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1.
Regul Toxicol Pharmacol ; 126: 105029, 2021 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-34455009

RESUMO

In drug development, nonclinical safety assessment is pivotal for human risk assessment and support of clinical development. Selecting the relevant/appropriate animal species for toxicity testing increases the likelihood of detecting potential effects in humans, and although recent regulatory guidelines state the need to justify or dis-qualify animal species for toxicity testing, individual companies have developed decision-processes most appropriate for their molecules, experience and 3Rs policies. These generally revolve around similarity of metabolic profiles between toxicology species/humans and relevant pharmacological activity in at least one species for New Chemical Entities (NCEs), whilst for large molecules (biologics) the key aspect is similarity/presence of the intended human target epitope. To explore current industry practice, a questionnaire was developed to capture relevant information around process, documentation and tools/factors used for species selection. Collated results from 14 companies (Contract Research Organisations and pharmaceutical companies) are presented, along with some case-examples or over-riding principles from individual companies. As the process and justification of species selection is expected to be a topic for continued emphasis, this information could be adapted towards a harmonized approach or best practice for industry consideration.


Assuntos
Avaliação Pré-Clínica de Medicamentos/métodos , Indústria Farmacêutica/métodos , Modelos Animais , Testes de Toxicidade/métodos , Produtos Biológicos/toxicidade , Indústria Farmacêutica/normas , Especificidade da Espécie , Testes de Toxicidade/normas
2.
Mol Immunol ; 39(1-2): 1-8, 2002 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-12213321

RESUMO

CS1 is a novel member of the CD2 subset of immunoglobulin superfamily (IgSF) expressed on NK, T and stimulated B cells. The cytoplasmic domain of CS1 contains immunoreceptor tyrosine-based switch motif (ITSM) which is present in 2B4, SLAM and CD84. The signaling adaptor molecule SAP/SH2D1A, the defective gene in X-linked lymphoproliferative disease (XLPD), binds to ITSM and regulates immune cell function. However, recent studies indicate that CS1 may be regulated by a SAP-independent mechanism. In this study, we have examined the ligand specificity of CS1 and the effect of CS1 interaction with its ligand on the cytolytic activity of YT, a human NK cell line. Recombinant fusion protein, CS1-Ig, containing the CS1 extracellular domain and Fc portion of the human IgG bound cells transfected with CS1. CS1-Ig did not show any binding to cells expressing other members of the CD2 family. The cytolytic activity of YT was enhanced in presence of soluble CS1-Ig fusion protein. These results demonstrate that CS1 is a self-ligand and homophilic interaction of CS1 regulates NK cell cytolytic activity.


Assuntos
Células Matadoras Naturais/imunologia , Receptores Imunológicos/fisiologia , Motivos de Aminoácidos , Linhagem Celular , Citotoxicidade Imunológica , Humanos , Receptores Imunológicos/química , Receptores Imunológicos/imunologia , Proteínas Recombinantes de Fusão/metabolismo , Família de Moléculas de Sinalização da Ativação Linfocitária
3.
Mol Immunol ; 40(16): 1157-63, 2004 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-15104121

RESUMO

Natural killer cell functions are regulated by signals through activating and inhibitory receptors. These receptors belong to the immunoglobulin superfamily or the lectin superfamily. We have previously identified a lectin-like transcript, LLT1, expressed in human NK cells. In the present study, we have generated a monoclonal antibody, L9.7, that specifically binds LLT1 receptor and studied the functional role of LLT1 in human NK cells. Binding of mAb L9.7 to surface LLT1 induced IFN-gamma production, but did not modulate cytotoxicity by YT cells, a human NK cell line. We further demonstrate that in resting NK cells as well as in IL-2 activated NK cells LLT1 induced IFN-gamma production, but not cytotoxicity. Excess amounts of L9.7 mAb failed to increase natural or antibody-dependent cell-mediated cytolytic activity, whereas minimal amounts achieved maximal production of IFN-gamma by YT and activated NK cells. These findings further support the separation of signaling pathways that regulate cytotoxicity and IFN-gamma production in resting as well as activated NK cells.


