences in drug metabolism and disposition can confound the extrapolation of in vivo PK data to man and also profoundly compromise drug efficacy studies owing to differences in pharmacokinetics, in metabolites produced (which are often pharmacologically active), and in differential activation of the transcription factors constitutive androstane receptor (CAR) and pregnane X receptor (PXR), which regulate the expression of such enzymes as P450s and drug transporters. These differences have gained additional importance as a consequence of the use of genetically modified mouse models for drug-efficacy testing and also patient-derived xenografts to predict individual patient responses to anticancer drugs. A number of humanized mouse models for cytochrome P450s, CAR, and PXR have been reported. However, the utility of these models has been compromised by the redundancy in P450 reactions across gene families, whereby the remaining murine P450s can metabolize the compounds being tested. To remove this confounding factor and create a mouse model that more closely reflects human pathways of drug disposition, we substituted 33 murine P450s from the major gene families involved in drug disposition, together with Car and Pxr, for human CAR, PXR, CYP1A1, CYP1A2, CYP2C9, CYP2D6, CYP3A4, and CYP3A7. We also created a mouse line in which 34 P450s were deleted from the mouse genome. Using model compounds and anticancer drugs, we demonstrated how these mouse lines can be applied to predict drug-drug interactions in patients and discuss here their potential application in the more informed design of clinical trials and the personalized treatment of cancer.

Evaluation of the yield of post-clipping angiography and nationwide current practice.

BACKGROUND: Surgical treatment of intracranial saccular aneurysms aims to prevent (re)hemorrhage by complete occlusion of the aneurysmal lumen. It is unclear whether routine postoperative imaging, to assess aneurysmal occlusion, is necessary since intraoperative assessment by the neurosurgeon may be sufficient. We assessed routine clinical protocols for post-clipping imaging in the Netherlands and determined whether intraoperative assessment of aneurysm clippings sufficiently predicts aneurysm residuals. METHODS: A survey was conducted to assess postoperative imaging protocols in centers performing clipping of intracranial aneurysms in the Netherlands (n = 9). Furthermore, a retrospective single-center cohort study was performed to determine the predictive value of intraoperative assessment of aneurysm occlusion in relation to postoperative digital subtraction angiography (DSA) findings, between 2009 and 2017. RESULTS: No center performed intraoperative DSA in a hybrid OR, routinely. Respectively, four (44.4%), seven (77.8%), and three (33.3%) centers did not routinely perform early postoperative imaging, late follow-up imaging, or any routine imaging at all. Regarding our retrospective study, 106 patients with 132 clipped aneurysms were included. There were 23 residuals ≥ 1 mm (17.4%), of which 10 (43.5%) were unexpected. For the presence of these residuals, intraoperative assessment showed a sensitivity of 56.5%, a specificity of 86.2%, a positive predictive value of 46.4%, and a negative predictive value of 90.4%. CONCLUSIONS: There is lack of consensus regarding the post-clipping imaging strategy in the Netherlands. Since intraoperative assessment is shown to be insufficient to predict postoperative aneurysm residuals, we advocate routine postoperative imaging after aneurysm clipping unless this is not warranted on the basis of patient age, clinical condition, and/or comorbidity.

The pharmacokinetics and metabolism of diclofenac in chimeric humanized and murinized FRG mice.

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.

The pharmacokinetics and metabolism of lumiracoxib in chimeric humanized and murinized FRG mice.

The pharmacokinetics and metabolism of lumiracoxib were studied, after administration of single 10mg/kg oral doses to chimeric liver-humanized and murinized FRG mice. In the chimeric humanized mice, lumiracoxib reached peak observed concentrations in the blood of 1.10±0.08µg/mL at 0.25-0.5h post-dose with an AUCinf of 1.74±0.52µgh/mL and an effective half-life for the drug of 1.42±0.72h (n=3). In the case of the murinized animals peak observed concentrations in the blood were determined as 1.15±0.08µg/mL at 0.25h post-dose with an AUCinf of 1.94±0.22µgh/mL and an effective half-life of 1.28±0.02h (n=3). Analysis of blood indicated only the presence of unchanged lumiracoxib. 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 obtained in humanized mice were different compared to murinized animals with e.g., a higher proportion of the dose detected in the form of acyl glucuronide metabolites and much reduced amounts of 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 useful approach to assessing the biotransformation of such compounds in humans.

