Paolo Gravina

Novel pharmacokinetic behavior of intravenous busulfan in children with thalassemia undergoing hematopoietic stem cell transplantation: a prospective evaluation of pharmacokinetic and pharmacodynamic profile with therapeutic drug monitoring

We prospectively studied the pharmacokinetics (PK) and clinical outcomes of intravenous busulfan (Bu) in 71 children with preexisting liver damage who underwent hematopoietic stem cell transplantation for thalassemia. Intravenous Bu was administered every 6 hours as part of a conditioning regimen with PK-based dose adjustment to target a conservative area under the concentration-versus-time curve (AUC) range (900-1350 microMol*min). The first-dose Bu clearance (CL) was significantly higher than the subsequent daily CL that remained unchanged in the ensuing days. One-third of patients required dose escalation based on dose 1 AUC, whereas dose reduction was needed in the subsequent days. At doses 5, 9, and 13, 78%, 81%, and 87% of patients, respectively, achieved the target range of AUC. A population PK analysis confirmed that the first-dose CL was 20% higher and that body weight was the most important covariate to explain PK variability. Patients with variant GSTA1*B had a 10% lower Bu CL than wild-type. These results suggest that the disease-specific behavior of intravenous Bu PK should be considered for PK-guided dose adjustment in patients with thalassemia, and the use of a conservative AUC range resulted in low toxicity, good engraftment, and good survival rate.

Valproic acid induces apoptosis, p16INK4A upregulation and sensitization to chemotherapy in human melanoma cells.

It is known that melanoma develops as a consequence of multifactorial alterations. To date several studies indicate the effective implication of p16 as a tumor suppressor gene with a major role in either the development or progression of human melanoma. Deregulation of melanoma cell growth has been widely associated with mutations in the p16-cyclin D/cdk4-pRb pathway. Recently anticancer therapies are focused on restoration of p16 CDK inhibitory function and other proteins unregulated in melanoma cell cycle pathway (e.g., c-myc, p27). A combined strategy for restoration of normal homeostasis in the melanoma skin with targeted delivery of apoptosis-inducing agents does not seems to be far obtain. New class of antitumoral agents are emerging: histone deacetylase (HDAC) inhibitors have attracted much interest because of their ability to arrest cell growth, induce cell differentiation, and in some cases, induce apoptosis of cancer cells. Recently, attention has been focused on the ability of HDAC inhibitors to induce perturbation in cell cycle regulatory protein (e.g., p21CIP1) and down-regulation of survival signalling pathway. In the present study, we have examined the effect of valproic acid (VPA) on M14 human melanoma cell line. Here we observed that VPA induces cell cycle arrest and apoptosis sensitising melanoma cells to cis-platin and etoposide treatment. IC50 dose (2,99 mM) of VPA was able to induce G1 arrest (up to 75 %) in association with up-regulation of p16, p21 and cyclin-D1 related to Rb ipo-phosphorilation. In addition VPA activated apoptosis (50 %) in M14 cells, when given alone or in combination with antitumoral agents. The ability of valproic acid to re-established the G1 pathway in melanoma cells suggests a potential application of VPA in melanoma therapeutic protocols.

Genetic polymorphisms of glutathione S-transferases GSTM1, GSTT1, GSTP1 and GSTA1 as risk factors for schizophrenia.

Oxidative damage is thought to play a role in the predisposition to schizophrenia. We determined if the polymorphisms of the GSTP1, GSTM1, GSTT1 and GSTA1 genes, which affect the activity of these enzymes against oxidative stress, have a role as susceptibility genes for schizophrenia, analyzing 138 schizophrenic patients and 133 healthy controls. We found that the combination of the absence of GSTM1 gene with the of the GSTM1 gene with the polymorphism GSTA1*B/*B, and the presence of the GSTT1 gene, represents a risk factor for schizophrenia, indicating that the combination of different GST polymorphisms has a role in the predisposition to schizophrenia, probably affecting the capacity of the cell to detoxify the oxidized metabolites of catecholamines.

Effects of dutasteride on the expression of genes related to androgen metabolism and related pathway in human prostate cancer cell lines

SummaryAndrogens play an important role in controlling the growth of the normal prostate gland and in the pathogenesis of benign prostate hyperplasia, and prostate cancer. Although testosterone is the main androgen secreted from the testes, dihydrotestosterone (DHT), a more potent androgen converted from testosterone by 5α-reductase isozymes, type I and II, is the major androgen in the prostate cells. The aim of this study is to investigate the cellular and molecular effects of dutasteride, a potent inhibitor of 5α-reductase type I and type II, in androgen-responsive (LNCaP) and androgen-unresponsive (DU145) human prostate cancer(PCa) cell lines. The expression pattern of 190 genes, selected on the basis of their proved or potential role in prostate cancerogenesis related to androgen signalling, were analysed using a low density home-made oligoarray (AndroChip 2). Our results show that dutasteride reduces cell viability and cell proliferation in both cell lines tested. AndroChip 2 gene signature identified in LNCaP a total of 11 genes differentially expressed (FC ≥ ±1.5). Eight of these genes, were overexpressed and three were underexpressed. Overexpressed genes included genes encoding for proteins involved in biosynthesis and metabolism of androgen (HSD17B1;HSD17B3;CYP11B2), androgen receptor and androgen receptor co-regulators (AR;CCND1), and signal transduction(ERBB2; V-CAM; SOS1) whereas, underexpressed genes (KLK3; KLK2; DHCR24) were androgen-regulated genes (ARGs). No differentially expressed genes were scored in DU145. Microarray data were confirmed by quantitative real-time PCR assay (QRT-PCR). These data offer a selective genomic signature for dutasteride treatment in prostate epithelial cells and provide important insights in prostate cancer pathophysiology.

