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1.
Chem Pharm Bull (Tokyo) ; 68(3): 201-211, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32115527

RESUMO

Auristatins are important payloads used in antibody drug conjugates (ADCs), and the most well-known compound family member, monomethyl auristatin (MMAE), is used in two Food and Drug Administration (FDA)-approved ADCs, Adcetris® and Polivy®. Multiple other auristatin-based ADCs are currently being evaluated in human clinical trials and further studies on this class of molecule are underway by several academic and industrial research groups. Our group's main focus is to investigate the structure-activity relationships (SAR) of novel auristatins with the goal of applying these to next generation ADCs. Modifications of the auristatin backbone scaffold have been widely reported in the chemical literature focusing on the terminal subunits: P1 (N-terminus) and P5 (C-terminus). Our approach was to modulate the activity and hydrophilic character through modifications of the central subunits P2-P3-P4 and thorough SAR study on the P5 subunit. Novel hydrophilic auristatins were observed to have greater potency in vitro and displayed enhanced in vivo antitumor activity when conjugated via protease-cleavable linkers and delivered intracellularly. Analysis of ADC aggregation also indicated that novel hydrophilic payloads enabled the synthesis of high-drug-to-antibody ratio (DAR) ADCs that were resistant to aggregation. Modification of the central peptide subunits also resulted in auristatins with potent cytotoxic activity in vitro and these azide-modified auristatins contain a handle for linker attachment from the central portion of the auristatin backbone.


Assuntos
Aminobenzoatos/química , Antineoplásicos/química , Oligopeptídeos/química , Aminobenzoatos/farmacologia , Animais , Antineoplásicos/farmacologia , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Humanos , Interações Hidrofóbicas e Hidrofílicas , Imunoconjugados , Estrutura Molecular , Oligopeptídeos/farmacologia
2.
J Fish Biol ; 95(5): 1320-1330, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31515796

RESUMO

The effects of common anaesthetics on the hue, saturation and brightness measurements of the poeciliid fish Girardinus metallicus were investigated in two experiments. For both experiments the coloration of four body regions was measured from digital images of the same males obtained under three conditions: (1) control (in a water-filled chamber); (2) anaesthetised with MS-222; and (3) anaesthetised with eugenol (clove oil). In experiment 1 anaesthetised fish were photographed out of water. In experiment 2 all photographs were taken in a water-filled chamber. Anaesthetics altered coloration in both experiments. In the more methodologically consistent experiment 2 we found significantly different hue, increased saturation and decreased brightness in anaesthetic v. control conditions, consistent with darkening caused by the anaesthetics. The body regions differed in coloration consistent with countershading but did not differentially change in response to anaesthesia. These findings suggest that photographing fish in a water-filled chamber without anaesthetic is preferable for obtaining digital images for colour analysis and that multiple body regions of fish should be measured when assessing coloration patterns meaningful in behavioural contexts, to account for the gradients caused by countershading. We are encouraged that some researchers employ such methods already and caution against using anaesthetics except when absolutely necessary for immobilisation.


Assuntos
Aminobenzoatos/farmacologia , Anestésicos/farmacologia , Óleo de Cravo/farmacologia , Ciprinodontiformes/fisiologia , Animais , Cor , Ciprinodontiformes/anatomia & histologia , Masculino
3.
Toxicol In Vitro ; 61: 104638, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31476374

RESUMO

The inclusion of a read-out to detect functional consequences of craniofacial alterations in the zebrafish embryotoxicity test will allow to evaluate these alterations which are difficult to assess morphologically, and to detect alterations in cranial nerves functions leading to impairment of jaw movements. In this study we have established an ingestion test in zebrafish larvae younger than 120 hpf. To overcome the challenge of evaluating larvae which still do not present independent feeding behaviour, we have tested the ability of 72, 96 or 102 hpf larvae to ingest food mixed with fluorescent microspheres under several conditions (dark/light, with/without shaking) to find the best experimental set-up for the test. We have included the investigation of two substances as potential positive controls: ketoconazole and tricaine. Ketoconazole 10 µM exposure during development produced significant embryotoxic effects including a characteristic craniofacial alteration pattern consisting in impaired development of brain, nasal cavity, mouth opening and jaw, as well as a significant decrease in food intake. Tricaine exposure at 380 µM during the food availability period significantly decreased the food intake. The method proposed will be a useful alternative tool to animal testing to detect compounds inducing adverse effects on craniofacial development.


