20-Hydroxyecdysone induces the expression of one beta-tubulin gene in Drosophila Kc cells.

The expression of 56D and 60C beta-tubulin genes has been examined in Drosophila melanogaster Kc cells in response to the insect moulting hormone, 20-hydroxyecdysone (20-OH-E). Northern blots probed with beta-tubulin subclones show that the 56D beta-tubulin gene encodes a 1.8 kb mRNA whose abundance is not affected by 20-OH-E. The 60C gene probe detects two mRNAs: one of 1.8 kb present in untreated and 20-OH-E-treated cells, and one of 2.6 kb present only in 20-OH-E-treated cells; using a 60C 3'-specific probe, only the 2.6 kb is revealed. Hybrid selection translation experiment demonstrates that a 20-OH-E-inducible mRNA homologous to the 60C gene encodes a beta-tubulin subunit (P4); this subunit is the so-called beta 3-tubulin. Translation of size-fractionated mRNA shows that the 20-OH-E-induced beta 3-tubulin subunit is encoded, in treated cells, by the 2.6 kb mRNA.

Effect of ketoconazole on the pharmacokinetics and safety of telithromycin and clarithromycin in older subjects with renal impairment.

OBJECTIVE: The objective of this study was to determine the effect of multiple impairments in drug elimination on the pharmacokinetics and pharmacodynamics (effect on QTc interval), using clarithromycin as a comparator. METHODS: Thirty-two subjects aged > or = 60 years with renal impairment who were otherwise medically stable were recruited into this parallel-group study. Following stratification according to creatinine clearance (CL(CR)), subjects were randomized to a five-day treatment with ketoconazole (400 mg once daily) alone, or a five-day treatment with ketoconazole (400 mg once daily) and telithromycin (800 mg once daily) given concomitantly or a five-day treatment with ketoconazole (400 mg once daily) and clarithromycin (500 mg twice daily) given concomitantly. Steady-state pharmacokinetics and safety, including serial electrocardiograms, were assessed. RESULTS: In subjects with CL(CR) 30 - 80 ml/min, the mean maximal telithromycin concentration at steady state (C(max),ss) was 3.6 mg/l and the steady state area under the plasma concentration-time curve from time zero to 24 hours (AUC(0-24 h) ss) was 33.4 mg x h/l. The mean C(max), ss and AUC(0-12 h)ss for clarithromycin were 6.2 mg/l and 56.1 mg x h/l, respectively. The increases in telithromycin C(max) ss and AUC(0-24 h) ss compared to corresponding data for healthy young subjects were 1.6- and 2.7-fold, respectively, whereas corresponding increases for clarithromycin were 2.2- and 3.3-fold, respectively. In the telithromycin plus ketoconazole group deltaQTc values were equal or < 60 ms. All QTc values were equal or < 450 ms in males and equal or < 470 ms in females. CONCLUSIONS: The increase in telithromycin plasma concentrations during ketoconazole-mediated inhibition of CYP3A4 in subjects aged 60 years or older with renal impairment was similar to that for clarithromycin under the same conditions. Telithromycin was well tolerated and produced no clinically significant prolongations in the QTc interval.

Receptor-based model accounts for phlebotomy-induced changes in erythropoietin pharmacokinetics.

