Enrico Domenici

Reversible and Covalent Binding of Drugs to Human Serum Albumin: Methodological Approaches and Physiological Relevance

Human serum albumin (HSA) plays a fundamental role in the transport of drugs, metabolites, and endogenous ligands. Binding to HSA controls the free, active concentration of a drug, provides a reservoir for a long duration of action, and ultimately affects drug absorption, metabolism, distribution and excretion. The free concentration of a drug can also be affected by interaction with co-administered drugs or by pathological conditions that can modify to a significant extent the binding properties of the carrier, resulting in important clinical impacts for drugs that have a relatively narrow therapeutic index. This manuscript will review the physiological role of albumin in the human body and the pharmacological consequences of drug-albumin binding, and then focus on the structure and the properties of the protein binding sites, as studied by different methodologies. Among these, biochromatography on immobilized albumin has been shown to be a rapid and effective tool for the characterization of albumin binding sites and their enantioselectivity, and for the study of the changes in the binding properties of the protein arising by interaction between different ligands. We will discuss the potential offered by the combined use of circular dichroism on the same protein/drug system in solution, not only for the determination of binding parameters and the detection of displacement phenomena, but also for the identification of conformational features underlying binding stereoselectivity. In particular, the essential role of these methodologies in the study of the enantioselective phenomena occurring in the HSA binding of chiral drugs will be addressed. The effect of reversible or covalent binding of drugs will also be discussed and examples of physiological relevance reported.

Chronic Social Stress Inhibits Cell Proliferation in the Adult Medial Prefrontal Cortex: Hemispheric Asymmetry and Reversal by Fluoxetine Treatment

Profound neuroplastic changes have been demonstrated in various limbic structures after chronic stress exposure and antidepressant treatment in animal models of mood disorders. Here, we examined in rats the effect of chronic social stress and concomitant antidepressant treatment on cell proliferation in the medial prefrontal cortex (mPFC). We also examined possible hemispheric differences. Animals were subjected to 5 weeks of daily social defeat by an aggressive conspecific and received concomitant, daily, oral fluoxetine (10 mg/kg) during the last 4 weeks. Bromodeoxyuridine (BrdU) labeling and quantitative stereological techniques were used to evaluate the treatment effects on proliferation and survival of newborn cells in limbic structures such as the mPFC and the hippocampal dentate gyrus, in comparison with nonlimbic structures such as the primary motor cortex and the subventricular zone. Phenotypic analysis showed that neurogenesis dominated the dentate gyrus, whereas in the mPFC most newborn cells were glia, with smaller numbers of endothelial cells. Chronic stress significantly suppressed cytogenesis in the mPFC and neurogenesis in the dentate gyrus, but had minor effect in nonlimbic structures. Fluoxetine treatment counteracted the inhibitory effect of stress. Hemispheric comparison revealed that the rate of cytogenesis was significantly higher in the left mPFC of control animals, whereas stress inverted this asymmetry, yielding a significantly higher incidence of newborn cells in the right mPFC. Fluoxetine treatment abolished hemispheric asymmetry in both control and stressed animals. These pronounced changes in gliogenesis after chronic stress exposure may relate to the abnormalities of glial cell numbers reported in the frontolimbic areas of depressed patients.

Plasma protein biomarkers for depression and schizophrenia by multi analyte profiling of case-control collections.

Despite significant research efforts aimed at understanding the neurobiological underpinnings of psychiatric disorders, the diagnosis and the evaluation of treatment of these disorders are still based solely on relatively subjective assessment of symptoms. Therefore, biological markers which could improve the current classification of psychiatry disorders, and in perspective stratify patients on a biological basis into more homogeneous clinically distinct subgroups, are highly needed. In order to identify novel candidate biological markers for major depression and schizophrenia, we have applied a focused proteomic approach using plasma samples from a large case-control collection. Patients were diagnosed according to DSM criteria using structured interviews and a number of additional clinical variables and demographic information were assessed. Plasma samples from 245 depressed patients, 229 schizophrenic patients and 254 controls were submitted to multi analyte profiling allowing the evaluation of up to 79 proteins, including a series of cytokines, chemokines and neurotrophins previously suggested to be involved in the pathophysiology of depression and schizophrenia. Univariate data analysis showed more significant p-values than would be expected by chance and highlighted several proteins belonging to pathways or mechanisms previously suspected to be involved in the pathophysiology of major depression or schizophrenia, such as insulin and MMP-9 for depression, and BDNF, EGF and a number of chemokines for schizophrenia. Multivariate analysis was carried out to improve the differentiation of cases from controls and identify the most informative panel of markers. The results illustrate the potential of plasma biomarker profiling for psychiatric disorders, when conducted in large collections. The study highlighted a set of analytes as candidate biomarker signatures for depression and schizophrenia, warranting further investigation in independent collections.