Assuntos
Aminobenzoatos/toxicidade , Anormalidades Craniofaciais/induzido quimicamente , Embrião não Mamífero/anormalidades , Cetoconazol/toxicidade , Teratogênios/toxicidade , Testes de Toxicidade/métodos , Peixe-Zebra/anormalidades , Alternativas aos Testes com Animais , Animais , Ingestão de Alimentos/efeitos dos fármacos
4.
Int J Radiat Oncol Biol Phys ; 105(4): 861-874, 2019 11 15.
Artigo em Inglês | MEDLINE | ID: mdl-31419512

RESUMO

PURPOSE: Telomerase is reactivated in non-small cell lung cancer (NSCLC), and it increases cell resistance to irradiation through protecting damaged telomeres and enhancing DNA damage repair. We investigated the radiosensitizing effect of BIBR1532, a highly selective telomerase inhibitor, and its corresponding mechanism in NSCLC. METHODS AND MATERIALS: Cell proliferation, telomerase activity, and telomere dysfunction-induced foci were measured with CCK-8 assay, real-time fluorescent quantitative polymerase chain reaction, and immunofluorescence. The effect of BIBR1532 on the response of NSCLC cells to radiation was analyzed using clonogenic survival and xenograft tumor assays. Cell death and cell senescence induced by BIBR1532 or ionizing radiation (IR), or both, were detected with western blotting, flow cytometry, and senescence-association ß-galactosidase staining assay. RESULTS: We observed dose-dependent direct cytotoxicity of BIBR1532 at relatively high concentrations in NSCLC cells. Low concentrations of BIBR1532 did not appear toxic to NSCLC cells; however, they substantially increased the therapeutic efficacy of IR in vitro by enhancing IR-induced apoptosis, senescence, and mitotic catastrophe. Moreover, in a mouse xenograft model, BIBR1532 treatment synergized with IR at nontoxic dose levels promoted the antitumor efficacy of IR without toxicity to hematologic and internal organs. Mechanistically, lower concentrations of BIBR1532 effectively inhibited telomerase activity and increased IR-induced telomere dysfunction, resulting in disruption of chromosomal stability and inhibition of the ATM/CHK1 (ataxia-telangiectasia-mutated/Checkpoint kinase 1) pathway, which impaired DNA damage repair. CONCLUSIONS: Our findings demonstrate that disturbances in telomerase function by nontoxic dose levels of BIBR1532 effectively enhance the radiosensitivity of NSCLC cells. This finding provides a rationale for the clinical assessment of BIBR1532 as a radiosensitizer.


Assuntos
Aminobenzoatos/farmacologia , Carcinoma Pulmonar de Células não Pequenas/radioterapia , Neoplasias Pulmonares/radioterapia , Naftalenos/farmacologia , Tolerância a Radiação/efeitos dos fármacos , Radiossensibilizantes/farmacologia , Telomerase/antagonistas & inibidores , Aminobenzoatos/administração & dosagem , Animais , Proteínas Mutadas de Ataxia Telangiectasia/antagonistas & inibidores , Carcinoma Pulmonar de Células não Pequenas/enzimologia , Morte Celular , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Senescência Celular/efeitos dos fármacos , Dano ao DNA/efeitos dos fármacos , Reparo do DNA/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ativação Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/administração & dosagem , Inibidores Enzimáticos/farmacologia , Reativadores Enzimáticos/farmacologia , Feminino , Humanos , Neoplasias Pulmonares/enzimologia , Camundongos , Camundongos Nus , Naftalenos/administração & dosagem , Fosforilação/efeitos dos fármacos , Sincalida , Telomerase/metabolismo , Telômero/efeitos dos fármacos , Homeostase do Telômero/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto
5.
mBio ; 10(4)2019 08 13.
Artigo em Inglês | MEDLINE | ID: mdl-31409675

RESUMO

Biosynthetic gene clusters (BGCs) are organized groups of genes involved in the production of specialized metabolites. Typically, one BGC is responsible for the production of one or several similar compounds with bioactivities that usually only vary in terms of strength and/or specificity. Here we show that the previously described ferroverdins and bagremycins, which are families of metabolites with different bioactivities, are produced from the same BGC, whereby the fate of the biosynthetic pathway depends on iron availability. Under conditions of iron depletion, the monomeric bagremycins are formed, representing amino-aromatic antibiotics resulting from the condensation of 3-amino-4-hydroxybenzoic acid with p-vinylphenol. Conversely, when iron is abundantly available, the biosynthetic pathway additionally produces a molecule based on p-vinylphenyl-3-nitroso-4-hydroxybenzoate, which complexes iron to form the trimeric ferroverdins that have anticholesterol activity. Thus, our work shows a unique exception to the concept that BGCs should only produce a single family of molecules with one type of bioactivity and that in fact different bioactive molecules may be produced depending on the environmental conditions.IMPORTANCE Access to whole-genome sequences has exposed the general incidence of the so-called cryptic biosynthetic gene clusters (BGCs), thereby renewing their interest for natural product discovery. As a consequence, genome mining is the often first approach implemented to assess the potential of a microorganism for producing novel bioactive metabolites. By revealing a new level of complexity of natural product biosynthesis, we further illustrate the difficulty of estimation of the panel of molecules associated with a BGC based on genomic information alone. Indeed, we found that the same gene cluster is responsible for the production of compounds which differ in terms of structure and bioactivity. The production of these different compounds responds to different environmental triggers, which suggests that multiplication of culture conditions is essential for revealing the entire panel of molecules made by a single BGC.


