Drug target identification for neuronal apoptosis through a genome scale screening.

During normal nervous system development, physiologically appropriate neuronal apoptosis contributes to a sculpting process that removes approximately one-half of all neurons born during neurogenesis. However, neuronal apoptosis subsequent to this developmental window is physiologically inappropriate for most systems and can contribute to neurodegenerative diseases. Neuronal apoptosis is characterized by specific morphological events and requires the activation of an intrinsic transcriptional program. With the completion of genome sequencing in humans and model organisms, and the advent of DNA microarray technology, the transcriptional cascades and networks regulating neuronal apoptosis are being elucidated providing new potential pharmacological targets. This review will introduce the reader to this genomic approach and illustrate with a few examples a methodological strategy for the rational selection of pharmacological targets and the development of neuroprotective agents.

Mitochondrial cytochrome c oxidase subunit III is selectively down-regulated by aluminum exposure in PC12S cells.

Aluminum (Al) has been implicated in several neurological diseases including dialysis dementia and Alzheimer's disease (AD). One possible mechanism of Al neurotoxicity could involve alteration of mitochondrial gene expression. We exposed PC12 cells to 0.1-100 microM AlCl3 for 6h at pH 7.4. Internalized Al, measured by atomic absorption spectrometry, was linearly proportional to the extracellular Al concentration. Northern blot analyses showed that cytochrome c oxidase subunit III (COX III) mRNA was significantly reduced by 70% after addition of 1 microM AlCl3. Higher concentrations of AlCl3 did not show a significant further effect. These results suggest that Al neurotoxicity involves a specific impairment of cytochrome c oxidase.

Nitric oxide synthase induction in astroglial cell cultures: effect on heat shock protein 70 synthesis and oxidant/antioxidant balance.

Glial cells in the nervous system can produce nitric oxide in response to cytokines. This production is mediated by the inducible isoform of nitric oxide synthase. Radical oxygen species (ROS) and nitric oxide (NO) derivatives have been claimed to play a crucial role in many different processes, both physiological such as neuromodulation, synaptic plasticity, response to glutamate, and pathological such as ischemia and various neurodegenerative disorders. In the present study we investigated the effects of NO synthase (iNOS) induction in astrocyte cultures on the synthesis of heat shock proteins, the activity of respiratory chain complexes and the oxidant/antioxidant balance. Treatment of astrocyte cultures for 18 hr with LPS and INFgamma produced a dose dependent increase of iNOS associated with an increased synthesis of hsp70 stress proteins. This effect was abolished by the NO synthase inhibitor L-NMMA and significantly decreased by addition of SOD/CAT in the medium. Time course experiments showed that iNOS induced protein expression increased significantly by 2 hr after treatment with LPS and INFgamma and reached a plateau at 18 hr; hsp70 protein synthesis peaked around 18 and 36 hr after the same treatment. Addition to astrocytes of the NO donor sodium nitroprusside resulted in a dose dependent increase in hsp70 protein that was comparable to that found after a mild heat shock. Additionally, a decrease in cytochrome oxidase activity, a marked decrease in ATP and protein sulfhydryl contents, an increase in the activity of the antioxidant enzymes mt-SOD and catalase were found which were abolished by L-NMMA. These findings suggest the importance of mitochondrial energy impairment as a critical determinant of the susceptibility of astrocytes to neurotoxic processes and point to a possible pivotal role of hsp70 in the signalling pathways of stress tolerance.

Structural features of the rat GFAP gene and identification of a novel alternative transcript.

