Clinical trials for drug approval : a pilot study of the view of doctors at Tokushima University Hospital

The development of new and useful pharmaceutical drugs is essential in order to improve the quality of drug therapeutics. Clinical trials play a central role in drug development. Over time, the clinical trial infrastructure has improved and is now integrating the contribution of clinical research coordinators (CRC). Nevertheless, the attitude of doctors towards clinical trials still favors conventional/historical methodologies. In the present study, we explored the view of doctors towards clinical trials for drug development, in order to improve communication among participants, sponsors, and investigators. A questionnaire was designed for this pilot study. The questionnaire included general attitudes, difficult points, the benefit of doctors in participating as investigators, special attention requirements, and the expected role of CRC in clinical trials for drug approval. In addition, the appropriate use of the outpatient clinic was examined. The questionnaire was provided to doctors in each department of Tokushima University Hospital in 2000 and 2004. Because of the small number of subjects included in this pilot study, no statistical analysis is presented. A total of 89 (81%) and62 (56%) doctors among 110 responded to the survey in 2000 and 2004, respectively. Inquiries about the familiarity of the physicians with clinical trials for drug approval revealed that 84% in 2000 and 66% in 2004 were aware of such trials. The attitude towards participating as investigators in the clinical trials was favorable, with a response of 66% in 2000 and 58% in 2004. Patients’ refusal and the informed consent process were considered difficult areas by many doctors. Expected roles of CRC included activities based on the nurse’s specialty. Although many doctors agreed to take care of the study participants separately from the clinical practice, they lacked the time to do so. In spite of the doctors’ workload reduction by introduction of the CRC concept, their views regarding clinical trials for drug approval remain conventional. Further refinement in the support process by CRC should be considered in our hospital, and the views of the doctors should be investigated in a larger study, in order to promote clinical trials for drug approval in Japan

Cathepsin E Deficiency Impairs Autophagic Proteolysis in Macrophages

Cathepsin E is an endosomal aspartic proteinase that is predominantly expressed in immune-related cells. Recently, we showed that macrophages derived from cathepsin E-deficient (CatE-/-) mice display accumulation of lysosomal membrane proteins and abnormal membrane trafficking. In this study, we demonstrated that CatE-/- macrophages exhibit abnormalities in autophagy, a bulk degradation system for aggregated proteins and damaged organelles. CatE-/- macrophages showed increased accumulation of autophagy marker proteins such as LC3 and p62, and polyubiquitinated proteins. Cathepsin E deficiency also altered autophagy-related signaling pathways such as those mediated by the mammalian target of rapamycin (mTOR), Akt, and extracellular signal-related kinase (ERK). Furthermore, immunofluorescence microscopy analyses showed that LC3-positive vesicles were merged with acidic compartments in wild-type macrophages, but not in CatE-/- macrophages, indicating inhibition of fusion of autophagosome with lysosomes in CatE-/- cells. Delayed degradation of LC3 protein was also observed under starvation-induced conditions. Since the autophagy system is involved in the degradation of damaged mitochondria, we examined the accumulation of damaged mitochondria in CatE-/- macrophages. Several mitochondrial abnormalities such as decreased intracellular ATP levels, depolarized mitochondrial membrane potential, and decreased mitochondrial oxygen consumption were observed. Such mitochondrial dysfunction likely led to the accompanying oxidative stress. In fact, CatE-/- macrophages showed increased reactive oxygen species (ROS) production and up-regulation of oxidized peroxiredoxin-6, but decreased antioxidant glutathione. These results indicate that cathepsin E deficiency causes autophagy impairment concomitantly with increased aberrant mitochondria as well as increased oxidative stress

Comparison of Akt/mTOR and ERK signaling pathways in wild-type and <i>CatE</i>^{<i>−/−</i>} macrophages.

<p>(<b>A</b>) The cell lysates (100 µg protein for each) derived from wild-type (+/+) and <i>CatE</i>^{<i>−/−</i>} macrophages (−/−) were subjected to SDS-PAGE followed by western blotting with specific antibodies to mTOR, p-mTOR, Akt, p-Akt(Thr308), p-Akt(Ser473), ERK, p-ERK, S-6, p-S6, AMPK, p-AMPK and actin. The data indicate the representative western blotting of 3 independent experiments. (<b>B</b>) Densitometric analysis for the quantification of each protein in the cell lysate of both cell types. The arbitrary density unit was defined as the relative chemiluminescence intensity per mm² measured by LAS1000. The data are indicated as the mean ± SD values from 3 independent experiments. *<i>P</i> < 0.05 for the indicated comparisons.</p

Immunofluorescence microscopy of LC3 and LysoTracker in wild-type and <i>CatE</i>^{<i>-/</i><i>-</i>}macrophages.