[Modular training in practical medicine: electronic evaluation of student education in general practice]. / Blockpraktikum Allgemeinmedizin: Elektronische Evaluation des studentischen Unterrichts in Allgemeinpraxen.

Effective from spring 2004, new regulations for undergraduate medical education in Germany require a two-week practical training in general practice. Similar to other forms of medical education, this practical training should be regularly evaluated by students. With regard to special conditions of the training, we preferred a web based evaluation. Since adequate models were not available, we designed, implemented and tested an electronic way of evaluation. The following aspects turned out to be of special importance: teamwork, time, data protection and cost. Meanwhile, the evaluation is established and still accessible as demo-version for visitors of the home page. This electronic evaluation of medical training in general practice is highly appropriate for a timely evaluation allowing us to obtain a comparison between students' expectations and actual experience as well as a continuous supervision and to provide feedback to the participating practices. This is an important step for quality assurance of medical education in practices inside and outside the university.

[Medical care research based on family doctor routine data--are interface-communicated treatment data feasible?]. / Versorgungsforschung mit hausärztlichen Routinedaten--Sind Behandlungsdatenträger-(BDT-)Exporte geeignet?

AIMS OF THE STUDY: As part of an ongoing project on the utilisation of generic drugs in general practice we aimed at determining whether the transfer of prescriptions and patient characteristics from doctors' computerized medical records via the BDT (Behandlungsdatenträger) interface was feasible, and whether these data are suitable for research in pharmacoepidemiology. METHODS: All 1,395 general practitioners from 6 regions in Germany were invited to participate in the 'generics project'; 232 (17 %) agreed. The 17 software companies whose systems were used by the participating practices were asked to grant access to the BDT interface. For a prescription survey, doctors were supposed to export BDT files from two 3-month periods each in 2000 and 2001. Data were anonymised and relevant information extracted with a special programme. RESULTS: So far, BDT data are available from 79 practices. They are suitable for practice- and patient-related prescribing analyses. By filter modifications, additional information (such as diagnoses, referrals, clinical findings or accounting codes) can be obtained. The procedure was well accepted if doctors and practice staff were assisted by computer experts. Some difficulties, however, were encountered in obtaining access to the BDT-interface from the software companies. Lack of standardisation of the BDT interface required additional conditioning of the data. CONCLUSION: The BDT interface offers an opportunity to export computerised patient records without the requirement of additional documentation. If routine data are more readily available for health services research, a standardised data structure and open access must be assured e. g. by centralised certification via the Federal statutory health organisation.

Notch signaling is required for arterial-venous differentiation during embryonic vascular development.

Recent evidence indicates that acquisition of artery or vein identity during vascular development is governed, in part, by genetic mechanisms. The artery-specific expression of a number of Notch signaling genes in mouse and zebrafish suggests that this pathway may play a role in arterial-venous cell fate determination during vascular development. We show that loss of Notch signaling in zebrafish embryos leads to molecular defects in arterial-venous differentiation, including loss of artery-specific markers and ectopic expression of venous markers within the dorsal aorta. Conversely, we find that ectopic activation of Notch signaling leads to repression of venous cell fate. Finally, embryos lacking Notch function exhibit defects in blood vessel formation similar to those associated with improper arterial-venous specification. Our results suggest that Notch signaling is required for the proper development of arterial and venous blood vessels, and that a major role of Notch signaling in blood vessels is to repress venous differentiation within developing arteries. Movies available on-line

An instructive function for Notch in promoting gliogenesis in the zebrafish retina.