The glial fibrillary acidic protein (GFAP) is expressed in a cell-specific manner and represents the major subunit of intermediate filaments of astroglial cells. The knowledge of the gene structure is an important step for further understanding the mechanisms of cell-specific expression. In the present study, we report the complete sequence of the rat GFAP gene and provide evidence for the existence, in the rat brain, of a novel alternative transcript. Since three different transcripts, indicated as GFAPalpha, beta, and gamma, have been previously reported (Feinstein et al. [1992] J. Neurosci. Res. 32:1-14; Zelenika et al. [1995] Mol. Brain Res. 30:251-258), we called this novel mRNA isoform GFAPdelta. It is generated by the alternative splicing of a novel exon located in the classic seventh intron. This alternative exon (called VII+) contains a 101-bp coding sequence in frame with exon VII and interrupted by a stop codon TAA at position +5451. Therefore, the novel GFAPdelta transcript encodes for an hypothetical GFAP where the forty-two carboxy-terminal amino acids encoded by exon VIII and IX are replaced by thirty-three amino acids encoded by exon VII+. Northern blot analysis with a specific probe for exon VII+ revealed a 4.2-kb mRNA, expressed in several brain areas, but absent in extracerebral tissues (lung, heart, kidney, liver, spleen). The previously discovered GFAP isoforms (alpha, beta, and gamma) produce hypothetical translation products differing in the amino-terminal Head domain. The present data suggest, for the first time, the possible existence of GFAP isoforms differing in the carboxy-terminal Tail domain.

Effect of postischaemic hypothermia on the mitochondrial damage induced by ischaemia and reperfusion in the gerbil.

In order to test the effect of hypothermia on mitochondrial function damage following cerebral ischaemia/reperfusion, Mongolian gerbils were submitted to 30 min bilateral carotid occlusion and 2 h of reperfusion at 37 degreesC or 30 degreesC. After normothermic (37 degreesC) ischaemia/reperfusion, significant decreases in mitochondrial state 3 (+ADP) oxygen consumption (-42.2%), complex II-III activity in synaptosomes (-31.7%) and complex IV were measured, in both free mitochondria and synaptosomes (-30.3% and -27. 8% respectively). However, following hypothermic (30 degreesC) reperfusion, both respiration rates and all enzyme activities remained at levels not significantly different from those in the sham operated controls.

Treatments for newly diagnosed multiple myeloma: analysis of survival data and cost-effectiveness evaluation.

The main therapeutic options currently available to induce remission in newly diagnosed cases of multiple myeloma include: i) melphalan at conventional doses without concurrent administration of interferon; ii) melphalan at conventional dose combined with interferon; iii) autologous bone marrow transplantation (ABMT). We conducted an analysis of the survival data reported in five large-scale published clinical trials and we evaluated the cost-effectiveness ratio. We determined the mean lifetime survival (MLS) for each treatment group using the Gompertz model. The cost data of patients given ABMT or standard chemotherapy were estimated from published information. The values of MLS were 3.47 years per patient for melphalan at conventional doses without interferon, 3.74 years for melphalan at conventional doses combined with interferon, and 7.28 years for ABMT. As compared with conventional melphalan treatment, ABMT yielded a significantly better survival. Survival after melphalan combined with interferon was not significantly different from that following melphalan alone. Using melphalan at conventional doses without interferon as reference term, the marginal cost-effectiveness ratio of ABMT was of about $26,000 per life year gained. For the induction treatment in patients with newly diagnosed myeloma, ABMT seems to be more effective and more cost-effective than the standard treatment with melphalan at conventional doses.

Regulation of cytochrome c oxidase and FoF1-ATPase subunits expression in rat brain during aging.

In the present study we analyzed the age-dependent changes of mRNA levels for cytochrome c oxidase and FoF1-ATP synthase subunits in rat cerebral cortex and cerebellum. To establish whether the regulation of expression is transcriptional or post-transcriptional, the results were compared to those related to protein subunits levels, of the same enzymatic complexes, previously observed. The different patterns of age-related changes of mRNA subunits, in particular the lower increments, compared with those related to protein subunits, indicate that post-transcriptional mechanisms of regulation might be involved in the coordinated expression of the various subunits of each complex. Northern blotting analyses of RNA from the cerebellum of rats at the various ages, showed also differences in age-dependent patterns of transcription between cerebral cortex and cerebellum. Moreover, the major age-dependent changes of mitochondrial-encoded subunits, compared with the nuclear-encoded ones, previously observed at proteins level, occur also during transcription.

Effect of nitric oxide synthase induction on the expression of mitochondrial respiratory chain enzyme subunits in mixed cortical and astroglial cell cultures.