Assuntos
Aminobenzoatos/metabolismo , Antibacterianos/metabolismo , Vias Biossintéticas/genética , Compostos Ferrosos/metabolismo , Quelantes de Ferro/metabolismo , Família Multigênica , Compostos Nitrosos/metabolismo , Aminobenzoatos/química , Antibacterianos/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Compostos Ferrosos/química , Genoma Bacteriano/genética , Ferro/metabolismo , Quelantes de Ferro/química , Estrutura Molecular , Compostos Nitrosos/química , Filogenia , Streptomyces/classificação , Streptomyces/genética , Streptomyces/metabolismo
6.
Expert Opin Drug Deliv ; 16(8): 783-793, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31327255

RESUMO

Introduction: Compared to the antibody and drug components of an ADC, the linker part has been somewhat neglected. However, its importance for the reduction of failures in ADC approvals is increasingly recognized. Next of being a stable glue between drug and antibody, an ideal linker should improve the manufacturability and widen the therapeutic window of ADCs. Areas covered: The biopharmaceutical company LinXis started an ADC development program in which platinum(II) is the key element of the first metal-organic linker. The cationic complex [ethylenediamineplatinum(II)]2+, herein called 'Lx®', is used successfully for conjugation of drugs to antibodies. Expert opinion: Based on lessons learned from ADC development, Lx linker technology fulfills most of the desirable linker characteristics. Lx allows large-scale cost-effective manufacturing of ADCs via a straightforward two-step 'plug-and-play' process. First clinical candidate trastuzumab-Lx-auristatin F shows favorable preclinical safety as well as outstanding in vivo tumor targeting performance and therapeutic efficacy.


Assuntos
Aminobenzoatos/química , Antineoplásicos/química , Imunoconjugados/química , Oligopeptídeos/química , Compostos Organoplatínicos/química , Trastuzumab/química , Aminobenzoatos/uso terapêutico , Animais , Antineoplásicos/uso terapêutico , Imunoconjugados/uso terapêutico , Neoplasias/tratamento farmacológico , Oligopeptídeos/uso terapêutico , Compostos Organoplatínicos/uso terapêutico , Trastuzumab/uso terapêutico
7.
J Zoo Wildl Med ; 50(1): 89-95, 2019 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-31120666

RESUMO

Although tricaine methanesulfonate (MS-222) immersion has historically been standard of care for fish and anuran euthanasia, recent research has proven it insufficient for euthanasia of goldfish. To assess appropriateness for humane euthanasia of anurans, this study evaluated the efficacy of MS-222 in Smokey Jungle Frogs (Leptodactylus pentadactylus). Eighteen frogs (21-33 g) were exposed to one of three MS-222 concentrations via partial immersion: 2.5 g/L for 90 min (M2.5/90), 5 g/L for 60 min (M5/60), or 10 g/L for 60 min (M10/60). Physiologic parameters and times to loss of spontaneous movement, righting reflex, and noxious stimulus response were recorded. Following exposure, frogs were rinsed with dechlorinated water, and time to cessation of heart beat was recorded. Survival in M2.5/90, M5/60, and M10/60 was one of six, zero of six, and zero of six, respectively. In M2.5/90, three of six frogs had continued purposeful, spontaneous movement throughout exposure. In M5/60 and M10/60, median (range) time to initial loss of movement was 14.3 (5.5-30.0) and 7.6 (4.8-19.7) min, respectively. Twelve of 18 frogs among all groups demonstrated a median (range) of two (one to six) episodes of regained consciousness with purposeful, spontaneous movement following loss of noxious stimulus response. Median (range) time to heart beat cessation in M2.5/90, M5/60, and M10/60 was 150 (135-210), 157.5 (60-225), and 90 (75-210) min, respectively. Although death was achieved in 17 of 18 frogs, given the repeated events of regained consciousness, MS-222 immersion when used at concentrations ≤10 g/L did not result in rapid and distress-free death and is not sufficient for humane euthanasia in this species.