Valproic acid induces neuroendocrine differentiation and UGT2B7 up-regulation in human prostate carcinoma cell line

Prostate cancer originates as an androgen-dependent hyperproliferation of the epithelial cells of the gland and it evolves in an androgen-independent, highly aggressive cancer for which no successful therapy is available to date. Neuroendocrine (NE) differentiation plays an important role in the progression of prostate cancer to an androgen-independent state with profound impact on prostate cancer (CaP) therapies. Actually, new approaches on treating advanced prostate cancer are focused on modulators of epigenetic transcriptional regulation. A new class of antitumoral agents is emerging: histone deacetylase (HDAC) inhibitors are interesting for their ability to arrest cell growth, to induce cell differentiation, and in some cases, to induce apoptosis of cancer cells. We studied the effect of valproic acid (VPA), an inhibitor of HDAC, in the human prostate androgen-dependent cancer cell line LNCaP. We observed that VPA promotes neuroendocrine-like differentiation associated with an increase in the expression of neuron-specific enolase, a decrease in prostate-specific antigen, and a down-regulation of androgen receptor protein, suggesting a modulation in the responsiveness to androgen therapy. Furthermore, selective gene expression profiling using a low-density microarray showed that VPA was able to modulate the expression of different androgen metabolism genes. We observed a down-regulation of androgen receptor coregulator (ARA24) and prostate-specific antigen, and an up-regulation of some of the UDP-glucuronosyltransferases (UGT2B11 and UGT2B7) implicated in catabolism of dihydrotestosterone (DHT) was detected. Even though UGT2B7 has only about one-tenth to one-hundredth the activity of UGT2B15 and 2B17 toward active androgens and we did not found any modulation in gene expression of these enzymes, it can be hypothesized that VPA might enhance DHT catabolism in this in vitro model and induces NE differentiation. Our data seem to raise concern about CaP treatment with VPA.

Delusion Symptoms and Response to Antipsychotic Treatment are Associated with the 5-HT2A Receptor Polymorphism (102T/C) in Alzheimer's Disease: A 3-Year Follow-up Longitudinal Study

Although the etiology of psychotic symptoms (hallucinations and delusions) in Alzheimers disease is still not known, alterations in serotonergic neurotransmission have been proposed. In a 3-year follow-up study, we evaluated the association of serotonin (5-HT) receptor 5-HT2a 102T/C polymorphism (allelic variants CC, CT and TT) with psychotic symptom severity and response to treatment with atypical antipsychotics (risperidone, olanzapine and quietapine) in 80 patients with a diagnosis of probable Alzheimers disease. The Neuropsychiatric Inventory (NPI) was administered to determine the frequency and severity (FxS) of psychotic and other behavioral symptoms. There was a significant difference in the NPI FxS delusion score among the three variants of the 5-HT2a 102T/C polymorphism, with patients carrying the TT genotype the most delusional during the follow-up period. In particular, NPI FxS delusion score was higher in TT than in CC genotype at year 2. Moreover, patients with delusion symptoms carrying the CT and TT genotypes were resistant to the treatment with antipsychotic drugs. Thus our study, although at preliminary level, suggests that the presence of T allele of the 102T/C polymorphism in patients with Alzheimers disease is associated with both increased presence of delusion symptoms and treatment-resistance to second generation antipsychotic drugs.

Paroxetine rapidly modulates the expression of brain-derived neurotrophic factor mRNA and protein in a human glioblastoma-astrocytoma cell line.

Neuronal upregulation of the brain-derived neurotrophic factor (BDNF) gene appears to be a crucial factor for the efficacy of antidepressants. However, besides neurons, little information is present on the modulation of BDNF by antidepressants at RNA and protein levels in other cell types of the central nervous system. Glial cells are able to store and release BDNF, and it has been hypothesized that glial dysfunction may contribute to the etiopathogenesis of depression. Thus, in this study we used the human glioblastoma-astrocytoma cell line U87 exposed to the antidepressant drug paroxetine, and evaluated BDNF mRNA and protein expression. In addition, since the BDNF gene can be posttranscriptionally modulated by a family of microRNA, we also evaluated the levels for one of these microRNA (miR-30a-5p) in the U87 cell line during paroxetine treatment. We found that paroxetine treatment rapidly increased BDNF in U87 cells, resulting from an induction of BDNF mRNA expression and de novo protein synthesis, and that these increases occurred in a time-dependent manner. Paroxetine effects were evident at 6 h of incubation for BDNF mRNA and at 12 h for BDNF protein. In addition, the transcriptional BDNF inhibitor miR-30a-5p was also overexpressed at 6 and 12 h of paroxetine incubation. These findings indicate that the U87 cell line, an in vitro model of glial cells, rapidly responds to paroxetine by increasing BDNF production, and that these effects are potentially limited by microRNA induction. These data may contribute to explain the action of paroxetine on cells of nonneuronal origin.

