[Comparison of medical and economic benefits of antipsychotics in the treatment of schizophrenia in France]. / Comparaison des bénéfices médico-économiques des antipsychotiques dans la prise en charge de la schizophrénie en France.

INTRODUCTION: The course of schizophrenia can vary widely, and patients experience remission phases alternating with relapse episodes, which generally lead to hospitalisation and have a significant impact on the burden of disease. The prevalence of schizophrenia in France is estimated to be approximately 600,000 people, with an incidence of 10,000 new patients per year. Patients with schizophrenia represent the largest group of hospitalised patients in French public institutions and specialised centres, and the French authorities recognise that the management of schizophrenia is a major public health concern. The Haute Autorité de Santé (HAS) and most of the evidence-based guidelines for the maintenance treatment of schizophrenia recommend long-acting injectable (LAI) antipsychotics to be used predominantly in the prevention of relapse for non-compliant patients; however, in clinical practice, the use of LAIs remains low. OBJECTIVE: This analysis aimed to estimate and to compare the cost-effectiveness of the most common antipsychotic strategies in France in the management of schizophrenia. METHODS: A Markov model was developed to simulate the progression of a cohort of patients with schizophrenia through four health states (stable treated, stable non-treated, relapse and death) and considered up to three lines of treatment to account for changes in treatment management. Antipsychotics including aripiprazole LAI (ALAI), olanzapine LAI (OLAI), paliperidone LAI (PLAI), risperidone LAI (RLAI), haloperidol decanoate (HD) and oral olanzapine (OO) were compared in terms of costs and clinical outcomes. Thus, costs, quality-adjusted life-years (QALYs) and number of relapses were assessed over five years based on three-month cycles from a French health insurance perspective with a discount rate of 4 %. Patients were considered to be stabilised after clinical decompensation and would enter the model at an initiation phase, followed by a prevention of relapse phase if successful. Data (e.g. relapse or discontinuation rates) for the initiation phase came from randomised clinical trials, whereas relapse rates in the prevention phase were derived from hospitalisation risks based on French real-life data in order to capture adherence effects. Safety and utility data were derived from international publications. Additionally costs were retrieved from French health insurance databases and publications. Robustness of results was assessed through deterministic and probabilistic sensitivity analyses. RESULTS: First and second generations of LAIs were found to have similar costs over five years; i.e. approximately € 55,000, except for PLAI which was associated with a discounted cost of € 50,880. Oral antipsychotics were found to be less costly (i.e. OO cost € 50,379 after five years) but associated with a lower number of QALYs gained and relapse avoided. PLAI and RLAI were associated with the greatest number of QALYs gained; i.e. PLAI dominated ALAI, OLAI and HD and was associated with an incremental costs-effectiveness ratio (ICER) of € 2411 per QALY gained versus OO. Finally, PLAI and OLAI were associated with the lowest number of relapses; i.e. PLAI dominated RLAI, ALAI and HLAI and was associated with an ICER of € 1782 per avoided relapse compared to OO. OO and HD were found to have led to the highest number of relapses. CONCLUSION: This analysis, to the best of our knowledge, is the first of its kind to assess the cost-effectiveness of antipsychotics based on French observational data. PLAI was associated with the highest probability of being the optimal treatment from the French health insurance perspective.

Negative cyclic AMP response elements in the promoter of the L-type pyruvate kinase gene.

L-type pyruvate kinase gene expression is modulated by hormonal and nutritional conditions. Here, we show by transient transfections in hepatocytes in primary culture that both the glucose response element and the contiguous hepatocyte nuclear factor 4 (HNF4) binding site (L3) of the promoter were negative cyclic AMP (cAMP) response elements and that cAMP-dependent inhibition through L3 requires HNF4 binding. Another HNF4 binding site-dependent construct was also inhibited by cAMP. However, HNF4 mutants whose putative PKA-dependent phosphorylation sites have been mutated still conferred cAMP-sensitive transactivation of a L3-dependent reporter gene. Overexpression of the CREB binding protein (CBP) increased the HNF4-dependent transactivation but this effect remained sensitive to cAMP inhibition.

Chicken ovalbumin upstream promoter-transcription factor II, a new partner of the glucose response element of the L-type pyruvate kinase gene, acts as an inhibitor of the glucose response.