Assuntos
Aminobenzoatos/uso terapêutico , Anestésicos/uso terapêutico , Anuros/fisiologia , Eutanásia Animal/métodos , Animais , Relação Dose-Resposta a Droga , Imersão
8.
J Zoo Wildl Med ; 50(1): 96-106, 2019 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-31120667

RESUMO

Despite extensive literature examining American horseshoe crab physiology, there are comparatively few publications addressing their medical care. Establishing anesthesia protocols for horseshoe crabs is integral to limiting the potential stress and pain associated with invasive procedures and for advancing euthanasia techniques. The objective of this study was to compare the effects of two immersion anesthetics, tricaine methanesulfonate (MS-222) at 1 g/L (buffered with sodium carbonate) and 2-phenoxyethanol (2-PE) at 2 mL/L, on horseshoe crabs. Twenty horseshoe crabs were assigned to one of two anesthetic treatment groups and individually anesthetized in natural seawater. Water quality, cardiac contractility, and hemolymph gas analytes were measured prior to anesthesia and at 30 min Animals were monitored via heart rate, gilling rate, and sedation score every 5 min until recovered. Transcarapacial ultrasonography was used to obtain heart rate, gilling rate, and percent fractional shortening. Light or surgical anesthesia was produced in 10/10 animals in the 2-PE group and 8/10 animals in the MS-222 group. There was no significant difference in sedation scores, induction time (median 15 min), or recovery time (median 20.5 min). Gilling rate and cardiac contractility decreased during anesthesia, whereas heart rate did not. Hemolymph pH and pO2 were not different among treatment groups or time points. Baseline pCO2 was higher than pCO2 at 30 min for both groups but significantly elevated only in the MS-222 group. This is attributed to increased activity during the handling of awake animals. Invasive blood pressure obtained via cardiac catheterization in two animals was markedly decreased during surgical anesthesia. In conclusion, 2-PE and MS-222 provided effective anesthesia with clinically useful induction and recovery times. 2-PE provided a subjectively more reliable and smoother anesthesia compared to MS-222.


Assuntos
Aminobenzoatos/efeitos adversos , Anestesia/veterinária , Anestésicos/efeitos adversos , Etilenoglicóis/efeitos adversos , Caranguejos Ferradura/efeitos dos fármacos , Anestesia/métodos , Animais , Feminino , Caranguejos Ferradura/fisiologia , Imersão , Masculino , North Carolina , Distribuição Aleatória
9.
J Zoo Wildl Med ; 50(1): 282-286, 2019 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-31120693

RESUMO

This communication briefly describes the use of tricaine methanesulfonate (MS222) to induce chemical restraint/general anesthesia of a Mexican axolotl (Ambystoma mexicanum) for the endoscopic retrieval of a gastric foreign body. There is very little published scientific literature concerning the anesthesia of Mexican axolotls. The anesthesia used in this case was an immersion bath of tricaine methanesulfonate where the concentration of tricaine methanesulfonate was gradually increased to 500 mg/L (ppm) over a 15-min period. A loss of righting reflex was observed within 3 min of attaining the final concentration of the anesthetic bath. The first voluntary movements following the transfer to a freshwater bath occurred within 7 min. The recovery was uneventful. Tricaine methanesulfonate in this case proved to be an effective anesthetic agent for a short, minimally invasive procedure.


Assuntos
Ambystoma mexicanum/lesões , Aminobenzoatos/administração & dosagem , Anestesia Geral/veterinária , Anestésicos/administração & dosagem , Endoscopia/veterinária , Corpos Estranhos/veterinária , Ambystoma mexicanum/cirurgia , Animais , Corpos Estranhos/cirurgia , Imersão , Imobilização/veterinária , Resultado do Tratamento
10.
Mol Cancer Ther ; 18(6): 1104-1114, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30962319

RESUMO

Although inhibiting EGFR-mediated signaling proved to be effective in treating certain types of cancers, a quickly evolved mechanism that either restores the EGFR signaling or activates an alternative pathway for driving the proliferation and survival of malignant cells limits the efficacy and utility of the approach via suppressing the EGFR functionality. Given the fact that overexpression of EGFR is commonly seen in many cancers, an EGFR-targeting antibody-drug conjugate (ADC) can selectively kill cancer cells independently of blocking EGFR-mediated signaling. Herein, we describe SHR-A1307, a novel anti-EGFR ADC, generated from an anti-EGFR antibody with prolonged half-life, and conjugated with a proprietary toxin payload that has increased index of EGFR targeting-dependent versus EGFR targeting-independent cytotoxicity. SHR-A1307 demonstrated strong and sustained antitumor activities in EGFR-positive tumors harboring different oncogenic mutations on EGFR, KRAS, or PIK3CA. Antitumor efficacy of SHR-A1307 correlated with EGFR expression levels in vitro and in vivo, regardless of the mutation status of EGFR signaling mediators and a resultant resistance to EGFR signaling inhibitors. Cynomolgus monkey toxicology study showed that SHR-A1307 is well tolerated with a wide therapeutic index. SHR-A1307 is a promising therapeutic option for EGFR-expressing cancers, including those resistant or refractory to the EGFR pathway inhibitors.