In the present study we evaluated the effects of NO synthase (NOS) induction on the regulation of cytochrome c oxidase (CO) and F0F1-ATPase subunit expression in astroglial and mixed cortical cell cultures. In mixed cortical cell cultures, 18 h of treatment with lipopolysaccharide (LPS, 0.1 microgram/mL) plus interferon-gamma (INF-gamma, 10 U/mL) caused an increase of mRNAs for CO-I, F0F1-ATPase 6 and also for iNOS at 20 DIV. The induction of both CO-I and F0F1-ATPase 6 was abolished by the NOS inhibitor N-monomethyl-L-arginine (NMMA) or by the enzymatic scavenger superoxide dismutase/catalase (SOD/CAT). In primary astroglial cell cultures, treatment for 18 h with increasing concentrations of LPS and INF gamma, produced an increase in the amount of mitochondrial encoded CO-I and -II subunits, with no significant modifications of nuclear encoded subunit IV. An increase was also observed at level of transcription for CO-I and -II, and F0F1-ATPase 6 mRNAs. These effects were abolished by addition of NMMA or SOD/CAT. mRNA induction of CO-I was higher in mixed cortical than in astroglial cell cultures while that of F0F1-ATPase 6 was similar in both cell types. These results suggest that the expression of mitochondrial encoded subunits (CO-I, CO-II and F0F1-ATPase 6) is up-regulated in response to oxygen and NO reactive species. The activity of cytochrome c oxidase decreased after LPS/INF gamma treatment in both astroglial and mixed cortical cultures. The activity of ATP synthase was unmodified, while ATP content drastically decreased after LPS/INF gamma treatment, in both astroglial and mixed cortical cultures. The enzymatic activities of catalase and Mn-SOD (mitochondrial) showed a significant increase after LPS/INF gamma treatment, which was abolished by NMMA.

Treatments for newly diagnosed advanced ovarian cancer: analysis of survival data and cost-effectiveness evaluation.

The main therapeutic options currently available for patients with newly diagnosed advanced ovarian cancer include: (i) cisplatin-based chemotherapy at conventional doses without paclitaxel, (ii) paclitaxel+cisplatin at conventional doses and (iii) high-dose chemotherapy with autologous hematopoietic rescue. After conducting a literature search to identify large-scale clinical trials based on these three therapeutic modalities, we carried out an analysis of the survival data and evaluated the cost-effectiveness ratio where appropriate. Cost data were obtained from published information. Effectiveness was estimated by determining the values of mean lifetime survival (MLS). Our analysis included a total of 15 clinical trials. The values of MLS were 3.05 years per patient for cisplatin-based chemotherapy at conventional doses without paclitaxel (1931 patients), 2.95 years per patient for chemotherapy with paclitaxel+cisplatin at conventional doses (184 patients) and 5.76 years per patient for high-dose chemotherapy with autologous hematopoietic rescue (53 patients). As compared with cisplatin-based chemotherapy without paclitaxel, high-dose treatments with hematopoietic rescue yielded a significantly better survival. Using cisplatin-based chemotherapy as a reference term, the incremental cost-effectiveness ratio for high-dose treatments was $25641 per life year gained (discounted dollars per discounted life year gained). Sensitivity testing suggested that the ratio remained below $50000 under most circumstances. We conclude that in the treatment of patients with advanced ovarian cancer, high-dose chemotherapy with hematopoietic rescue seems to be more effective and more cost-effective than standard treatments with cisplatin-based regimens at conventional doses.

A retrospective cost-effectiveness analysis of interferon as adjuvant therapy in high-risk resected cutaneous melanoma.

To assess the cost per life year gained of alpha interferon (IFN) as adjuvant therapy for patients with high-risk resected melanoma, we conducted a retrospective, incremental cost-effectiveness analysis on clinical data from a previously published ECOG trial [9]. The Gompertz model was used to estimate the total lifetime values of patient-years of subjects receiving IFN in comparison with subjects given no adjuvant treatment. The ECOG trial involved 143 patients treated with high-dose IFN and 137 given no adjuvant treatment. Estimated drug expenditures were based on the assumption of a cost of $109.25 per 10 MU of IFN. Our analysis of the ECOG results showed that the adjuvant treatment of 100 subjects with high-dose IFN improved survival expectancy by 133.6 discounted life years or 308 undiscounted life years. The use of IFN (compared with no adjuvant treatment) implied an incremental cost of $16,467 per discounted life year saved (95% CI of $4752-50,000) or $7143 per undiscounted life year saved (95% CI of $3226-33,846). Sensitivity testing, in which variations were introduced in the main factors influencing cost and effectiveness, showed that this value always remained below $50,000. Our pharmacoeconomic analysis indicates that adjuvant treatment with high-dose IFN in patients with high-risk resected melanoma implies a favourable cost-effectiveness ratio. Because two other studies showed no significant survival benefit in patients receiving adjuvant IFN at lower values of total dose per patient, the controversy remains and confirmation data are needed for the ECOG trial's results. If these clinical results are confirmed, our analysis shows that the dosage of IFN given in this trial has a favourable pharmacoeconomic profile.

