RESUMO
Animals frequently eat less after a test-article treatment in nonclinical toxicological studies, and it can be difficult to distinguish test article-derived toxicities from secondary changes related to this reduced food intake. Therefore, in this study, we restricted the food intake of cynomolgus monkeys (Cambodian, male, n=2 or 3, 48 ± 3 months old) to 25% of the control for two weeks and evaluated the effects on toxicological parameters (general conditions, body weight, electrocardiography, urinalysis, hematology, blood chemistry, bone marrow analysis, pathological examination). After 2 weeks, the monkeys exhibited decreases in bone marrow erythropoiesis (e.g., decreases in reticulocytes and bone marrow erythrocytes), as well as glycogenesis induction (e.g., increase in aspartate aminotransferase (AST)) and malnutrition (e.g., decrease in triglyceride and systemic adipocytes atrophy). Additionally, histopathological analysis revealed granuloma and inflammatory cell infiltration in coronary fat, which had never been found in previous food restriction studies using other animal species. These findings will enable researchers to more accurately evaluate the toxicological risks of test articles that simultaneously induce food intake reduction.
Assuntos
Ingestão de Alimentos , Alimentos , Masculino , Animais , Macaca fascicularis , Peso Corporal , EletrocardiografiaRESUMO
Therapeutic antibodies sometimes elicit anti-drug antibodies (ADAs) that can affect efficacy and safety. Engineered antibodies that contain artificial amino acid sequences are potentially highly immunogenic, but this is currently difficult to predict. Therefore, it is important to efficiently assess immunogenicity during the development of complex antibody-based formats. Here, we present an in vitro peripheral blood mononuclear cell-based assay that can be used to assess immunogenicity potential within 3 days. This method involves examining the frequency and function of interleukin (IL)-2-secreting CD4+ T cells induced by therapeutic antibodies. IL-2-secreting CD4+ T cells seem to be functionally relevant to the immunogenic potential due to their proliferative activity and the expression of several cytokines. The rates of the donors responding to low and high immunogenic proteins, mAb1, and keyhole limpet hemocyanin were 1.3% and 93.5%, respectively. Seven antibodies with known rates of immunogenicity (etanercept, emicizumab, abciximab, romosozumab, blosozumab, humanized anti-human A33 antibody, and bococizumab) induced responses in 1.9%, 3.8%, 6.4%, 10.0%, 29.2%, 43.8%, and 89.5% of donors, respectively. These data are comparable with ADA incidences in clinical settings. Our results show that this assay can contribute to the swift assessment and mechanistic understanding of the immunogenicity of therapeutic antibodies.
Assuntos
Interleucina-2 , Linfócitos T , Interleucina-2/farmacologia , Leucócitos Mononucleares/metabolismo , Citocinas/metabolismo , Linfócitos T CD4-PositivosRESUMO
Agonistic antibodies targeting CD137 have been clinically unsuccessful due to systemic toxicity. Because conferring tumor selectivity through tumor-associated antigen limits its clinical use to cancers that highly express such antigens, we exploited extracellular adenosine triphosphate (exATP), which is a hallmark of the tumor microenvironment and highly elevated in solid tumors, as a broadly tumor-selective switch. We generated a novel anti-CD137 switch antibody, STA551, which exerts agonistic activity only in the presence of exATP. STA551 demonstrated potent and broad antitumor efficacy against all mouse and human tumors tested and a wide therapeutic window without systemic immune activation in mice. STA551 was well tolerated even at 150 mg/kg/week in cynomolgus monkeys. These results provide a strong rationale for the clinical testing of STA551 against a broad variety of cancers regardless of antigen expression, and for the further application of this novel platform to other targets in cancer therapy. SIGNIFICANCE: Reported CD137 agonists suffer from either systemic toxicity or limited efficacy against antigen-specific cancers. STA551, an antibody designed to agonize CD137 only in the presence of extracellular ATP, inhibited tumor growth in a broad variety of cancer models without any systemic toxicity or dependence on antigen expression.See related commentary by Keenan and Fong, p. 20.This article is highlighted in the In This Issue feature, p. 1.