Assuntos
Aminobenzoatos/imunologia , Anticorpos Monoclonais Humanizados/imunologia , Antineoplásicos Imunológicos/imunologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Imunoconjugados/farmacologia , Imunoconjugados/uso terapêutico , Neoplasias/tratamento farmacológico , Oligopeptídeos/imunologia , Animais , Anticorpos Monoclonais Humanizados/administração & dosagem , Antineoplásicos Imunológicos/administração & dosagem , Linhagem Celular Tumoral , Sistemas de Liberação de Medicamentos/métodos , Receptores ErbB/imunologia , Feminino , Células HEK293 , Humanos , Macaca fascicularis , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Transfecção , Carga Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto
11.
ACS Sens ; 4(4): 1090-1096, 2019 04 26.
Artigo em Inglês | MEDLINE | ID: mdl-30945529

RESUMO

Telomerase is a universal biomarker of malignant tumors. Sensitive and reliable analysis for telomerase activity is of vital importance for both early diagnosis and therapy of malignant tumors. Herein, a novel fluorescent strategy was proposed for sensitive and label-free detection of telomerase activity. One highlight of this strategy is that an exponential signal amplification can be triggered by a very short telomerase extension product (TEP). Without adding dATP, the designed telomerase primer can be easily controlled to extend five bases (GGGTT) to give short TEP with definite length. The resulting short TEP can then be constructed as a circular rolling circle amplification (RCA) template and thus initiate a nicking enzyme-mediated exponential RCA, producing G-rich amplification products that can be sensitively probed via specific binding between the fluorescent dye Thioflavin T (ThT) and the nucleic acid G-quadruplexes. Elevated telomerase translocation efficiency, combined with exponential signal amplification and specific probing of RCA products by ThT, endow the sensing platform with extraordinarily high detection sensitivity. The requirement for short TEP increases the possibility to analyze telomerase with low activity. The proposed sensing platform can achieve sensitive telomerase activity detection in individual cells, even with the interference of accumulated normal cells. It was also demonstrated to show excellent capability in screening for the inhibitors of telomerase. Therefore, the proposed sensing platform has great potential for not only clinical diagnosis but also anticancer drug development.


Assuntos
Ensaios Enzimáticos/métodos , Técnicas de Amplificação de Ácido Nucleico/métodos , Espectrometria de Fluorescência/métodos , Telomerase/análise , Aminobenzoatos/química , Benzotiazóis/química , Biomarcadores Tumorais/análise , Linhagem Celular Tumoral , DNA/química , DNA/genética , Inibidores Enzimáticos/química , Corantes Fluorescentes/química , Quadruplex G , Humanos , Naftalenos/química , Análise de Célula Única/métodos , Telomerase/antagonistas & inibidores
12.
J Am Assoc Lab Anim Sci ; 58(3): 390-396, 2019 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-30925946

RESUMO

MS222 is a compound used in anesthetizing vertebrates, including fish and frogs. Several side effects of this anesthetic have been reported, but its effect on hemostasis has not been studied. In our laboratory, we have used zebrafish for more than 2 decades as a model system to study hemostasis. During this period, we have had trouble in collecting blood from anesthetized zebrafish and observed more rapid blood clotting than in nonanesthetized counterparts. However, no systematic studies regarding the effect of MS222 on zebrafish hemostasis are available. In this study, we performed various assays such as gill bleeding, measurement of Hct, total blood cell counts, thrombocyte counts, thrombocyte aggregation, and coagula- tion and measured the amount of blood collected. We found that Hct values, the amount of blood collected, bleeding, and coagulation differed significantly between anesthetized and nonanesthetized fish. Our results suggest that blood collected after MS222 anesthesia of zebrafish has altered hemostasis.


Assuntos
Aminobenzoatos/farmacologia , Anestésicos/farmacologia , Hemostasia/efeitos dos fármacos , Peixe-Zebra/sangue , Anestesia , Animais , Contagem de Células Sanguíneas/veterinária , Coagulação Sanguínea/efeitos dos fármacos
13.
Chemistry ; 25(30): 7232-7242, 2019 May 28.
Artigo em Inglês | MEDLINE | ID: mdl-30730065

RESUMO

Overuse and misuse of antibacterial drugs has resulted in bacteria resistance and in an increase in mortality rates due to bacterial infections. Therefore, there is an imperative necessity of new antibacterial drugs. Bio-organometallic derivatives of antibacterial agents offer an opportunity to discover new active antibacterial drugs. These compounds are well-characterized products and, in several examples, their antibacterial activities have been studied. Both inhibition of the antibacterial activity and strong increase in the antibiotic activity of the parent drug have been found. The synthesis of the main classes of bio-organometallic derivatives of these drugs, as well as examples of the use of structure-activity relation (SAR) studies to increase the activity and to understand the mode of action of bio-organometallic antimicrobial peptides (BOAMPs) and platensimicyn bio-organometallic mimics is presented in this article.


