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Artigo em Inglês | MEDLINE | ID: mdl-31150018


Collection of an adequate amount of autologous haematopoietic stem progenitor cells (HSPC) is required for ex vivo manipulation and successful engraftment for certain inherited disorders. Fifty-seven paediatric patients (age 0.5-11.4 years) underwent a bone marrow harvest for the purpose of HSPC gene therapy (GT), including adenosine deaminase-severe combined immunodeficiency (ADA-SCID), Wiskott-Aldrich syndrome (WAS) and metachromatic leukodystrophy (MLD) patients. Total nucleated cells and the percentage and absolute counts of CD34+ cells were calculated at defined steps of the procedure (harvest, CD34+ cell purification, transduction with the gene transfer vector and infusion of the medicinal product). A minimum CD34+ cell dose for infusion was 2 × 106/kg, with an optimal target at 5-10 × 106/kg. Median volume of bone marrow harvested was 34.2 ml/kg (range 14.2-56.6). The number of CD34+ cells collected correlated inversely with weight and age in all patients and particularly in the MLD children group. All patients reached the minimum target dose for infusion: median dose of CD34+ cells/kg infused was 10.3 × 106/kg (3.7-25.9), with no difference among the three groups. Bone marrow harvest of volumes > 30 ml/kg in infants and children with ADA-SCID, WAS and MLD is well tolerated and allows obtaining an adequate dose of a medicinal product for HSPC-GT.

Lancet Haematol ; 6(5): e239-e253, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30981783


BACKGROUND: Wiskott-Aldrich syndrome is a rare, life-threatening, X-linked primary immunodeficiency characterised by microthrombocytopenia, infections, eczema, autoimmunity, and malignant disease. Lentiviral vector-mediated haemopoietic stem/progenitor cell (HSPC) gene therapy is a potentially curative treatment that represents an alternative to allogeneic HSPC transplantation. Here, we report safety and efficacy data from an interim analysis of patients with severe Wiskott-Aldrich syndrome who received lentiviral vector-derived gene therapy. METHODS: We did a non-randomised, open-label, phase 1/2 clinical study in paediatric patients with severe Wiskott-Aldrich syndrome, defined by either WAS gene mutation or absent Wiskott-Aldrich syndrome protein (WASP) expression or a Zhu clinical score of 3 or higher. We included patients who had no HLA-identical sibling donor available or, for children younger than 5 years of age, no suitable 10/10 matched unrelated donor or 6/6 unrelated cord blood donor. After treatment with rituximab and a reduced-intensity conditioning regimen of busulfan and fludarabine, patients received one intravenous infusion of autologous CD34+ cells genetically modified with a lentiviral vector encoding for human WAS cDNA. The primary safety endpoints were safety of the conditioning regimen and safety of lentiviral gene transfer into HSPCs. The primary efficacy endpoints were overall survival, sustained engraftment of genetically corrected HSPCs, expression of vector-derived WASP, improved T-cell function, antigen-specific responses to vaccinations, and improved platelet count and mean platelet volume normalisation. This interim analysis was done when the first six patients treated had completed at least 3 years of follow-up. The planned analyses are presented for the intention-to-treat population. This trial is registered with (number NCT01515462) and EudraCT (number 2009-017346-32). FINDINGS: Between April 20, 2010, and Feb 26, 2015, nine patients (all male) were enrolled of whom one was excluded after screening; the age range of the eight treated children was 1·1-12·4 years. At the time of the interim analysis (data cutoff April 29, 2016), median follow-up was 3·6 years (range 0·5-5·6). Overall survival was 100%. Engraftment of genetically corrected HSPCs was successful and sustained in all patients. The fraction of WASP-positive lymphocytes increased from a median of 3·9% (range 1·8-35·6) before gene therapy to 66·7% (55·7-98·6) at 12 months after gene therapy, whereas WASP-positive platelets increased from 19·1% (range 4·1-31·0) to 76·6% (53·1-98·4). Improvement of immune function was shown by normalisation of in-vitro T-cell function and successful discontinuation of immunoglobulin supplementation in seven patients with follow-up longer than 1 year, followed by positive antigen-specific response to vaccination. Severe infections fell from 2·38 (95% CI 1·44-3·72) per patient-year of observation (PYO) in the year before gene therapy to 0·31 (0·04-1·11) per PYO in the second year after gene therapy and 0·17 (0·00-0·93) per PYO in the third year after gene therapy. Before gene therapy, platelet counts were lower than 20 × 109 per L in seven of eight patients. At the last follow-up visit, the platelet count had increased to 20-50 × 109 per L in one patient, 50-100 × 109 per L in five patients, and more than 100 × 109 per L in two patients, which resulted in independence from platelet transfusions and absence of severe bleeding events. 27 serious adverse events in six patients occurred after gene therapy, 23 (85%) of which were infectious (pyrexia [five events in three patients], device-related infections, including one case of sepsis [four events in three patients], and gastroenteritis, including one case due to rotavirus [three events in two patients]); these occurred mainly in the first 6 months of follow-up. No adverse reactions to the investigational drug product and no abnormal clonal proliferation or leukaemia were reported after gene therapy. INTERPRETATION: Data from this study show that gene therapy provides a valuable treatment option for patients with severe Wiskott-Aldrich syndrome, particularly for those who do not have a suitable HSPC donor available. FUNDING: Italian Telethon Foundation, GlaxoSmithKline, and Orchard Therapeutics.