Assuntos
Trifosfato de Adenosina , Neoplasias , Animais , Anticorpos Monoclonais/farmacologia , Antígenos de Neoplasias , Imunoterapia , Camundongos , Neoplasias/tratamento farmacológico , Microambiente Tumoral , Membro 9 da Superfamília de Receptores de Fatores de Necrose TumoralRESUMO
Heat shock protein 90 (Hsp90) is a constitutively expressed molecular chaperone and plays an important role in the folding of client proteins with key regulatory roles in growth, survival, differentiation and metastasis. Because inhibition of Hsp90 degrades multiple oncogenic client proteins, it is considered to be an attractive anticancer therapy, and clinical trials of several Hsp90 inhibitors have been carried out. In the present study, two structurally distinct Hsp90 inhibitors, CH5164840 and CH5449302, were orally administered to beagle dogs to evaluate systemic toxicity. CH5164840 induced symptoms that suggest visual disorder, and ophthalmological observation and electroretinography (ERG) revealed loss of pupillary light reflex and abnormal waveforms, respectively. Histopathological examination showed changes in the photoreceptor cell layer and the outer nuclear layer of retina. On the other hand, while there were no clinical symptoms related to visual disorder, animals treated with CH5449302 showed similar abnormalities of ERG responses and histopathological changes in the photoreceptor cell layer and the outer nuclear layer of retina. The visual symptoms and abnormalities of ERG responses were noted at an earlier stage or lower dose than other toxicities in both compounds. Considering that two structurally distinct Hsp90 inhibitors induced a retinal toxicity in dogs after repeated administration, and that visual disorders were also reported in some clinical trials of Hsp90 inhibitors, it would seem highly likely that Hsp90 inhibition induces retinal toxicity. Also, our study indicated that a detailed ocular examination to evaluate the safety of Hsp90 inhibitors would be useful in both preclinical and clinical studies.
Assuntos
Benzoquinonas/toxicidade , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Compostos Heterocíclicos com 3 Anéis/toxicidade , Lactamas Macrocíclicas/toxicidade , Retina/efeitos dos fármacos , Triazinas/toxicidade , Administração Oral , Animais , Benzoquinonas/administração & dosagem , Cães , Eletrorretinografia , Compostos Heterocíclicos com 3 Anéis/administração & dosagem , Lactamas Macrocíclicas/administração & dosagem , Peso Molecular , Células Fotorreceptoras de Vertebrados/efeitos dos fármacos , Reflexo Pupilar/efeitos dos fármacos , Triazinas/administração & dosagem , Transtornos da Visão/induzido quimicamenteRESUMO
Some therapeutic antibodies as anticancer agents exert their effects through the host immune system, but the factors that predict their cytotoxicity, including complement-dependent cytotoxicity (CDC), are unclear. In the present study, we attempted to elucidate some of these factors in a preclinical model. CDC-related mesangiolysis caused by administration of the anti-Thy-1.1 antibody can be studied in the rat anti-Thy-1 glomerulonephritis model, so the model was used in this study. Three animals each were sacrificed at 0.5, 1, 8, 24 and 48 hours after i.v. administration of the anti-Thy-1.1 antibody at 1mg/kg. The distribution of the Thy-1.1 antigen and 2 membrane complement regulatory proteins (mCRPs), Crry and CD55, in three non-treated animals and the distribution of the injected antibody and C3 in the model was studied by immunohistochemistry. In the mesangial cells of the kidney, both expression of the antigen and distribution of the antibody with C3 deposition were observed with weak expression of mCRPs. There was also antigen and antibody distribution in the medullary cells of the adrenal gland and in the lymphocytes of the thymus but no C3 deposition, which was thought to be related to high expression of mCRPs. The antigen was observed in several other organs and tissues without distribution of the antibody. Cell death was only observed in the mesangial cells. These results clearly demonstrate that activation of CDC is regulated by several factors, such as distribution of the target molecule, antibody distribution and the balance among the molecules of the CDC cascade and mCRPs.
RESUMO
Dimethyl sulfoxide (DMSO) is a very common organic solvent used for dissolving lipophilic substances, for example for in vitro cell-based assays. At the same time, DMSO is known to be cytotoxic at high concentrations. Therefore, it is important to define threshold concentrations of DMSO for cells but relevant data at the molecular level are very limited. We have focused on conducting microarray analyses of human and rat hepatocytes treated with more than 100 chemicals in attempts to identify candidate biomarker genes. In the present study, the effects of DMSO on gene expression and cytotoxicity were assessed in human cryopreserved hepatocytes and rat primary cultured hepatocytes. A cytotoxicity test with lactate dehydrogenase (LDH) activity demonstrated DMSO to be noncytotoxic up to a concentration of 2% (v/v) in both cases and there were only few effects on the gene expression profiles up to 0.5% (v/v). The observed differences from controls were considered to be of little toxicological importance, but still need to be taken into account in interpretation of findings when DMSO is used at high concentration.
Assuntos
Dimetil Sulfóxido/toxicidade , Expressão Gênica/efeitos dos fármacos , Hepatócitos/efeitos dos fármacos , Solventes/toxicidade , Animais , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Perfilação da Expressão Gênica , Hepatócitos/metabolismo , Humanos , L-Lactato Desidrogenase/metabolismo , Masculino , Análise de Sequência com Séries de Oligonucleotídeos , Ratos , Ratos Sprague-DawleyRESUMO
The study examined the feasibility of screening for hepatotoxicity by an in vitro gene expression analysis using rat primary hepatocytes and Affymetrix Rat Toxicology U34 arrays. Hepatocytes were exposed for 6 or 24 h to eight drugs, with different mechanisms of hepatotoxicity, at one third of the cytotoxic concentration TC50, i.e. acetaminophen, cyclophosphamide, clofibrate, chlorpromazine, lithocholic acid, cisplatin, diclofenac and disulfiram. The types of transcriptional changes observed in this study were generally consistent with previously reported in vivo data, although there were some differences. In hierarchical cluster analysis, drugs formed clusters depending on their mode of toxicity against cells. The number of transcripts affected by the cholestatic hepatotoxicants (lithocholic acid and chlorpromazine) or the drugs that rarely cause of hepatotoxicity (cisplatin, diclofenac and disulfiram) were limited compared with the other drugs (acetaminophen, clobifibrate and cyclophosphamide), where they did not induce transcriptional changes apparently related to toxicity. It is concluded that in vitro gene expression analysis of hepatocytes using microarray is a useful tool for evaluating the toxicological profile of drugs and in screening for the direct toxicity of drugs against hepatocytes.