Transcription of the L-type pyruvate kinase (L-PK) gene is induced by glucose in the presence of insulin and repressed by glucagon via cyclic AMP. The DNA regulatory sequence responsible for mediating glucose and cyclic AMP responses, called glucose response element (GlRE), consists of two degenerated E boxes spaced by 5 base pairs and is able to bind basic helix-loop-helix/leucine zipper proteins, in particular the upstream stimulatory factors (USFs). From ex vivo and in vivo experiments, it appears that USFs are required for correct response of the L-PK gene to glucose, but their expression and binding activity are not known to be regulated by glucose. A genetic screen in yeast has allowed us to identify a novel transcriptional factor binding to the GlRE, i.e. the chicken ovalbumin upstream promoter-transcription factor II (COUP-TFII). Binding of COUP-TFII to the GlRE was confirmed by electrophoretic mobility shift assays, and COUP-TFII-containing complexes were detectable in liver nuclear extracts. Neither abundance nor binding activity of COUP-TFII appeared to be significantly regulated by diets. In footprinting experiments, two COUP-TFII-binding sites overlapping the E boxes were detected. Overexpression of COUP-TFII abrogated the USF-dependent transactivation of an artificial GlRE-dependent promoter in COS cells and the glucose responsiveness of the L-PK promoter in hepatocytes in primary culture. In addition, a mutated GlRE with increased affinity for USF and very low affinity for COUP-TFII conferred a dramatically decreased glucose responsiveness on the L-PK promoter in hepatocytes in primary culture by increasing activity of the reporter gene in low glucose condition. We propose that COUP-TFII could be a negative regulatory component of the glucose sensor complex assembled on the GlRE of the L-PK gene and most likely of other glucose-responsive genes as well.

Control of adipocyte differentiation by CCAAT/enhancer binding protein alpha (C/EBP alpha).

Upon differentiation of 3T3-L1 preadipocytes into adipocytes transcription of many adipose-specific genes is coordinately activated. A differentiation-induced factor, later identified as C/EBP alpha, binds to and transactivates the promoters of these genes. Vector-directed expression of antisense C/EBP alpha RNA in preadipocytes blocked expression of C/EBP alpha, as well as adipose-specific mRNAs, and also prevented cytoplasmic triglyceride accumulation. Rescue of the 'adipocyte phenotype' was accomplished by transfection of the antisense cells with a complementary sense C/EBP alpha RNA expression vector. Using an IPTG-inducible double-vector LacSwitch C/EBP alpha expression system, it was found that differentiation can be induced without exogenous hormone inducers. These findings indicate that C/EBP alpha is not only required, but is sufficient, to trigger differentiation of 3T3-L1 preadipocytes. The C/EBP alpha gene promoter possesses a C/EBP binding site through which C/EBP alpha autoactivates its own expression. A nuclear protein referred to as CUP (C/EBP undifferentiated protein) that binds to a bipartite element in the C/EBP alpha promoter just 5' to the C/EBP binding site has been purified and characterized. During differentiation of preadipocytes, expression of CUP activity decreases as expression of C/EBP alpha increases. Evidence suggests that a CUP-containing protein complex bridges between the CUP (repression) and C/EBP (autoactivation) elements in the promoter and may maintains the C/EBP alpha gene in the repressed state prior to differentiation.

CCAAT/enhancer binding protein alpha (C/EBP alpha) undifferentiated protein: a developmentally regulated nuclear protein that binds to the C/EBP alpha gene promoter.

During differentiation of 3T3-L1 preadipocytes into adipocytes, transcription of the C/EBP alpha (CCA-AT/enhancer binding protein alpha) gene is activated. The promoter of the C/EBP alpha gene contains a bipartite cis element with binding sites for C/EBP alpha undifferentiated protein (CUP) and an Sp1-like GT box binding protein. Binding of CUP to this element is markedly enhanced by its interaction with the Sp1-like protein. CUP, purified approximately 100,000-fold from HeLa cell nuclear extracts, appears to be composed of at least two types of subunit. Evidence is presented that a CUP-containing protein complex bridges between the CUP/Sp1-like GT box element and a downstream cis element, which contains a C/EBP binding site. During differentiation of 3T3-L1 preadipocytes into adipocytes, CUP activity or expression decreases as expression of C/EBP alpha increases. It is suggested that bridging by the CUP-containing protein complex may play a role in transcriptional regulation of the C/EBP alpha gene.

Trans-acting factors involved in adipogenic differentiation.

The differentiation of preadipocytes into adipocytes in culture is accompanied by the coordinate transcriptional activation of adipose-specific genes. Recent studies have identified cis-acting elements that are involved in activating (or derepressing) the transcription of many of these genes during differentiation. The identification of key trans-acting nuclear factors that interact with certain of these elements makes it possible to formulate models for the regulatory network governing the adipogenic differentiation program.

Elements responsible for hormonal control and tissue specificity of L-type pyruvate kinase gene expression in transgenic mice.