Cost effectiveness of adjuvant intraportal chemotherapy in patients with colorectal cancer.

Recent studies have shown that the analysis of published survival curves allows cost-effectiveness evaluations in which two treatments are compared with each other in terms of cost per life-year saved. In patients with colorectal cancer, the administration of adjuvant intraportal chemotherapy (with mitomycin and fluorouracil) has been reported to improve long-term survival in comparison with patients who are not given this treatment. To assess the pharmacoceconomic profile of this adjuvant chemotherapy, we carried out an incremental cost-effectiveness analysis in which we used the Gompertz model to estimate lifetime patient years gained by patients given this chemotherapy in comparison with controls. Using the data of a published controlled long-term trial involving 252 patients treated with intraportal chemotherapy and 253 controls who were given no such therapy, we estimated that the adjuvant treatment improved life expectancy by 89 discounted patient years (or 218 undiscounted patient years) every 100 patients. Cost of chemotherapy was calculated as $107,720 for every 100 patients. On the basis of these data, the administration of adjuvant intraportal chemotherapy was found to imply an incremental cost of $1,210 per discounted life-year saved or $494 per undiscounted life-year saved. The cost-effectiveness ratio of adjuvant intraportal chemotherapy in patients with colorectal cancer seems to be particularly favorable in comparison with estimates of cost per life-year saved previously obtained in many other areas of pharmacological intervention. Even though systemic fluorouracil + levamisole is the form of adjuvant chemotherapy most widely used in these patients, intraportal chemotherapy has the best cost-effectiveness profile.

Pharmacoeconomic profile of paclitaxel as a first-line treatment for patients with advanced ovarian carcinoma. A lifetime cost-effectiveness analysis.

BACKGROUND: Analysis of published survival curves has recently been proposed as a method for conducting incremental cost-effectiveness analysis in which two treatments are compared with each other in terms of cost per year of life gained. In patients with advanced ovarian carcinoma, the combination of paclitaxel and cisplatin has been reported to improve survival more significantly than standard therapy with cyclophosphamide and cisplatin. However, the high cost of paclitaxel indicates a need for an evaluation of the pharmacoeconomic profile of these treatments. METHODS: The authors conducted an incremental cost-effectiveness analysis to assess the paclitaxel-based regimen in terms of cost per year of life gained. The analysis utilized data from a published controlled long term trial involving 184 patients treated with paclitaxel and cisplatin and 202 patients treated with cyclophosphamide and cisplatin. Gompertz' law was employed to obtain the lifetime estimate of the years gained by patients given the first treatment in comparison with patients given the second. RESULTS: The paclitaxel-based treatment was found to improve life expectancy by 46 years for every 100 patients. Costs of chemotherapy were higher in the paclitaxel group than in the standard-therapy group (the cost difference was $901,723 for every 100 patients; costs for treating febrile neutropenia induced by chemotherapy were taken into account). On the basis of these data of cost and effectiveness, the administration of paclitaxel and cisplatin was found to imply a cost per year of life gained of $19,603 more than standard chemotherapy. CONCLUSIONS: The pharmacoeconomic profile of paclitaxel compares favorably with economic data previously calculated for other types of pharmacologic treatment.

G-CSF for the prophylaxis of neutropenic fever in patients with small cell lung cancer receiving myelosuppressive antineoplastic chemotherapy: meta-analysis and pharmacoeconomic evaluation.