Assuntos
Perfilação da Expressão Gênica/métodos , Hepatócitos/efeitos dos fármacos , Análise de Sequência com Séries de Oligonucleotídeos , RNA Mensageiro/metabolismo , Testes de Toxicidade/métodos , Transcrição Gênica/efeitos dos fármacos , Acetaminofen/toxicidade , Animais , Técnicas de Cultura de Células , Células Cultivadas , Clofibrato/toxicidade , Análise por Conglomerados , Relação Dose-Resposta a Droga , Estudos de Viabilidade , Hepatócitos/metabolismo , Masculino , Ratos , Ratos Sprague-Dawley , Fatores de TempoRESUMO
Rat primary renal cortical tubular cells were exposed to seven test substances, some with, and some without, known direct renal tubular cell toxicity. Cells were exposed to the substances at either one-third or one-tenth of the TC50 for cytotoxicity for 6 h or 24 h, so as not to induce cytotoxicity but to cause some transcriptional changes. Transcriptional profiles were investigated by using the Affymetrix Rat Toxicology U34 arrays, containing probes for more than 850 genes and ESTs. Four direct toxicants, cisplatin (CDDP), its less nephrotoxic analogue carboplatin (CBDCA), cephaloridine and gentamicin, were grouped together in a hierarchical clustering. In addition, the four direct toxicants affected more than 32 transcripts at their subcytotoxic concentrations at either 6 h or 24 h exposure. On the other hand, diclofenac, cyclosporine A and zinc, which are not considered to be directly toxic to tubules, affected less than 12 transcripts. Decreased Map3k12 and increased Hmox1 were commonly observed among the four direct toxicants, which appeared to be responses to cellular damage. Two platinum complexes, CDDP and CBDCA, induced similar changes, regardless of exposure duration or concentration. The types of transcriptional changes observed in this study were consistent with previously reported in vivo data, although there were some differences. These observations suggest that an in vitro gene expression analysis approach using GeneChip is feasible for screening for direct tubular toxicity of drugs and may help to clarify the underlying mechanisms of tubular toxicity.
Assuntos
Perfilação da Expressão Gênica/métodos , Túbulos Renais/efeitos dos fármacos , Análise de Sequência com Séries de Oligonucleotídeos , RNA Mensageiro/metabolismo , Testes de Toxicidade/métodos , Transcrição Gênica/efeitos dos fármacos , Animais , Carboplatina/toxicidade , Técnicas de Cultura de Células , Células Cultivadas , Cefaloridina/toxicidade , Cisplatino/toxicidade , Análise por Conglomerados , Relação Dose-Resposta a Droga , Estudos de Viabilidade , Gentamicinas/toxicidade , Túbulos Renais/metabolismo , Masculino , Ratos , Ratos Sprague-Dawley , Fatores de TempoRESUMO
Design, synthesis, and in vitro and in vivo evaluation of a series of antipsoriatic antedrugs having 16-en-22-oxa-vitamin D3 are described. Among the seven compounds examined, two are promising: ester 5c and amide 5f, both of which exhibit greater potent antiproliferation activity with lessened calcemic activity than the presently prescribed maxacalcitol (2).
Assuntos
Colecalciferol/química , Colecalciferol/uso terapêutico , Desenho de Fármacos , Psoríase/tratamento farmacológico , Animais , Colecalciferol/síntese química , Colecalciferol/farmacologia , Estrutura Molecular , Ratos , Relação Estrutura-AtividadeRESUMO
A series of 16-en-22-oxa-derivatives of vitamin D3 based on the structure of maxacalcitol (2) were prepared. Maxacalcitol is currently used topically for the treatment of psoriasis and is recognized as the most successful antedrug of natural vitamin D(3) because it retains the original antiproliferative activity of calcitriol without increased calcemic activity. We introduced 16-olefinic functionality to accelerate the oxidative metabolism of the drug in liver, presumed to be essential for the reduction of calcemic activity, and modified the side-chain moiety by placing the 22-oxygen on the more labile allylic carbon center. Novel 22-oxa analogs (7a-i), carrying either the 24-alkynyl bond or 24-hydroxy functionality in addition to the 16-double bond were synthesized and their pharmacokinetics were evaluated.