Genome-wide association study of recurrent major depressive disorder in two European case-control cohorts.

Major depressive disorder (MDD) is a highly prevalent disorder with substantial heritability. Heritability has been shown to be substantial and higher in the variant of MDD characterized by recurrent episodes of depression. Genetic studies have thus far failed to identify clear and consistent evidence of genetic risk factors for MDD. We conducted a genome-wide association study (GWAS) in two independent datasets. The first GWAS was performed on 1022 recurrent MDD patients and 1000 controls genotyped on the Illumina 550 platform. The second was conducted on 492 recurrent MDD patients and 1052 controls selected from a population-based collection, genotyped on the Affymetrix 5.0 platform. Neither GWAS identified any SNP that achieved GWAS significance. We obtained imputed genotypes at the Illumina loci for the individuals genotyped on the Affymetrix platform, and performed a meta-analysis of the two GWASs for this common set of approximately half a million SNPs. The meta-analysis did not yield genome-wide significant results either. The results from our study suggest that SNPs with substantial odds ratio are unlikely to exist for MDD, at least in our datasets and among the relatively common SNPs genotyped or tagged by the half-million-loci arrays. Meta-analysis of larger datasets is warranted to identify SNPs with smaller effects or with rarer allele frequencies that contribute to the risk of MDD.

Pharmacologically Diverse Antidepressants Rapidly Activate Brain-Derived Neurotrophic Factor Receptor TrkB and Induce Phospholipase-Cγ Signaling Pathways in Mouse Brain

Previous studies suggest that brain-derived neurotrophic factor and its receptor TrkB are critically involved in the therapeutic actions of antidepressant drugs. We have previously shown that the antidepressants imipramine and fluoxetine produce a rapid autophosphorylation of TrkB in the rodent brain. In the present study, we have further examined the biochemical and functional characteristics of antidepressant-induced TrkB activation in vivo. We show that all the antidepressants examined, including inhibitors of monoamine transporters and metabolism, activate TrkB rapidly in the rodent anterior cingulate cortex and hippocampus. Furthermore, the results indicate that acute and long-term antidepressant treatments induce TrkB-mediated activation of phospholipase-Cγ1 (PLCγ1) and increase the phosphorylation of cAMP-related element binding protein, a major transcription factor mediating neuronal plasticity. In contrast, we have not observed any modulation of the phosphorylation of TrkB Shc binding site, phosphorylation of mitogen-activated protein kinase or AKT by antidepressants. We also show that in the forced swim test, the behavioral effects of specific serotonergic antidepressant citalopram, but not those of the specific noradrenergic antidepressant reboxetine, are crucially dependent on TrkB signaling. Finally, brain monoamines seem to be critical mediators of antidepressant-induced TrkB activation, as antidepressants reboxetine and citalopram do not produce TrkB activation in the brains of serotonin- or norepinephrine-depleted mice. In conclusion, our data suggest that rapid activation of the TrkB neurotrophin receptor and PLCγ1 signaling is a common mechanism for all antidepressant drugs.

Synthesis and chromatographic properties of an HPLC chiral stationary phase based upon human serum albumin

SummaryA new HPLC stationary phase was synthesized by thein situ covalent immobilization of human serum albumin (HSA). The protein was immobilized on a commerically available diol column which had been activated with 1,1-carbonyldiimidazole. Initial chromatographic studies show that this phase can be used for chiral separations of enantiomeric solutes and that these separations may reflectin vitro binding to the HSA. The effects of mobile phase composition and temperature on the stereochemical resolutions are reported.

Oxidative stress-related biomarkers in autism: systematic review and meta-analyses.