Assuntos
Antibacterianos/síntese química , Materiais Biocompatíveis/síntese química , Compostos Organometálicos/síntese química , Adamantano/síntese química , Adamantano/farmacologia , Aminobenzoatos/síntese química , Aminobenzoatos/farmacologia , Anilidas/síntese química , Anilidas/farmacologia , Antibacterianos/farmacologia , Peptídeos Catiônicos Antimicrobianos/síntese química , Peptídeos Catiônicos Antimicrobianos/farmacologia , Materiais Biocompatíveis/farmacologia , Materiais Biomiméticos/síntese química , Materiais Biomiméticos/farmacologia , Humanos , Metais/química , Estrutura Molecular , Compostos Organometálicos/farmacologia , Relação Estrutura-Atividade
14.
Biomed Chromatogr ; 33(5): e4512, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30793335

RESUMO

Tricaine methanesulfonate is one of most commonly used anesthetics in fish during blood sampling, artificial propagation and long-distance transportation. In this study, an accurate method for the quantitative determination of tricaine in fish samples by a stable isotope dilution assay coupled with high-performance liquid chromatography-triple quadrupole mass spectrometry was developed. Tricaine-D5 was synthesized and used as an isotopically labeled internal standard for the determination of tricaine. The analytical performance of the method was validated for tricaine determination in marine fish and freshwater fish. The determination of tricaine was linear in the range of 2.0-200.0 µg L-1 . The limit of detection and limit of quantitation for fish muscle tissues were 1.0 and 4.0 µg kg-1 , respectively. Good recoveries were obtained in the range of 92.08-97.50%. The inter- and intra-assay relative standard deviations (RSD values) were investigated, and the values were 0.39-3.01 and 0.85-2.77%, respectively. The values of CCα and CCß were 10.21-10.43 and 10.42-10.87 µg kg-1 , respectively. The clearance of MS-222 from grass carp was further studied using our method. The results demonstrate that MS-222 could be well absorbed and rapidly eliminated after bath administration.


Assuntos
Aminobenzoatos/análise , Cromatografia Líquida de Alta Pressão/métodos , Resíduos de Drogas/análise , Alimentos Marinhos/análise , Espectrometria de Massas em Tandem/métodos , Aminobenzoatos/química , Animais , Carpas , Marcação por Isótopo , Limite de Detecção , Modelos Lineares , Reprodutibilidade dos Testes
15.
Eur J Pharmacol ; 846: 49-62, 2019 Mar 05.
Artigo em Inglês | MEDLINE | ID: mdl-30658112

RESUMO

Telomerase-mediated immortalization and proliferation of tumor cells is a promising anti-cancer treatment strategy and development of potent telomerase inhibitors is believed to open new window of treatments in human malignancies. In the present study, we found that BIBR1532, a small molecule inhibitor of human telomerase, exerted cytotoxic effects on a panel of human cancer cells spanning from solid tumors to hematologic malignancies; however, as compared with solid tumors, leukemic cells were more sensitive to this inhibitor. This was independent of molecular status of p53 in the leukemic cells. The results of a miRNA PCR array revealed that BIBR1532-induced cytotoxic effects in NB4, the most sensitive cell line, was coupled with alteration in a substantial number of cancer-related miRNAs. Interestingly, most of these miRNAs were found to act as tumor suppressors with validated targets in cell cycle or nuclear factor (NF)-κB-mediated apoptosis. In accordance with a bioinformatics analysis, our experimental studies showed that BIBR1532-induced apoptosis is mediated, at least partly, by inhibition of NF-κB. Moreover, we found that the alteration in the expression of miRNAs was coupled with the alteration in the cell cycle progression. To sum up with, a straightforward interpretation of our results is that telomerase inhibition using BIBR1532 not only induced CDKN1A-mediated G1 arrest in NB4, but also resulted in a caspase-3-dependent apoptotic cell death mostly through suppression of NF-κB axis.