Assuntos
Interferon gama/biossíntese , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Lectinas Tipo C/fisiologia , Receptores de Superfície Celular/fisiologia , Anticorpos Monoclonais/metabolismo , Linhagem Celular Tumoral , Testes Imunológicos de Citotoxicidade , Dimerização , Relação Dose-Resposta Imunológica , Humanos , Interferon gama/metabolismo , Lectinas Tipo C/metabolismo , Ativação Linfocitária , Receptores de Superfície Celular/metabolismo
4.
J Med Chem ; 47(27): 6854-63, 2004 Dec 30.
Artigo em Inglês | MEDLINE | ID: mdl-15615534

RESUMO

A dozen 24-sulfoximine analogues of the hormone 1alpha,25-dihydroxyvitamin D(3) were prepared, differing not only at the stereogenic sulfoximine stereocenter but also at the A-ring. Although these sulfoximines were not active transcriptionally and were only very weakly antiproliferative, some of them are powerful hydroxylase enzyme inhibitors. Specifically, 24-(S)-NH phenyl sulfoximine 3a is an extremely potent CYP24 inhibitor (IC(50) = 7.4 nM) having low calcemic activity. In addition, this compound shows high selectivity toward the CYP24 enzyme in comparison to CYP27A1 (IC(50) > 1000 nM) and CYP27B (IC(50) = 554 nM).


Assuntos
Calcitriol/análogos & derivados , Cálcio/urina , Inibidores das Enzimas do Citocromo P-450 , Esteroide Hidroxilases/antagonistas & inibidores , Sulfonas/síntese química , Animais , Calcitriol/farmacologia , Masculino , Ratos , Ratos Endogâmicos F344 , Relação Estrutura-Atividade , Sulfonas/farmacologia , Vitamina D3 24-Hidroxilase
6.
Int J Cancer ; 119(8): 1819-28, 2006 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-16708384

RESUMO

1Alpha,25-dihydroxyvitamin D3 (1,25D3) displays potent antiproliferative activity in a variety of tumor model systems and is currently under investigation in clinical trials in cancer. Studies were initiated to explore its potential in nonsmall cell lung cancer (NSCLC), as effective approaches to the treatment of that disease are needed. In evaluating factors that may affect activity in NSCLC, the authors found that CYP24 (25-hydroxyvitamin D3-24-hydroxylase), the enzyme that catabolizes 1,25D3, is frequently expressed in NSCLC cell lines but not in the nontumorigenic bronchial epithelial cell line, Beas2B. CYP24 expression by RT-PCR was also detected in 10/18 primary lung tumors but in only 1/11 normal lung tissue specimens. Tumor-specific CYP24 upregulation was confirmed at the protein level via immunoblot analysis of patient-matched normal lung tissue and lung tumor extracts. Enzymatically active CYP24 is expected to desensitize NSCLC cells to 1,25D3. The authors therefore implemented a high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) assay for 1,25D3 and its CYP24-generated metabolites to determine whether NSCLC cells express active enzyme. Analysis of NSCLC cell cultures revealed time-dependent loss of 1,25D3 coincident with the appearance of CYP24-generated metabolites. MK-24(S)-S(O)(NH)-Ph-1, a specific inhibitor of CYP24, slowed the loss of 1,25D3 and increased 1,25D3 half-life. Furthermore, combination of 1,25D3 with MK-24(S)-S(O)(NH)-Ph-1 resulted in a significant decrease in the concentration of 1,25D3 required to achieve maximum growth inhibition in NSCLC cells. These data suggest that increased CYP24 expression in lung tumors restricts 1,25D3 activity and support the preclinical evaluation of CYP24 inhibitors for lung cancer treatment.


Assuntos
Carcinoma Pulmonar de Células não Pequenas/enzimologia , Carcinoma Pulmonar de Células não Pequenas/patologia , Esteroide Hidroxilases/metabolismo , Vitamina D/metabolismo , Idoso , Carcinoma Pulmonar de Células não Pequenas/genética , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Feminino , Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Pessoa de Meia-Idade , RNA Mensageiro/genética , Esteroide Hidroxilases/antagonistas & inibidores , Esteroide Hidroxilases/genética , Vitamina D/farmacologia , Vitamina D3 24-Hidroxilase
7.
Immunology ; 109(3): 432-9, 2003 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-12807490