L-type pyruvate kinase (L-PK) is a key enzyme of the glycolytic pathway specifically expressed in the liver and, to a lesser degree, in the small intestine and kidney. One important characteristic of L-PK gene expression is its strong activation by glucose and insulin and its complete inhibition by fasting or glucagon treatment. Having previously established that the entire rat L-PK gene plus 3.2 kbp of 5'-flanking region functions in mice in a tissue-specific and hormonally regulated manner, various deletions of these 3.2 kbp of 5'-flanking regions were tested in transgenic animals to map the cis-acting elements involved in transcriptional gene regulation. Our experiments indicate that the proximal region between -183 and +11 confers tissue specificity and contains all the information necessary for dietary and hormonal control of L-PK gene expression in vivo. We found, however, that the transcriptional activity generated by this proximal promoter fragment can be modulated by distal sequences in a tissue-specific manner. (i) Sequences between bp -183 and -392 seem to play a dual role in the liver and small intestine; they induce L-PK expression in the liver but repress it in the small intestine. (ii) Sequences from bp -392 up to -1170 do not seem to have any additional effect on promoter activity. (iii) Between bp -1170 and -2080, we found a putative extinguisher whose transcriptional inhibitory effect is much more marked in the small intestine than in the liver. (iv) Finally, between bp -2080 and -3200, we identified an activating sequence required for full expression of the gene in the liver.

cis-acting DNA elements regulating expression of the liver pyruvate kinase gene in hepatocytes and hepatoma cells. Evidence for tissue-specific activators and extinguisher.

To identify the DNA sequences that cis-regulate the expression of the rat liver pyruvate kinase (L-PK) genes, a series of constructs in which the chloramphenicol acetyltransferase reporter genes is driven by various deleted fragments of the 3200 base pairs (bp) upstream of the L-PK gene cap site have been assayed for transient expression after introduction into hepatoma HepG2 cells, rat hepatocytes in primary culture, fibroblast LTK- cells, myogenic C2C12 cells, and CHO cells. Four distinct regulatory domains have been characterized. A proximal promoter region containing a binding site for the hepatocyte nuclear factor 1 (HNF1) which is sufficient to confer liver specificity, even in the presence of a ubiquitous enhancer. A distal promoter region (-96 to -283 bp) containing binding sites for the liver-specific factor A1 (LFA1), the ubiquitous nuclear factor 1 (NF1), the major late transcriptional factor (MLTF), and so far unidentified proteins binding to the L5-PK region which is essential to maximally activate expression of the construct in HepG2 cells. An extinguisher region, located between positions -2082 and -1170 bp, which decreases efficiency of the L-PK promoter in HepG2 cells, but not in hepatocytes in primary culture. Finally, a far upstream region (-2900 to -2500 bp) which seems to correspond to a liver-specific DNase I hypersensitive site and which behaves in HepG2 cells as an activating sequence efficient in the absence of the extinguisher.

Positive and negative regulation of gene expression by insulin and glucagon: the model of L-type pyruvate kinase gene.

L-type pyruvate kinase gene regulation is an excellent model of gene control by hormones and diet. In vivo and ex vivo experiments allowed us to established that thyroid hormones and glucocorticoids act on pyruvate kinase gene expression at the post-transcriptional level. In contrast, glucose and insulin together stimulate transcription of this gene while glucagon inhibits it. Insulin or glucose are individually inefficient and glucagon-dependent transcriptional inhibition seems to be dominant in insulin + glucose-dependent activation. A 14-kbp fragment encompassing the entire pyruvate kinase gene and 3.2-kbp of 5' flanking sequences is expressed in transgenic mice exactly like the endogenous gene; the 3.2-kbp upstream region is sufficient to confer this tissue-specific and hormone/diet-regulated expression to reporter genes. In vivo, DNAse I hypersensitivity analysis revealed the presence of 3 liver-specific groups of hypersensitive sites (HSS). The proximal sites, between + 1 and -183 bp with respect to the start site of transcription, were, in addition, transcription-dependent. The nature and functional role of proteins binding to this proximal upstream sequence were analyzed by in vitro binding and cell free transcription experiments. The existence of more upstream cis-acting elements was investigated by transient transfection assays using differentiated hepatoma cell lines and hepatocytes in primary culture. These experiments permitted the detection of an extinguisher active in hepatoma Hep G2 cells but not in hepatocytes, and of an activating element which could correspond to a distal HSS. Unfortunately, this investigation has not yet allowed us to determine with accuracy the DNA elements responsible for response to diet and hormones.

Expression of the rat L-type pyruvate kinase gene from its dual erythroid- and liver-specific promoter in transgenic mice.