Assuntos
Aminobenzoatos/uso terapêutico , Leucemia Promielocítica Aguda/tratamento farmacológico , MicroRNAs/metabolismo , Naftalenos/uso terapêutico , Telomerase/antagonistas & inibidores , Aminobenzoatos/farmacologia , Apoptose/efeitos dos fármacos , Caspase 3/metabolismo , Linhagem Celular Tumoral , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Pontos de Checagem da Fase G1 do Ciclo Celular , Humanos , NF-kappa B/metabolismo , Naftalenos/farmacologia
16.
PLoS One ; 14(1): e0211080, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30668593

RESUMO

Large numbers of lumpfish are produced for the Norwegian salmon industry and are used to combat sea lice infestations. Periodically high mortality of farmed lumpfish demonstrates the need to improve farming conditions and animal welfare. As part of such efforts, the present work tested the efficacy of three anaesthetic chemicals on lumpfish fries (average weight of 0.97 g). The anaesthetic impact from salinity (15 ppt-18 ppt), temperature (12°C versus 7 and 18°C), and fasting conditions (three days) was also examined. Surgical anaesthesia was induced within 3 to 5 min (preferred time) at concentrations of 18 mg/L (Aqui-S), 37.5 mg/L (Benzoak), and 60 mg/L (buffered MS-222). Safety margins were regarded as low when using Aqui-S; therefore, this chemical was not considered suitable for prolonged exposures. The lumpfish made a rapid recovery from both Benzoak and MS-222 even after 20 min of exposure. A 6°C increase in exposure temperature (reaching 18°C) was found to delay or inhibit recovery. The effect of a 5°C decrease (down to 7°C) significantly reduced induction time for MS-222 and was insignificant for Aqui-S, while it prolonged Benzoak induction time significantly and gave a longer recovery period. Fasting resulted in 70% recovery after 20 min of Aqui-S exposure compared to 0% in fed fish but had only minor effects on Benzoak and MS-222. Use of brackish water (15 ppt-18 ppt) gave 20% recovery from Aqui-S and significantly shorter recovery time from MS-222 exposure, while the effects on Benzoak were insignificant.


Assuntos
Aminobenzoatos/farmacologia , Anestésicos/farmacologia , Doenças dos Peixes/tratamento farmacológico , Perciformes/crescimento & desenvolvimento , Animais , Doenças dos Peixes/fisiopatologia
17.
Adv Physiol Educ ; 43(1): 69-75, 2019 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-30694709

RESUMO

Frogs are routinely used in physiology teaching laboratories to demonstrate important physiological processes. There have been recent directives that promote the use of the anesthetic MS-222 (tricaine methanesulfonate), rather than lowering body temperature with a cold water bath to prepare reptiles and amphibians for physiological experiments or euthanasia. Indeed, the most recent edition of the American Veterinary Medical Association (AVMA) Guidelines for the Euthanasia of Animals proclaims that chilling in water is not an appropriate method and advocates for the usage of MS-222 or other anesthetics. However, prominent researchers have responded to this position by highlighting evidence that cooling ectothermic vertebrates is, in fact, an effective and appropriate method. Furthermore, MS-222 is a known voltage-gated Na+ channel blocker, and this anesthetic's impact on the physiology of excitable tissues suggests that its use might be incompatible with experiments on nerve and muscle tissues. In the present study, I examined the effects of MS-222 at a concentration of 1.5 g/l on nerve, skeletal muscle, and cardiac muscle physiology of frogs. I found that immersion of frogs in this anesthetic blocked basic nerve and muscle physiology, making the frogs unsuitable for laboratory experiments. Applying MS-222 directly to the sciatic nerve dramatically blocked normal excitation-contraction coupling in skeletal muscle preparations, and direct application to the heart caused the organs to stop contracting. Based on these results, I conclude that MS-222 at the concentration studied may be incompatible with physiological preparations that rely on electrically excitable tissues for their normal function. Physiology educators who must use MS-222 with frogs should empirically determine an appropriate dosage and recovery time before using the anesthetic in the teaching laboratory.


Assuntos
Aminobenzoatos/farmacologia , Anestésicos/farmacologia , Contração Muscular/fisiologia , Músculo Esquelético/fisiologia , Fisiologia/educação , Nervo Isquiático/fisiologia , Animais , Humanos , Contração Muscular/efeitos dos fármacos , Músculo Esquelético/efeitos dos fármacos , Rana pipiens , Nervo Isquiático/efeitos dos fármacos
18.
MAbs ; 11(1): 153-165, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30365359

RESUMO

HER2-ECD (human epidermal growth factor receptor 2 - extracellular domain) is a prominent therapeutic target validated for treating HER2-positive breast and gastric cancer, but HER2-specific therapeutic options for treating advanced gastric cancer remain limited. We have developed antibody-drug conjugates (ADCs), comprising IgG1 linked via valine-citrulline to monomethyl auristatin E, with potential to treat HER2-positive gastric cancer in humans. The antibodies optimally selected from the ADC discovery platform, which was developed to discover antibody candidates suitable for immunoconjugates from synthetic antibody libraries designed using antibody-antigen interaction principles, were demonstrated to be superior immunoconjugate targeting modules in terms of efficacy and off-target toxicity. In comparison with the two control humanized antibodies (trastuzumab and H32) derived from murine antibody repertoires, the antibodies derived from the synthetic antibody libraries had enhanced receptor-mediated internalization rate, which could result in ADCs with optimal efficacies. Along with the ADCs, two other forms of immunoconjugates (scFv-PE38KDEL and IgG1-AL1-PE38KDEL) were used to test the antibodies for delivering cytotoxic payloads to xenograft tumor models in vivo and to cultured cells in vitro. The in vivo experiments with the three forms of immunoconjugates revealed minimal off-target toxicities of the selected antibodies from the synthetic antibody libraries; the off-target toxicities of the control antibodies could have resulted from the antibodies' propensity to target the liver in the animal models. Our ADC discovery platform and the knowledge gained from our in vivo tests on xenograft models with the three forms of immunoconjugates could be useful to anyone developing optimal ADC cancer therapeutics.