Previous clinical studies have demonstrated two distinctive pharmacokinetic behaviors of erythropoietin (EPO): changes in pharmacokinetics (PK) after a period of rhEPO treatment and nonlinear pharmacokinetics. The objective of this work was to study the temporal changes in EPO's PK following phlebotomy in order to propose possible mechanisms for this behavior. Five healthy adult sheep were phlebotomized on two separate occasions 4-6 weeks apart to hemoglobin levels of PK 3-4 g/dL. PK parameters were estimated from the concentration-time profiles obtained following repeated intravenous bolus PK studies using tracer doses of biologically active 125I-rhEPO. Based on the changes in clearances, a PK model was derived to provide a mechanistic receptor-based description of the observed phenomena. Phlebotomy resulted in a rapid increase in the EPO plasma concentration, which peaked at 760 +/- 430 mU/mL (mean +/- SD) at 1.8 +/- 0.65 days, and which coincided with a transient reduction in EPO clearance from prephlebotomy values, i.e., from 45.6 +/- 11.2 mL/hr/kg to 24.3 +/- 9.7 mL/hr/kg. As plasma EPO levels returned toward baseline levels in the next few days, a subsequent increase in EPO clearance was noted. EPO clearance peaked at 90.2 +/- 26.2 mL/hr/kg at 8.5 +/- 3.3 days and returned to baseline by 4-5 weeks postphlebotomy. The proposed model derived from these data includes positive feedback control of the EPO receptor (EPOR) pool. The model predicts that: 1) the initial reduction in EPO plasma clearance is due to a transient saturation of EPORs resulting from the phlebotomy-induced high EPO concentration; and 2) the EPOR pool is expandable not only to compensate for EPOR loss but also to adjust to a greater need for EPORs/progenitor cells to restore hemoglobin (Hb) concentration to normal levels.

Physiologically based and population PK modeling in optimizing drug development: A predict-learn-confirm analysis.

Physiologically based pharmacokinetic (PBPK) modeling and classical population pharmacokinetic (PK) model-based simulations are increasingly used to answer various drug development questions. In this study, we propose a methodology to optimize the development of drugs, primarily cleared by the kidney, using model-based approaches to determine the need for a dedicated renal impairment (RI) study. First, the impact of RI on drug exposure is simulated via PBPK modeling and then confirmed using classical population PK modeling of phase 2/3 data. This methodology was successfully evaluated and applied to an investigational agent, orteronel (nonsteroidal, reversible, selective 17,20-lyase inhibitor). A phase 1 RI study confirmed the accuracy of model-based predictions. Hence, for drugs eliminated primarily via renal clearance, this modeling approach can enable inclusion of patients with RI in phase 3 trials at appropriate doses, which may be an alternative to a dedicated RI study, or suggest that only a reduced-size study in severe RI may be sufficient.

Gene transfer to hepatocellular carcinoma: transduction efficacy and transgene expression kinetics by using retroviral and lentiviral vectors.

Gene therapy is an attractive therapy for hepatocarcinoma, and several approaches have been studied using murine leukemia virus-derived retroviruses. We compared gene transfer efficacy and transgene expression kinetics after transduction of hepatocarcinoma cell lines using enhanced green fluorescent protein (EGFP)-expressing murine leukemia virus-derived retroviral vectors and HIV-derived lentiviral vectors. First, we showed that both retroviral and lentiviral vectors efficiently transduce cycling hepatocarcinoma cell lines in vitro. However, after cell cycle arrest, transduction efficacy remained the same for lentiviral vectors but it decreased by 80% for retroviral vectors. Second, we studied EGFP expression kinetics using lentiviral vectors expressing EGFP under the control of cytomegalovirus (CMV) or phosphoglycerolkinase (PGK) promoter. We show that the CMV promoter allows a stronger EGFP expression than the PGK promoter. However, in contrast to PGK-driven EGFP expression, which persists up to 2 months after transduction, CMV-driven EGFP expression rapidly decreased with time. This phenomenon is due to promoter silencing, and EGFP expression can be restored in transduced cells by using transcription activators such as interleukin-6 or phorbol myristate acetate/ionomycin and, to a lesser extent, the demethylating agent 5'-azacytidine. Altogether, our results suggest that lentiviral vectors, which allow efficient transduction of hepatocarcinoma cell lines with a strong and a sustained expression according to the promoter used, are promising tools for gene therapy of hepatocarcinomas.

Intronic and 5' flanking sequences of the Drosophila beta 3 tubulin gene are essential to confer ecdysone responsiveness.