The Gal4-UAS technique has been used to misexpress a constitutively active Notch receptor variant (notch1a-intra) in the developing zebrafish retina. This is the first study to use this technique to misexpress genes and assess their function in neural development of the zebrafish. Expression of activated Notch1a either ubiquitously, driven by a heat-shock70 promoter, or in a spatially regulated manner, controlled by the deltaD promoter, causes a block in neuronal differentiation that affects all cell types. Developing cells take on either a glial fate or remain undifferentiated. A large number of cells eventually undergo apoptosis. These phenotypic effects of activated Notch1a are expressed cell autonomously. Cells within central regions of the retina adopt a glial fate if they express activated Notch1a in a time window that extends from 27 to 48 hours postfertilization. This period corresponds mainly to the time of origin of ganglion cells in the normal retina. Activation of notch1a at later stages results in defects in cell type specification that remain restricted to the ciliary marginal zone, whereas neuronal types are specified normally within the central region. These observations indicate that glial differentiation is initiated by Notch1a-intra expressing cells, which become postmitotic in the same time window. Our results strongly suggest that Notch1a instructs a certain cell population to enter gliogenesis, and keeps the remaining cells in an undifferentiated state. Some or all of these cells will eventually succumb to apoptosis.

Transcriptional activation by the PHD finger is inhibited through an adjacent leucine zipper that binds 14-3-3 proteins.

The PHD finger, a Cys(4)-His-Cys(3) zinc finger, is found in many regulatory proteins from plants or animals which are frequently associated with chromatin-mediated transcriptional regulation. We show here that the PHD finger activates transcription in yeast, plant and animal cells. In plant homeodomain transcription factors the PHD finger is combined with an upstream leucine zipper. Both domains together form a highly conserved 180 amino acid region called the ZIP/PHDf motif and transcriptional activity of the PHD finger is masked when embedded in this motif. Our results indicate that the ZIP/PHDf domain is a potential regulatory domain of PHDf-HD proteins. The leucine zipper upstream of the PHD finger interacts with 14-3-3GF14 mu from Arabidopsis thaliana and 14-3-3GF14-12 from maize via a leucine zipper conserved in helix 4 of various 14-3-3 proteins from plants and animals. PHD-type plant homeodomain proteins consequently may represent potential targets of 14-3-3 signalling.

Use of the Gal4-UAS technique for targeted gene expression in the zebrafish.

The most common way to analyze the function of cloned genes in zebrafish is to misexpress the gene product or an altered variant of it by mRNA injection. However, mRNA injection has several disadvantages. The GAL4-UAS system for targeted gene expression allows one to overcome some of these disadvantages. To test the GAL4-UAS system in zebrafish, we generated two different kinds of stable transgenic lines, carrying activator and effector constructs, respectively. In the activator lines the gene for the yeast transcriptional activator GAL4 is under the control of a given promoter, while in the effectors the gene of interest is fused to the sequence of the DNA-binding motif of GAL4 (UAS). Crosses of animals from the activator and effector lines show that effector genes are transcribed with the spatial pattern of the activators. This work smoothes the way for a novel method of misexpression of gene products in zebrafish in order to analyze the function of genes in developmental processes.

A measurement of attitudes about an activity program.

Staff and patient attitudes about an activity program were measured to highlight problems that existed in a paritcular milieu. Statements concerning the rationale for this activity program were compared to determine the similarities and differences between staff and patient perceptions of the program. To create a more therapeutic milieu, suggestions for openness and sharing were made based on the results of the attitude study. Their implementation would require staff and patients to interact on a more nearly equal basis. Such an approach is a bias toward the Maxwell Jones philosophy of therapeutic milieu, the direction in which the ward staff of this study wanted to go. This paper offers a way for the staff to reach their goal and suggests several paths they might take. The attitude questionnaire method used in this study might be a useful tool for those who are studying the therapeutic aspects of ward milieu.