The (AAT)n repeat of the cannabinoid CB1 receptor gene influences disease progression in relapsing multiple sclerosis

Background: Genetic and pharmacological inactivation of cannabinoid CB1 receptors (CB1Rs) exacerbates disease course in experimental autoimmune encephalomyelitis, suggesting that CB1Rs might play a role in the neurodegenerative damage associated with multiple sclerosis (MS). Objectives: To see whether CNR1 gene polymorphism could influence disease progression in relapsing–remitting MS. Methods: The genotype of 350 patients for the number of AAT repeats was characterized and correlation studies were performed with measures of disease severity and progression. Results: MS patients with the homozygous genotype for long AAT repeats in the CNR1 gene had more severe disease and higher risk of progression. These subjects had significantly higher scores on both the progression index and the MS severity scale. Furthermore, the percentage of patients with MS functional composite score progression or Bayesian Risk Estimate for MS (BREMS) score ≥2 (considered at very high risk of secondary progression) was significantly higher in the AAT long group than in the short group, while the frequency of patients with BREMS score ≤−0.63 (very likely to remain progression-free) was not significantly different between the two groups, although lower in the long group. Finally, the frequency of patients prescribed a second-line treatment was significantly higher among subjects of the AAT long group, providing a further, indirect indication of higher disease severity. Conclusions: The results of the present investigation point to CB1R as an important modulator of disease severity in relapsing MS subjects.

Thrombin-activatable fibrinolysis inhibitor polymorphisms and recurrent pregnancy loss.

OBJECTIVE To investigate the possible association between selected thrombin-activatable fibrinolysis inhibitor (TAFI) single nucleotide polymorphisms (SNPs) and recurrent pregnancy loss (RPL). DESIGN Case-control study. SETTING University hospital. PATIENT(S) One hundred fifty-eight women (86 cases and 72 controls). INTERVENTION(S) Determination of TAFI SNPs -438A/G, +505A/G, +1040T/C, +1542C/G, and +1583A/T by polymerase chain reaction (PCR) reactions and sequencing analysis performed on peripheral blood samples. MAIN OUTCOME MEASURE(S) Analysis of the genotype and allele frequencies of TAFI SNPs -438A/G, +505A/G, +1040T/C, +1542C/G, and +1583A/T in women with and without RPL. RESULT(S) Genotype and allele frequencies of TAFI +505 and +1583 SNPs were significantly different in women with RPL compared with control women. The frequencies of the +505A/A and +505G/G genotypes were 1.2% and 61.6% in women with RPL and 13.9% and 43.1% in control women, respectively. The frequencies of the +1583A/A and +1583T/T genotypes were 1.2% and 61.6% in women with RPL and 13.9% and 45.8% in control women, respectively. The genotype and allele frequencies at TAFI position -438, +1040, and +1542 were not significantly different between RPL and control women. CONCLUSION(S) The SNPs leading to increased TAFI levels are associated with a reduced risk of RPL. It is possible that TAFI is involved in RPL.

Association between a Genetic Variant of Type-1 Cannabinoid Receptor and Inflammatory Neurodegeneration in Multiple Sclerosis

Genetic ablation of type-1 cannabinoid receptors (CB1Rs) exacerbates the neurodegenerative damage of experimental autoimmune encephalomyelitis, the rodent model of multiple sclerosis (MS). To address the role on CB1Rs in the pathophysiology of human MS, we first investigated the impact of AAT trinucleotide short tandem repeat polymorphism of CNR1 gene on CB1R cell expression, and secondly on the inflammatory neurodegeneration process responsible for irreversible disability in MS patients. We found that MS patients with long AAT repeats within the CNR1 gene (≥12 in both alleles) had more pronounced neuronal degeneration in response to inflammatory white matter damage both in the optic nerve and in the cortex. Optical Coherence Tomography (OCT), in fact, showed more severe alterations of the retinal nerve fiber layer (RNFL) thickness and of the macular volume (MV) after an episode of optic neuritis in MS patients carrying the long AAT genotype of CNR1. MS patients with long AAT repeats also had magnetic resonance imaging (MRI) evidence of increased gray matter damage in response to inflammatory lesions of the white matter, especially in areas with a major role in cognition. In parallel, visual abilities evaluated at the low contrast acuity test, and cognitive performances were negatively influenced by the long AAT CNR1 genotype in our sample of MS patients. Our results demonstrate the biological relevance of the (AAT)n CNR1 repeats in the inflammatory neurodegenerative damage of MS.