<p>(<b>A</b>) Cells on glass cover-slips were preincubated with LysoTracker-Red for 30 min, subsequently fixed, permeabilized with 0.3% Tween-20 in PBS, and allowed to react with anti-LC3 antibody. The cells were then incubated with a fluorescence-labeled secondary antibody and visualized by confocal laser microscopy. (<b>B</b>) Based on the data from immunofluorescence microscopy, the number of LC3- or LysoTracker-positive vesicles per cell was counted. The data are shown as percent merged vesicles per cell and acquired from 3 independent experiments. *<i>P</i> < 0.05 for the indicated comparisons.</p

Mitochondrial membrane potential of wild-type and <i>CatE</i>^{<i>−/−</i>} macrophages.

<p>(<b>A</b>). Mitochondrial membrane potential was measured with JC-1. A suspension of peritoneal macrophages (2 × 10⁵ cells/100 μL) was incubated on ice for 15 min with JC-1 in PBS containing 2.5% FBS and 0.01% NaN₃. After washing, flow cytometric analyses were performed. (<b>B</b>) Comparison of the percentage of gated number of macrophages. Ten thousand cells were analyzed in each sample. The data are indicated as mean ± SD values of data from 4 independent experiments. *<i>P</i> < 0.05 for the indicated comparisons.</p

Two-dimensional gel maps of the cell lysates of macrophages.

<p>(<b>A</b>) Proteome analysis of wild-type and <i>CatE</i>^{<i>−/−</i>} macrophages revealed that almost all the spots located in the areas under pI 6.0–6.7 were identified annexin A₁, and those under pI 6.0–6.5 were identified the oxidized and reduced forms of peroxiredoxin-6 respectively. These data indicate the representative 2-D gel maps of 3 independent experiments. (<b>B</b>) Proteomic analysis with wild-type macrophages treated with H₂O₂ or untreated. </p

Intracellular ATP levels in wild-type and <i>CatE</i>^{<i>−/−</i>} macrophages.

<p>Macrophages (1 × 10⁵ cells) were cultured on 96-well plates at 37°C for 24 h. After replacement with fresh media, the culture plate was incubated at room temperature (25°C) for 30 min. ATP levels were determined by the manufacturer’s protocol of the CellTiter-Glo assay kit according to the manufacturer’s protocol. The concentration of intracellular ATP was determined by the titration of the control medium without cells plus 0.1−1.0 µM ATP.</p

Changes of LC3 in wild-type and <i>CatE</i>^{<i>−/−</i>} macrophages under starvation conditions.

<p>(<b>A</b>) Cultured macrophages derived from wild-type (+/+) and <i>CatE</i>^{<i>−/−</i>} macrophages (−/−) were incubated with Hank’s balanced salt solution for the indicated times (0, 1, 2, 3, and 4). The cell lysates (100 µg protein for each) were subjected to SDS-PAGE followed by western blotting with specific antibodies to LC3. (<b>B</b>) Densitometric analysis for the quantification of each protein in the cell lysate of both cell types. The data was defined as the relative chemiluminescence intensity per mm² measured by LAS1000. The data are indicated as the mean <u>+</u> SD values from 3 independent experiments. *<i>P</i> < 0.05 for the indicated comparisons. </p

Levels of oxidative burst, H₂O₂ production, and reduced glutathione in wild-type and <i>CatE</i>^{<i>−/−</i>} macrophages.

<p>(<b>A</b>) Levels of oxidative burst by macrophages. Macrophages in cell suspension (1 × 10⁷ cells/mL) were incubated with zymosan suspension at 37 °C for 30 min. Levels of oxidative burst of macrophages were determined by a luminophotometer. (<b>B</b>) The levels of H₂O₂ production of macrophages. Macrophages (1 × 10⁶ cells/50 μL) were cultured without any stimulation at 37°C for 1 h. Determination of H₂O₂ production into the culture media by macrophages was performed by an Amplex Redassay kit. (<b>C</b>) The GSH levels of cell lysates of macrophages were assayed with NADPH, measuring the development with DTNB at 412 nm with spectrophotometer. (D) Levels of H₂O₂ in the serum (50 μL) of mice were determined by an Amplex Redassay kit. The data are the means ± SD of values from 4 independent experiments. *<i>P</i> <0.05, **<i>P</i> <0.01, ***<i>P</i> <0.001.</p