The expression of the beta 3 tubulin gene is regulated, at the transcriptional level, by the steroid hormone ecdysone, in Drosophila Kc cells. Using a transient expression assay, we show that 360 bp from the first intron of the beta 3 tubulin gene, associated with the 5' flanking sequences, are essential to confer ecdysone inducibility on a minimum promoter driving the chloramphenicol acetyl transferase (CAT) gene. The 5' flanking region contains ecdysone-independent cis-positive elements located in proximity to the promoter. Deletion analysis of the 360 bp intronic region reveals that a fragment of 57 bp is crucial for the ecdysone response of the beta 3 tubulin gene. This fragment contains 5'-TGA(A/C)C-3' motifs homologous to ecdysone responsive elements (EcRE) half sites. Band shift assays show that this 57-bp fragment is bound by three specific complexes. One of them appears to be involved in the level of the ecdysone response.

Enhancer and silencer elements within the first intron mediate the transcriptional regulation of the beta 3 tubulin gene by 20-hydroxyecdysone in Drosophila Kc cells.

We have studied the transcriptional regulation of the beta 3 tubulin gene by the steroid hormone 20-hydroxyecdysone (20-E) in Drosophila Kc cells. A series of hybrid genes, with different fragments of the beta 3 tubulin gene driving the bacterial chloramphenicol acetyl transferase (CAT) gene were constructed. The promoter activity was assayed after transient expression in Kc cells, in the presence and the absence of 20-E. Constructs with 0.91 kb upstream from the transcription start site and 360 bp from the first large intron allowed the hormonal regulation, i.e. a repression in the absence of 20-E and a derepression-activation in the presence of the hormone. This 360 bp fragment contains several enhancers and silencer(s) sequences. The regulation of the expression of the beta 3 tubulin gene results from the combined activity of all the positive and negative regulatory sequences of the first intron, and a dialogue with the promoter sequences. The nucleotide sequence of this intronic regulatory-fragment has been established and we have identified several EcRE (ecdysone responsive element) consensus sequences.

Comparison of QTc data analysis methods recommended by the ICH E14 guidance and exposure-response analysis: case study of a thorough QT study of asenapine.

An assessment of the effects of asenapine on QTc interval in patients with schizophrenia revealed a discrepancy between the results obtained by two different methods: an intersection-union test (IUT) (as recommended in the International Conference on Harmonisation E14 guidance) and an exposure-response (E-R) analysis. Simulations were performed in order to understand and reconcile this discrepancy. Although estimates of the time-matched, placebo-corrected mean change in QTc from baseline (ddQTc) at peak plasma concentrations from the E-R analysis ranged from 2 to 5 ms per dose level, the IUT applied to simulated data from the E-R model yielded maximum ddQTc estimates of 7-10 ms for the various doses of asenapine. These results indicate that the IUT can produce biased estimates that may induce a high false-positive rate in individual thorough QTc trials. In such cases, simulations from an E-R model can aid in reconciling the results from the two methods and may support the use of E-R results as a basis for labeling.

Complex organization of CTF/NF-I, C/EBP, and HNF3 binding sites within the promoter of the liver-specific vitellogenin gene.

Vitellogenin genes are expressed specifically in the liver of female oviparous vertebrates under the strict control of estrogen. To explain this tissue-specific expression, we performed a detailed analysis of the Xenopus laevis vitellogenin gene B1 promoter by DNase I footprinting and gel mobility-shift assays. We characterized five binding sites for the ubiquitous factor CTF/NF-I. Two of these sites are close to the TATA-box, whereas the others are located on both sides of the estrogen responsive unit formed by two imperfect estrogen response elements. Moreover two liver-enriched factors, C/EBP and HNF3, were found to interact with multiple closely spaced proximal promoter elements in the first 100 base pairs upstream of the TATA-box. To confirm the physiological significance of this in vitro analysis, in vivo DNase I footprinting experiments were carried out using the ligation-mediated polymerase chain reaction technique. The various cis-elements characterized in vitro as binding sites for known transcription factors and more particularly for liver-enriched transcription factors are efficiently recognized in vivo as well, suggesting that they play an important role in the control of the liver-specific vitellogenin gene B1 expression.

Expression of a new beta tubulin subunit is induced by 20-hydroxyecdysone in Drosophila cultured cells.