Terapia Genética , Vetores Genéticos/genética , Células-Tronco Hematopoéticas/metabolismo , Lentivirus/genética , Síndrome de Wiskott-Aldrich/genética , Síndrome de Wiskott-Aldrich/terapia , Criança , Pré-Escolar , Feminino , Terapia Genética/métodos , Transplante de Células-Tronco Hematopoéticas/métodos , Humanos , Lactente , Itália , Masculino , Mutação , Linfócitos T/imunologia , Linfócitos T/metabolismo , Condicionamento Pré-Transplante/métodos , Resultado do Tratamento , Síndrome de Wiskott-Aldrich/sangue , Síndrome de Wiskott-Aldrich/diagnóstico , Proteína da Síndrome de Wiskott-Aldrich/genética
J Med Chem ; 50(7): 1571-83, 2007 Apr 05.
Artigo em Inglês | MEDLINE | ID: mdl-17352462


Pharmacophore-based structural identification, synthesis, and structure-activity relationships of a new class of muscarinic M3 receptor antagonists, the diaryl imidazolidin-2-one derivatives, are described. The versatility of the discovered scaffold allowed for several structural modifications that resulted in the discovery of two distinct classes of compounds, specifically a class of tertiary amine derivatives (potentially useful for the treatment of overactive bladder by oral administration) and a class of quaternary ammonium salt derivatives (potentially useful for the treatment of respiratory diseases by the inhalation route of administration). In this paper, we describe the synthesis and biological activity of tertiary amine derivatives. For these compounds, selectivity for the M3 receptor toward the M2 receptor was crucial, because the M2 receptor subtype is mainly responsible for adverse systemic side effects of currently marketed muscarinic antagonists. Compound 50 showed the highest selectivity versus M2 receptor, with binding affinity for M3 receptor Ki = 4.8 nM and for M2 receptor Ki = 1141 nM. Functional in vitro studies on selected compounds confirmed the antagonist activity toward the M3 receptor and functional selectivity toward the M2 receptor.

Imidazolidinas/síntese química , Receptor Muscarínico M3/antagonistas & inibidores , Administração Oral , Animais , Função Atrial/efeitos dos fármacos , Células CHO , Células CACO-2 , Permeabilidade da Membrana Celular , Cricetinae , Cricetulus , Feminino , Humanos , Imidazolidinas/química , Imidazolidinas/farmacologia , Técnicas In Vitro , Masculino , Camundongos , Microssomos/metabolismo , Modelos Moleculares , Contração Muscular/efeitos dos fármacos , Músculo Liso/efeitos dos fármacos , Músculo Liso/fisiologia , Ensaio Radioligante , Ratos , Receptor Muscarínico M2/antagonistas & inibidores , Relação Estrutura-Atividade , Bexiga Urinária/efeitos dos fármacos , Bexiga Urinária/fisiologia
J Med Chem ; 50(7): 1693-7, 2007 Apr 05.
Artigo em Inglês | MEDLINE | ID: mdl-17352463


Synthesis and biological activity of a novel class of quaternary ammonium salt muscarinic M3 receptor antagonists, showing high selectivity versus the M2 receptor, are described. Selected compounds exhibited potent anticholinergic properties, in isolated guinea-pig trachea, and good functional selectivity for trachea over atria. In vivo, the same compounds potently inhibited acetylcholine-induced bronchoconstriction after intratracheal administration in the guinea pig.