Autism spectrum disorders (ASDs) are rarely diagnosed in children younger than 2 years, because diagnosis is based entirely on behavioral tests. Oxidative damage may play a central role in this pathogenesis, together with the interconnected transmethylation cycle and transsulfuration pathway. In an attempt to clarify and quantify the relationship between oxidative stress-related blood biomarkers and ASDs, a systematic literature review was carried out. For each identified study, mean biomarker levels were compared in cases and controls providing a point estimate, the mean ratio, for each biomarker. After meta-analysis, the ASD patients showed decreased blood levels of reduced glutathione (27%), glutathione peroxidase (18%), methionine (13%), and cysteine (14%) and increased concentrations of oxidized glutathione (45%) relative to controls, whereas superoxide dismutase, homocysteine, and cystathionine showed no association with ASDs. For the C677T allele in the methylene tetrahydrofolate reductase gene (MTHFR), homozygous mutant subjects (TT) showed a meta-OR of 2.26 (95% CI 1.30-3.91) of being affected by ASD with respect to the homozygous nonmutant (CC). Case-control studies on blood levels of vitamins suggest a lack of association (folic acid and vitamin B12) or rare association (vitamins A, B6, C, D, E). Sparse results were available for other biomarkers (ceruloplasmin, catalase, cysteinylglycine, thiobarbituric acid-reactive substances, nitric oxide) and for polymorphisms in other genes. Existing evidence is heterogeneous and many studies are limited by small sample size and effects. In conclusion, existing evidence suggests a role for glutathione metabolism, the transmethylation cycle, and the transsulfuration pathway, although these findings should be interpreted with caution, and larger, more standardized studies are warranted.

Gene expression elucidates functional impact of polygenic risk for schizophrenia.

Over 100 genetic loci harbor schizophrenia-associated variants, yet how these variants confer liability is uncertain. The CommonMind Consortium sequenced RNA from dorsolateral prefrontal cortex of people with schizophrenia (N = 258) and control subjects (N = 279), creating a resource of gene expression and its genetic regulation. Using this resource, ∼20% of schizophrenia loci have variants that could contribute to altered gene expression and liability. In five loci, only a single gene was involved: FURIN, TSNARE1, CNTN4, CLCN3 or SNAP91. Altering expression of FURIN, TSNARE1 or CNTN4 changed neurodevelopment in zebrafish; knockdown of FURIN in human neural progenitor cells yielded abnormal migration. Of 693 genes showing significant case-versus-control differential expression, their fold changes were ≤ 1.33, and an independent cohort yielded similar results. Gene co-expression implicates a network relevant for schizophrenia. Our findings show that schizophrenia is polygenic and highlight the utility of this resource for mechanistic interpretations of genetic liability for brain diseases.

Effects of fluoxetine on behavioral deficits evoked by chronic social stress in rats

Recently, we described an advanced model of chronic social stress in male rats based on the resident intruder paradigm. In this model, rats subjected to daily social stress for 5 weeks showed behavioral changes resembling anhedonia and motivational deficits in humans. In the present study, male Wistar rats were subjected to 5 weeks of daily social defeat by an aggressive conspecific and concomitant treatment with the antidepressant drug fluoxetine (10 mg/kg) after the first week of stress. Compared with controls, rats exposed to chronic stress had significantly reduced locomotor and exploratory activity (rearing and sniffing) and diminished preference for sucrose solution. These effects were paralleled by decreased body weight gain, increased adrenal weights and decreased plasma levels of testosterone measured post mortem. The stress-induced effects on locomotor activity and rearing behavior were counteracted by fluoxetine treatment.

Global analysis of transcription kinetics during competence development in Streptococcus pneumoniae using high density DNA arrays.

The kinetics of global changes in transcription patterns during competence development in Streptococcus pneumoniae was analysed with high‐density arrays. Four thousand three hundred and one clones of a S. pneumoniae library, covering almost the entire genome, were amplified by PCR and gridded at high density onto nylon membranes. Competence was induced by the addition of CSP (competence stimulating peptide) to S. pneumoniae cultures grown to the early exponential phase. RNA was extracted from samples at 5 min intervals (for a period of 30 min) after the addition of CSP. Radiolabelled cDNA was generated from isolated total RNA by random priming and the probes were hybridized to identical high‐density arrays. Genes whose transcription was induced or repressed during competence were identified. Most of the genes previously known to be competence induced were detected together with several novel genes that all displayed the characteristic transient kinetics of competence‐induced genes. Among the newly identified genes many have suggested functions compatible with roles in genetic transformation. Some of them may represent new members of the early or late competence regulons showing competence specific consensus sequences in their promoter regions. Northern experiments and mutational analysis were used to confirm some of the results.