One polypeptide, P4, of molecular weight 55000 daltons and pHi, 5,1, is synthesized in Drosophila melanogaster Kc 0% cells only when they were treated by the insect moulting hormone, 20 hydroxyecdysone (20-HE). P4 is precipitated with vinblastine sulfate and migrates as a tubulin subunit. Immunoblot experiment confirms that P4 is a beta subunit of tubulin. This beta tubulin is recovered by in vitro translation only when mRNAs are extracted from treated cells. Thus, regulation of expression of this polypeptide is at the level of transcripts and is under a steroid hormone control. The 20-HE induced beta tubulin comigrates with the beta 3 subunit and like the latter, is specific to the mid period of embryogenesis.

A pharmacodynamic analysis of erythropoietin-stimulated reticulocyte response in phlebotomized sheep.

The pharmacodynamics (PD) of the reticulocyte response resulting from phlebotomy-induced erythropoietin (EPO) was investigated in adult sheep. The anemia caused by the controlled phlebotomy (Hb < 4 g/dl, t = 0) resulted in a rapid increase in EPO with peak concentrations from 200 to 1400 mU/ml at 0.5 to 3 days generating a delayed reticulocyte response with peak levels from 9.3 to 14.1% at 2.5 to 5.1 days. The PD EPO-reticulocyte relationship is well described by a simple kinetic model involving 3 relevant physiologic parameters: T(1) = lag-time (0.73 +/- 0.32 days, mean +/- S.D.), T(2) = reticulocyte maturation time (5.61 +/- 1.41 days), and k = EPO efficacy coefficient (0.052 +/- 0.048% g/dl mU/ml/day). Accordingly, 0.52% reticulocytes at 10 g/dl Hb level are generated per day at an EPO concentration of 100 mU/ml. The difference between the T(2) parameter in this study and the maturation time reported for humans may be due to interspecies differences or different technique and experimental conditions. The PD transduction appears largely linear in the observed EPO concentration range, indicating a full utilization of EPO without any significant PD saturation. Also, the EPO concentration versus time profiles resulting from the phlebotomy were similar to exogenous EPO profiles resulting from s.c. therapeutic dosing. This study supports the hypothesis that s.c. EPO dosing is more efficacious than i.v. dosing.

Fate of linear and supercoiled multinucleosomic templates during transcription.

Electron microscopy was used to monitor the fate of reconstituted nucleosome cores during in vitro transcription of long linear and supercoiled multinucleosomic templates by the prokaryotic T7 RNA polymerase and the eukaryotic RNA polymerase II. Transcription by T7 RNA polymerase disrupted the nucleosomal configuration in the transcribed region, while nucleosomes were preserved upstream of the transcription initiation site and in front of the polymerase. Nucleosome disruption was independent of the topology of the template, linear or supercoiled, and of the presence or absence of nucleosome positioning sequences in the transcribed region. In contrast, the nucleosomal configuration was preserved during transcription from the vitellogenin B1 promoter with RNA polymerase II in a rat liver total nuclear extract. However, the persistence of nucleosomes on the template was not RNA polymerase II-specific, but was dependent on another activity present in the nuclear extract. This was demonstrated by addition of the extract to the T7 RNA polymerase transcription reaction, which resulted in retention of the nucleosomal configuration. This nuclear activity, also found in HeLa cell nuclei, is heat sensitive and could not be substituted by nucleoplasmin, chromatin assembly factor (CAF-I) or a combination thereof. Altogether, these results identify a novel nuclear activity, called herein transcription-dependent chromatin stabilizing activity I or TCSA-I, which may be involved in a nucleosome transfer mechanism during transcription.

Regulatory elements in the first intron contribute to transcriptional regulation of the beta 3 tubulin gene by 20-hydroxyecdysone in Drosophila Kc cells.