The gene for the L-type pyruvate kinase possesses two promoters which are located 500 base pairs apart. The L promoter is specific to liver and regulated by hormones and diet; the L' promoter is specific to erythroid cells. We produced two series of transgenic mice carrying either the entire rat L-pyruvate kinase gene or a minigene devoid of exons two to nine, with 2.7 kilobases of flanking sequences 5' to the cap site of the L' promoter and 1.4 kilobases 3' to the downstream polyadenylation site. In both series the patterns of expression from the two promoters were similar to those of the endogenous rat gene. The rat L promoter was expressed strongly in liver and weakly in kidney and gut of adult transgenic mice. Moreover, it was regulated like the endogenous rat L-pyruvate kinase gene upon hormonal and nutritional adaptation: the level of L-pyruvate kinase mRNA was decreased dramatically by 24 h of starvation, while refeeding a carbohydrate-rich diet strongly stimulated expression of the transgenes. This stimulation was prevented by glucagon. Use of alternative polyadenylation sites in the last exon of the rat L-type pyruvate kinase gene was similar for both types of transgenes and similar to that in rat and not control mice, suggesting that the transgenes contain the sequences that control the choice of polyadenylation site. Transcription of the minigene was higher than that of the entire transgene, probably due to the high copy number of the minigene. At the protein level, rat L subunits encoded by the entire transgene were more abundant than mouse subunits in the liver of adult transgenic mice. In contrast, expression of the rat L' promoter in fetal liver was only 5% of that in fetal rat liver, and we were unable to detect rat L' subunits of pyruvate kinase enzyme in the red blood cells from transgenic mice. Our results suggest that the integrated DNA contains all elements necessary for tissue specificity (L' and L) as well as hormonal and nutritional control (L) of expression of the rat transgene. Nevertheless, a L'-specific activating element may be missing.

Proteins binding to the liver-specific pyruvate kinase gene promoter. A unique combination of known factors.

A 183 base-pair fragment of the liver-specific promoter of the L-type puruvate kinase (L-PK) gene has been shown by transfection assay to be sufficient to confer a tissue-specific expression to a reporter gene. The proteins binding in vitro to this fragment have been investigated by a combination of DNase I footprinting, gel retardation of synthetic oligonucleotides and ultraviolet cross-linking. Four proteins from liver nuclear extracts bind to the investigated fragment. They were called, from 3' to 5', L1 to L4 binding factors. The L1 site (nucleotides -95 to -66 with respect to the cap site) binds hepatocyte nuclear factor 1 (HNF1), a liver-specific protein. The L2 site (nucleotides -114 to -97) binds the ubiquitous nuclear factor 1 (NF1), or a related factor. The L3 site (nucleotides -144 to -126) binds liver factor A1 (LF-A1), another liver-specific protein. Finally, the L4 site (nucleotides -168 to -145) binds major late transcription factor (MLTF/USF/UEF), an ubiquitous protein. Each of these proteins has been detected in other liver-specific promoters, but their combination is unique to the liver-specific promoter of the L-PK gene.

Transfection of hepatic genes into adult rat hepatocytes in primary culture and their tissue-specific expression.

We describe in this paper a method for studying transient gene expression in a primary culture of adult rat hepatocytes. After isolation by collagenase perfusion, hepatocytes in a monolayer were transfected with foreign DNA by the calcium phosphate precipitation technique during the first 24 hours after plating. When they were transfected with a plasmid containing the gene for chloramphenicol acetyltransferase driven by the early promoter of simian virus 40, hepatocytes reproducibly expressed high levels of chloramphenicol acetyltransferase (CAT); this transient expression was much higher than that obtained with the rat hepatoma cell line H4II. Different medium conditions have been tested; an optimal level of CAT activity can be obtained using a serum-free, hormonally defined medium. Using these techniques, we have investigated the expression of liver-specific genes transferred into hepatocytes. We show that the L-pyruvate kinase promoter is active in these hepatocytes while it is silent in fibroblasts. Moreover, the use of serum-free medium may allow investigation of the role of hormones and nutrients in cells which respond normally to these effectors.

Regulation of genes for glycolytic enzymes in cultured rat hepatoma cell lines.