RESUMO

2B4 (CD244) is a member of the CD2 subset of the immunoglobulin superfamily and functions as a triggering molecule on natural killer (NK) cells. Previously, we have found that 2B4-mediated activation of NK cells involves complex interactions involving LAT, Ras, Raf, ERK and p38 and that cytolytic function and cytokine production may be regulated by distinct pathways. Here we assessed the role of protein kinase C (PKC) in 2B4-mediated cytotoxicity of YT cells, a human NK cell line. Our data indicate that PKC-delta is activated upon stimulation with monoclonal antibody against 2B4. Treatment with the PKC inhibitor, bisindolylmaleimide I (Gö6850), of YT cells or YT cells depleted of Ca2+-dependent isoforms of PKC prior to 2B4 stimulation, resulted in inhibition of natural cytotoxicity and redirected antibody-dependent cellular cytotoxicity. However, inhibition of PKC failed to block 2B4 stimulation of interferon-gamma secretion as opposed to pretreatment with LY294002, a phosphoinositide 3-kinase inhibitor. We also examined the effect of phorbol 12-myristate 13-acetate (PMA) induction on 2B4 gene transcription. PMA induction resulted in a more than two-fold increase of 2B4 transcription. However, when we introduced a three-base substitution mutation to disrupt the activator protein-1 binding site at (-106 to -100) in the 2B4 promoter, we found complete loss of transcriptional activity, including the two-fold increase due to PMA induction of PKC. The present study indicated that PKC may play an important role in 2B4 signalling and activator protein-1 activation.


Assuntos
Antígenos CD , Citotoxicidade Imunológica/imunologia , Células Matadoras Naturais/imunologia , Glicoproteínas de Membrana/imunologia , Proteína Quinase C/imunologia , Receptores Imunológicos , Fator de Transcrição AP-1/metabolismo , Citotoxicidade Celular Dependente de Anticorpos/imunologia , Humanos , Interferon gama/biossíntese , Transdução de Sinais/imunologia , Família de Moléculas de Sinalização da Ativação Linfocitária , Acetato de Tetradecanoilforbol/imunologia , Células Tumorais Cultivadas , Regulação para Cima/imunologia
8.
J Biol Chem ; 279(8): 6305-14, 2004 Feb 20.
Artigo em Inglês | MEDLINE | ID: mdl-14660610

RESUMO

Long chain fatty acids have recently emerged as critical signaling molecules in neuronal, cardiovascular, and renal processes, yet little is presently known about the precise mechanisms controlling their tissue distribution and bioactivation. We have identified a novel cytochrome P450, CYP2U1, which may play an important role in modulating the arachidonic acid signaling pathway. Northern blot and real-time PCR analysis demonstrated that CYP2U1 transcripts were most abundant in the thymus and the brain (cerebellum), indicating a specific physiological role for CYP2U1 in these tissues. Recombinant human CYP2U1 protein, expressed in baculovirus-infected Sf9 insect cells, was found to metabolize arachidonic acid exclusively to two region-specific products as determined by liquid chromatography-mass spectrometry. These metabolites were identified as 19- and 20-hydroxy-modified arachidonic acids by liquid chromatography-tandem mass spectrometry analysis. In addition to omega/omega-1 hydroxylation of arachidonic acid, CYP2U1 protein also catalyzed the hydroxylation of structurally related long chain fatty acid (docosahexaenoic acid) but not fatty acids such as lauric acid or linoleic acid. This is the first report of the cloning and functional expression of a new human member of P450 family 2, CYP2U1, which metabolizes long chain fatty acids. Based on the ability of CYP2U1 to generate bioactive eicosanoid derivatives, we postulate that CYP2U1 plays an important physiological role in fatty acid signaling processes in both cerebellum and thymus.


Assuntos
Encéfalo/enzimologia , Citocromo P-450 CYP4A/metabolismo , Sistema Enzimático do Citocromo P-450/biossíntese , Sistema Enzimático do Citocromo P-450/química , Ácidos Graxos/química , Oxigenases de Função Mista/biossíntese , Oxigenases de Função Mista/química , Timo/enzimologia , Sequência de Aminoácidos , Animais , Ácidos Araquidônicos/metabolismo , Baculoviridae , Sequência de Bases , Western Blotting , Catálise , Linhagem Celular , Cromatografia Líquida , Família 2 do Citocromo P450 , DNA Complementar/metabolismo , Etiquetas de Sequências Expressas , Humanos , Ácidos Hidroxieicosatetraenoicos/química , Immunoblotting , Insetos , Espectrometria de Massas , Microssomos/metabolismo , Dados de Sequência Molecular , Família Multigênica , Filogenia , Estrutura Terciária de Proteína , RNA Mensageiro/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Transdução de Sinais , Especificidade por Substrato , Timo/metabolismo , Fatores de Tempo , Distribuição Tecidual , Ácido alfa-Linolênico/química
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