We have studied the transcriptional regulation of the beta 3 tubulin gene by the steroid hormone 20-hydroxyecdysone (20-OH-E) in Drosophila Kc cells. A series of hybrid genes with varying tubulin gene lengths driving the bacterial chloramphenicol acetyl transferase (CAT) gene were constructed. The promoter activity was assayed after transient expression in Kc cells, in the presence or absence of 20-OH-E. We find that 0.91Kb upstream from the transcription start site contain one or several hormone independent positive cis-acting elements, responsible for the constitutive expression of the beta 3 tubulin gene. In the large (4.5 Kb) first intron of this gene, we identified additional hormone dependent negative and positive regulatory elements, which can act in both directions and in a position-independence manner. Then, the negative intron element(s), which repress the transcription in the absence of 20-OH-E has characteristics of silencer.

An integrated pharmacodynamic analysis of erythropoietin, reticulocyte, and hemoglobin responses in acute anemia.

PURPOSE: To determine by pharmacodynamic (PD) analysis physiologically relevant parameters of the cellular kinetics of erythropoiesis in acute anemia. METHODS: The PD relationships among erythropoietin (EPO), reticulocyte, and RBC (Hb) responses were investigated in young adult sheep in acute anemia induced twice by two controlled phlebotomies separated by a 4-week recovery period. RESULTS: The phlebotomies resulted in rapid increases in plasma EPO, with maximal levels occurring at 3 to 8 days, followed by a reticulocyte response with a delay of 0.5 to 1.5 days. The Hb returned to prephlebotomy base line at the end of the 4-week recovery period. The EPO, reticulocyte count, and Hb responses were well described by a PK/PD model (r = 0.975) with the following cellular kinetics parameters: the lag time between EPO activation of erythroid progenitor cells and reticulocyte formation; the reticulocyte-to-RBC maturation time; the reticulocyte and Hb formation efficacy coefficients, quantifying EPO's efficacy in stimulating the formation of reticulocytes and Hb, respectively; the C50 PK/PD transduction parameter defined as the EPO level resulting in half the maximum rate of erythropoiesis. CONCLUSION: Physiologically relevant cellular kinetics parameters can be obtained by an endogenous PK/PD analysis of phlebotomy data and are useful for elucidating the pathophysiologic etiology of various anemias.

20-OH-ecdysone regulates 60 C beta tubulin gene expression in Kc cells and during Drosophila development.

Cultured Kc cells of Drosophila melanogaster are sensitive to the insect moulting hormone, 20-hydroxy-ecdysone (20-OH-E). Morphological changes of Kc-treated cells were observed and electron microscopic analysis of pseudopodia shows a large increase in the number of microtubules, all arranged in the same orientation. The 60 C beta tubulin gene which is expressed only in 20-OH-E-treated cells encodes a 2.6-kb mRNA which is essentially cytoplasmic and polyadenylated. The corresponding premessenger is 7 kb in length and is absent in untreated cells. Two peaks of expression of the 60 C beta tubulin gene are observed during Drosophila development: at midembryogenesis (stage 8-13 h) and at the late third instar larvae-early pupae stage. By use of the Ecdysone 1 mutant, 60 C beta tubulin gene expression was demonstrated to be regulated in part by 20-OH-E during Drosophila development. Through these two complementary biological models of study, the mode and role of beta tubulin gene regulation are discussed.

Changes in erythropoietin pharmacokinetics following busulfan-induced bone marrow ablation in sheep: evidence for bone marrow as a major erythropoietin elimination pathway.

The contribution of the bone marrow to in vivo erythropoietin (EPO) elimination was evaluated by determining EPO pharmacokinetic (PK) parameters in five adult sheep in a paired manner before and after chemotherapy-induced marrow ablation. After busulfan-induced bone marrow ablation, EPO PK demonstrated progressive decreases in plasma clearance (CL), elimination half-life [t1/2(beta)], and volume of distribution at steady state (Vss) with concomitant increases in mean residence time (MRT). Eight days after beginning busulfan treatment, there were no further changes in CL, t1/2(beta), MRT, and Vss. Only 20% of baseline CL remained by day 8. The volume of distribution (Vc) and distribution half-life [t1/2(alpha)], in contrast, remained unchanged from baseline. White blood cell counts and reticulocytes gradually declined after the start of marrow ablation. Examination of bone marrow core biopsy samples obtained on day 10 revealed less than 10% of baseline marrow cellularity. No colony-forming unit erythroid (CFU-E) colonies were found after 6 days of incubation for bone marrow aspirates drawn at days 8 and 13 following busulfan treatment, whereas pre-busulfan aspirates yielded 29 CFU-E colonies per 10(5) cells in CFU-E cultures. Treatment of a sheep with 5-fluorouracil showed changes in PK parameters that were similar to the results from treatment with busulfan. The present study indicates that the bone marrow significantly contributes to the elimination of EPO in vivo.