Assuntos
Aminobenzoatos/farmacologia , Imunoconjugados/farmacologia , Terapia de Alvo Molecular/métodos , Oligopeptídeos/farmacologia , Receptor ErbB-2/antagonistas & inibidores , Neoplasias Gástricas/patologia , Animais , Anticorpos Monoclonais Humanizados/farmacologia , Antineoplásicos/farmacologia , Humanos , Camundongos , Ensaios Antitumorais Modelo de Xenoenxerto
19.
Fish Shellfish Immunol ; 84: 1170-1179, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30366089

RESUMO

Stress response has negative effect on fish in aquaculture and research, which can be alleviated with anesthetic. To determine the optimal anesthetic, we investigated the physiological response of crucian carp (Carassius auratus) treated with three different anti-stress treatments: MS-222, eugenol and percussive stunning. Stress responses were evaluated by analyzing serum cortisol level and gene expression in blood. We determined the optimal concentrations of MS-222 (100 mg L-1) and eugenol (20 mg L-1) by dose selection. We found that the control group had significantly higher cortisol levels (172.78 ±â€¯19.95 ng mL-1) compared to the MS-222 treated group (46.85 ±â€¯3.22 ng mL-1), the eugenol treated group (72.78 ±â€¯9.07 ng mL-1), and the stunning treatment group (82.78 ±â€¯8.16 ng mL-1). Transcriptome analysis revealed 1572 differentially expressed genes (DEGs), including 155 DEGs related to the stress response, mainly involved in oxidative-stress response, heat shock proteins, and cold shock domain-containing protein. The heat shock protein genes were the primary DEGs in response to stress. RT-qPCR analysis confirmed differential expression of Hsps. We analyzed the function of the DEGs, which were enriched in genes involved in cellular response to stress and antigen processing and presentation. Combining the results from biochemical, transcriptome, and gene expression analysis, our data suggest that eugenol is more effective than MS-222 and percussive stunning in alleviating stress in crucian carp.


Assuntos
Aminobenzoatos/farmacologia , Anestesia/veterinária , Anestésicos/farmacologia , Carpas/fisiologia , Eugenol/farmacologia , Hidrocortisona/sangue , Anestesia/métodos , Animais , Carpas/genética , Relação Dose-Resposta a Droga , Expressão Gênica/efeitos dos fármacos , Perfilação da Expressão Gênica/veterinária , Carpa Dourada/genética , Carpa Dourada/fisiologia , Longevidade/efeitos dos fármacos , Estresse Fisiológico
20.
J Am Assoc Lab Anim Sci ; 58(1): 58-64, 2019 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-30497541

RESUMO

Invertebrates are often overlooked as laboratory animals, yet they are commonly used in toxicology, developmental, cellular and molecular biology, and radiation studies with euthanasia as an endpoint. Little is known regarding appropriate euthanasia methods for invertebrate species, particularly for Artemia. Here, we evaluated the AVMA-recommended 2-step method of euthanasia in brine shrimp (Artemia franciscana). Artemia were exposed first to anesthetic solutions of 60% alcohol, 2.5 mg/L eugenol, or 4 g/L tricaine methanesulfonate (TMS) and then were transferred to euthanasia solutions of 70% alcohol, 95% alcohol, or 10% neutral buffered formalin. We examined time to anesthesia, behavioral response to anesthesia, anesthesia recovery, and time to euthanasia. Our results show that 2.5 mg/L eugenol and 4 g/L TMS inconsistently achieved anesthesia. Although 60% alcohol produced anesthesia, the time to anesthesia varied among replicate groups, and exposure resulted in an increase in abnormal behavior. We therefore do not recommend any of the tested anesthetic solutions for use in Artemia. Although all 3 euthanasia solutions were effective, more research is needed to provide recommendations regarding euthanasia for this species.


Assuntos
Álcoois/farmacologia , Aminobenzoatos/farmacologia , Anestesia , Artemia/efeitos dos fármacos , Eugenol/farmacologia , Aminobenzoatos/química , Anestésicos/farmacologia , Animais , Eugenol/administração & dosagem , Eutanásia Animal , Solventes/farmacologia
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