Broncodilatadores/química , Imidazolidinas/síntese química , Compostos de Amônio Quaternário/síntese química , Receptor Muscarínico M3/antagonistas & inibidores , Animais , Broncoconstrição/efeitos dos fármacos , Broncodilatadores/síntese química , Broncodilatadores/farmacologia , Células CHO , Cricetinae , Cricetulus , Cobaias , Humanos , Imidazolidinas/química , Imidazolidinas/farmacologia , Técnicas In Vitro , Masculino , Contração Muscular/efeitos dos fármacos , Músculo Liso/efeitos dos fármacos , Músculo Liso/fisiologia , Contração Miocárdica/efeitos dos fármacos , Compostos de Amônio Quaternário/química , Compostos de Amônio Quaternário/farmacologia , Ensaio Radioligante , Relação Estrutura-Atividade , Traqueia/efeitos dos fármacos , Traqueia/fisiologia
J Pharmacol Exp Ther ; 308(2): 454-61, 2004 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-14593080


(-)-cis-1-Methyl-7-[[4-(2,6-dichlorophenyl)piperidin-1-yl]methyl]-6,7,8,9-tetrahydro-5H-benzocyclohepten-5-ol (SB-612111) is a novel human opiate receptor-like orphan receptor (ORL-1) antagonist that has high affinity for the clonal human ORL-1 receptor (hORL-1 K(i) = 0.33 nM), selectivity versus mu-(174-fold), delta-(6391-fold), and kappa (486-fold)-opioid receptors and is able to inhibit nociceptin signaling via hORL-1 in a whole cell gene reporter assay. SB-612111 has no measurable antinociceptive effects in vivo in the mouse hot-plate test after intravenous administration but is able to antagonize the antimorphine action of nociceptin [ED(50) = 0.69 mg/kg, 95% confidence limit (CL) = 0.34-1.21]. SB-62111 administration can also reverse tolerance to morphine in this model, established via repeated morphine administration. In addition, intravenous SB-612111 can antagonize nociceptin-induced thermal hyperalgesia in a dose-dependent manner (ED(50) = 0.62 mg/kg i.v., 95% CL = 0.22-1.89) and is effective per se at reversing thermal hyperalgesia in the rat carrageenan inflammatory pain model. These data show that an ORL-1 receptor antagonist may be a useful adjunct to chronic pain therapy with opioids and can be used to treat conditions in which thermal hyperalgesia is a significant component of the pain response.

Cicloeptanos/farmacologia , Tolerância a Medicamentos/fisiologia , Morfina/efeitos adversos , Antagonistas de Entorpecentes , Piperidinas/farmacologia , Animais , Sítios de Ligação , Células CHO , Cricetinae , Cicloeptanos/uso terapêutico , Humanos , Morfina/uso terapêutico , Dor/tratamento farmacológico , Piperidinas/uso terapêutico , Receptores Opioides
J Neurosci Res ; 72(5): 622-8, 2003 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-12749027


Brain-derived neurotrophic factor (BDNF) promotes a variety of neuromodulatory processes during development as well as in adulthood. This neurotrophin has been associated with synaptic plasticity, suggesting that its regulation may represent one of the mechanisms through which psychotropic drugs alter brain function. Because reduced glutamatergic function represents a major feature of schizophrenia, we investigated the effects of the concomitant administration of haloperidol or olanzapine with the N-methyl-D-aspartate (NMDA) receptor antagonist MK-801 on BDNF expression. MK-801 reduces the hippocampal expression of the neurotrophin; this effect was exacerbated by haloperidol, but it was normalized by olanzapine. Our data reveal a fine tuning of BDNF biosynthesis and a differential modulation by antipsychotic drugs when NMDA-mediated transmission is reduced, suggesting that haloperidol and olanzapine can produce different effects on brain plasticity through the modulation of BDNF expression.

Antipsicóticos/farmacologia , Fator Neurotrófico Derivado do Encéfalo/efeitos dos fármacos , Encéfalo/efeitos dos fármacos , Ácido Glutâmico/metabolismo , Pirenzepina/análogos & derivados , Receptores de N-Metil-D-Aspartato/antagonistas & inibidores , Esquizofrenia/tratamento farmacológico , Animais , Benzodiazepinas , Encéfalo/metabolismo , Encéfalo/fisiopatologia , Fator Neurotrófico Derivado do Encéfalo/biossíntese , Fator Neurotrófico Derivado do Encéfalo/genética , Maleato de Dizocilpina/farmacologia , Relação Dose-Resposta a Droga , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/fisiologia , Antagonistas de Aminoácidos Excitatórios/farmacologia , Haloperidol/farmacologia , Masculino , Plasticidade Neuronal/efeitos dos fármacos , Plasticidade Neuronal/fisiologia , Olanzapina , Pirenzepina/farmacologia , RNA Mensageiro/efeitos dos fármacos , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Receptores de N-Metil-D-Aspartato/metabolismo , Esquizofrenia/metabolismo , Esquizofrenia/fisiopatologia , Transmissão Sináptica/efeitos dos fármacos , Transmissão Sináptica/fisiologia