Standard meta-analytical and pharmacoeconomic techniques were used to study the clinical effectiveness and the cost-effectiveness ratio of the prophylactic (or pre-emptive) administration of G-CSF to patients with small cell lung cancer treated with conventional myelosuppressive cytotoxic chemotherapy. In the first part of our study, we conducted a meta-analysis of the randomized clinical trials evaluating G-CSF for this clinical indication. Three trials were identified by our literature search and were included in the meta-analysis (overall number of patients = 606). The end-points for evaluating G-CSF included mortality from infection and the cumulative incidence of neutropenic fever over six cycles of chemotherapy. The results of our meta-analysis demonstrate that prophylactic G-CSF did not affect mortality but significantly reduced the incidence of neutropenic fever from 68.3% to 38.7% (pooled odds ratio = 0.29, 95% CI: 0.21-0.40; P < 0.001). In the second part of our study, we carried out a pharmacoeconomic analysis to estimate the cost-effectiveness ratio of pre-emptive G-CSF (i.e. the 'average' cost associated with the prevention of an episode of neutropenic fever). This cost-effectiveness ratio was US$ 14,372 using the Italian price of the drug converted into dollars, or US$ 41 088 using the US price. Finally, we estimated the revenue-neutral price of G-CSF based on American data of the cost-of-illness. The price ranged from US$ 395 to US$ 569 per cycle, a figure higher than the value (US$ 150) previously reported in the literature.

Cost-effectiveness of adjuvant chemotherapy with cyclophosphamide+methotrexate+fluorouracil in patients with node-positive breast cancer.

BACKGROUND: The analysis of published survival curves can be used as the basis for incremental cost-effectiveness analyses in which two treatments are compared with each other in terms of cost per life year saved. In patients with node-positive breast cancer adjuvant chemotherapy with cyclophosphamide+ methotrexate+fluorouracil has been reported to improve survival in comparison with patients who are not given this treatment. METHODS: To assess the pharmacoeconomic profile of this adjuvant chemotherapeutic regimen in terms of cost per life-year gained, we conducted an incremental cost-effectiveness analysis in which the Gompertz model was used to calculate the lifetime estimate of the patient-years gained by treated subjects compared to controls. RESULTS: Using data from a published, controlled long-term trial involving 207 patients treated with cyclophosphamide+methotrexate+fluorouracil and 179 controls, we estimated that this adjuvant chemotherapy improved life expectancy by 357 patient-years per 100 subjects. Direct costs, which were almost exclusively related to the administration of chemotherapy, were estimated to be US $159,516 per 100 patients. On the basis of these data, adjuvant chemotherapy was found to imply an incremental cost of US $447 per life-year saved. CONCLUSIONS: The cost-effectiveness ratio of adjuvant chemotherapy with cyclophosphamide+methotrexate+fluorouracil in patients with node-positive breast cancer seems to be particularly favourable in comparison with estimates of cost per life-year saved previously calculated for other types of pharmacological intervention.

Age-related chances of mitochondrial cytochrome C oxidase and F0F1-ATP synthase subunit contents in rat cerebral cortex.

The levels of subunits I, II/III, and IV of cytochrome c oxidase and of subunits alpha, beta and gamma of F(0)F(1)-ATP synthase in inner mitochondrial membrane proteins purified from cerebral cortex of rat at 2, 6, 12, 18, 24, 26 months of age were analyzed by Western blot. Age-related changes in the content of subunits, encoded either in mitochondrial or nuclear DNA, were observed.

Changes of mitochondrial cytochrome c oxidase and FoF1 ATP synthase subunits in rat cerebral cortex during aging.

The contents of subunits I, II/III, and IV of cytochrome c oxidase and of subunits alpha, beta and gamma of FoF1 ATP synthase in inner mitochondrial membrane proteins purified from cerebral cortex of rat at 2, 6, 12, 18, 24, and 26 months of age were analyzed by western blot. Age-related changes in the content of subunits, either of mitochondrial or nuclear origin, were observed. All the cytochrome c oxidase (COX) subunits examined showed an age-related increase from 2-month-old rats up to 24 months with a decrease at the oldest age (26 months). The same pattern of age-dependent changes was observed for gamma ATP synthase, while the alpha and beta subunits increased progressively up to 26 months.