We examined the control by hormones and culture conditions of the expression of pyruvate kinase L, aldolase B, and a liver-specific 5.4-kb mRNA species [Pichard, A. L. et al. (1985) Biochem. J. 226, 637-644] in three rat hepatoma cell lines, MH1C1, Fao and Faza. The expression level of these markers ranges from 2% (for pyruvate kinase L mRNA) to 10-12% (for 5.4-kb mRNA species) of the glucose-induced mRNA values found in rat liver. The mRNAs of the three liver-specific genes strongly decrease after treatment of the hepatoma cells with cyclic 8-bromo-AMP, cyclic dibutyryl-AMP or forkolin, pyruvate kinase L mRNA being the most sensitive to this inhibiting effect. In contrast, the concentration of pyruvate kinase L mRNA nuclear precursors is not modified by the cyclic AMP analogues, indicating that these agents do not act at the transcriptional level but, instead, probably destabilize the transcripts. Glucose or fructose does not modify the expression of these three marker genes in any of the studied cell lines. Insulin is inefficient in modifying concentrations of the mRNAs for pyruvate kinase L and aldolase B, alone or in the presence of carbohydrates. In contrast, it stimulates about fivefold the expression of the 5.4-kb mRNA species in the MH1C1 cell line; this stimulation is carbohydrate-independent. The hepatoma cell lines mimic, therefore, the effect of cyclic AMP on the inhibition in vivo of the expression of genes encoding glycolytic or lipogenic enzymes [Vaulont, S. et al. (1984) Biochem. Biophys. Res. Commun. 125, 135-147]. In contrast, the effect of carbohydrates [Munnich, A. et al. (1984) J. Biol. Chem. 259, 10228-10231] is undetectable. The insulin sensitivity of the liver-specific genes is conserved for the 5.4-kb mRNA species only, especially in the MH1C1 cell line, but not for the other investigated mRNAs, which seems to reflect a fundamental difference in the in vivo effect of insulin on these genes. Finally, S1 nuclease mapping of the start-site of pyruvate kinase L gene transcription shows that the normal site used in vivo is also used in the Fao and Faza lines while, in the MH1C1 line, it coexists with multiple aberrant upstream initiation sites.

Structure of the rat L-type pyruvate kinase gene.

The total sequence of a 13,021 base-pair (bp) genomic fragment containing the rat L-type pyruvate kinase (L-PK) gene was determined by "shot gun" sequencing. This fragment includes 8360 bp of the L-PK gene, plus 3193 bp of the 5'-flanking and 1468 bp of the 3'-flanking regions. Like the chicken PK-M1 gene, the rat L-PK gene exhibits a fully conserved exon-intron structure, with 11 exons and 10 introns. In the chicken M1 gene, the coding sequences are well conserved (about 70%), in particular at the level of the exons implicated in the formation of PK active sites, exons that are also partially homologous to the corresponding sequences of the yeast gene. Various types of repetitive sequences exist in the L-PK gene, especially two ID (identifier) sequences located in the second intron and the 11th exon. Elements very similar to the "cyclic AMP-dependent regulatory element" recently described in the phosphoenolpyruvate carboxykinase and somatostatin genes are found in the sequenced fragment, but far upstream (-2338) and downstream (+5788) from the cap site. Various sequences homologous to described regulatory elements (glucocorticoid regulatory elements, enhancers, potential Z-DNA) are also observed 5' and 3' of the cap site. A comparison of the 5'-flanking region of the L-PK gene with the same regions of liver-specific or non-specific, cyclic-AMP-responsive or non-responsive genes was also made. It revealed various potentially interesting features that will be used to guide a further functional study. The cap site was determined by primer extension and nuclease S1 mapping using either mature mRNA or precursor RNA as templates. With both templates the start site of transcription appeared to be microheterogeneous, 19 to 14 bp before the ATG translation initiation codon.

Tissue-specific heterogeneity of the 3'-untranslated region of L-type pyruvate kinase mRNAs.

A single L-type pyruvate kinase (PK) gene seems to exist per haploid genome. It is expressed in the liver, kidney and small intestine in the form of three mRNA species of 2, 2.2 and 3.2 X 10(3) bases (kb). All three species are polyadenylated and translatable into the same L-type subunit. Primer extension experiments demonstrate that all three PK mRNAs have the same 5' ends. Nuclease S1 protection experiments with various cDNA and 3' genomic probes indicate that the different mRNA species only differ by the length of their 3' noncoding region. The mechanism responsible for the production of the three transcripts seems to be the use of alternative unusual polyadenylation sites. Run-on assays with specific probes recognizing only the 3.2-kb or all three mRNA species show that the transcription proceeds across the gene with similar rate. This means that the process involved in generation of the three transcripts is a posttranscriptional event, probably due to different sites of endonucleolytic cleavage of primary transcripts extending 3' from the gene region encoding the mature mRNAs. The ratio between the different PK mRNA species is, to a certain extent, tissue-specific and changes with development. The role of an 'identifier sequence' located in the 3' noncoding sequence of the 3.2-kb species in such a tissue-specific use of alternative polyadenylation sites is discussed.