In Drosophila Kc cells 20-OHE induction of the 60C beta3 tubulin gene expression is a primary transcriptional event.

In Drosophila Kc cells, the 60C beta3 tubulin transcription unit, whose expression is induced by 20-hydroxyecdysone (20-OHE), has the same structure as in Drosophila. This gene is characterized by an unusual 5' intron of regulating importance, by an alternatively spliced second intron and by a long 3' transcribed but untranslated region. This gene codes for two beta3 tubulin isoforms with one amino acid difference. We have established that beta3 tubulin gene expression is transcriptionally regulated by the steroid hormone in a time and hormonal concentration-dependent fashion, without requirement of protein synthesis. This implies that this transcriptional induction is a primary event and that this gene is probably a direct target for the 20-OHE receptor.

Two functional forms of the Xenopus laevis estrogen receptor translated from a single mRNA species.

Steroid receptors are nuclear proteins that regulate gene transcription in a ligand-dependent manner. Over-expression of the Xenopus estrogen receptor in a vaccinia virus-derived expression system revealed that the receptor localized exclusively in the nucleus of the infected cells, irrespective of the presence or absence of the ligand. Furthermore, two forms of the receptor were produced, a full-length and a N-terminal truncated version, which are translated from a single mRNA species by the use of two AUG within the same reading frame. These 66- and 61-kDa receptors were also observed after in vitro translation of the mRNA as well as in primary Xenopus hepatocytes. Both forms are potent estrogen-dependent transcriptional activators in transient transfection experiments, as well as in in vitro transcription assays.

A differential pharmacokinetic analysis of the erythropoietin receptor population in newborn and adult sheep.

Strong evidence indicates that erythropoietin (Epo) is eliminated via Epo receptors (EpoR). Epo receptors may be classified as erythropoietic receptors that are largely located on erythroid progenitor cells in the bone marrow (BM) and nonerythropoietic receptors present in most tissues. Epo's elimination kinetics was studied using a very sensitive tracer interaction method (TIM) before and after chemical ablation of BM as an indirect way of evaluating the EpoR through an assortment of pharmacokinetic parameters (VM, KM, K, and CL) used in differentiating the EpoR population in newborn and adult sheep. TIM identified a parallel nonlinear Michaelis-Menten (VM and KM), and linear (K) elimination pathway and found the latter pathway to be significantly (p < 0.01) more dominant in lamb: K/(VM/KM + K) = 0.309 (25.3) versus 0.0895 (18.4) mean (CV%) lambs versus adult sheep. The significantly (p < 0.01) larger total clearance found for lambs indicates a larger nonhematopoietic tissue clearance of Epo (CL = 118 (10.9) ml/h/kg versus 67.8 (19.3) lamb versus adult sheep). The VM/KM ratio for the nonlinear pathway was not found to be significantly different (p > 0.05) between newborn and adults with values of 1.10 (15.8) and 1.30 (3.81) h-1, respectively. We proposed the hypothesis that the linear pathway is via nonhematopoietic EpoR. Assuming that Epo's elimination largely depends not only on erythropoietic EpoR but also on nonhematopoietic EpoR, this work shows a significant difference in the relative proportions of the two EpoR populations in lamb and adult sheep. The larger dominance of the nonhematopoietic EpoR in lamb supports the hypothesis that these receptors are more needed in early life, e.g., providing neuroprotection from perinatal hypoxemic-ischemic episodes.