RESUMO
In the study presented, a simple analytical method for the direct determination of glycine in immunoglobulins by hydrophilic interaction liquid chromatography was developed. The HPLC separation was performed using a SeQuant ZIC-HILIC column (250 mm × 4.6 mm i.d, 5 µm) with the isocratic mobile phase consisting of ammonium formate (20 mM) and acetonitrile (30:70, v/v), and the flow rate set at 0.8 mL/min. UV detection was carried out at a wavelength of 210 nm. The procedure was validated for specificity, precision, linearity, accuracy, limit of detection, limit of quantitation, and robustness. The calibration curve was found to be linear within the concentration range of 1.2-3.6 mg/mL. RSD values for intra-day and inter-day precision were in the range of 0.66 to 1.84%. The limit of quantification and limit of detection were 0.10 mg/mL and 0.03 mg/mL, respectively. The developed chromatographic method was applied for the glycine analysis in various immunoglobulins.
Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Glicina/análise , Imunoglobulinas/química , Calibragem , Interações Hidrofóbicas e Hidrofílicas , Limite de Detecção , Reprodutibilidade dos Testes , Espectrofotometria UltravioletaRESUMO
We report the effects of two anti-cancer drugs, PBT-4, an experimental antagonist to the pro-inflammatory hepoxilins, and Gleevec (STI-571), an anti-leukaemic drug, on eicosanoid tumour levels in immunodeficient mice (NU/NU) xenografted with the leukaemic cell line, U937 bcl-xL. After the tumours had grown to 80-100mm(3) volume, an 8-day treatment with the drugs was initiated and the animals were monitored for 28 days. On various days, tumours were removed for measurement of 24 omega-6 eicosanoids. The data show remarkable direct correlation between inhibition of tumour AA release and 12-LOX products (including 12-HETE and hepoxilins) during PBT or STI treatment with tumour growth suppression. These findings suggest that inhibition of AA release may represent a novel underlying mechanism of action of PBT-4 (and STI) in vivo in suppressing tumour growth. As the PBT wears off, AA and 12-LOX products rise rapidly (Day 18) leading to the observed tumour growth spurt.
Assuntos
Ácido 8,11,14-Eicosatrienoico/análogos & derivados , Antineoplásicos/farmacologia , Ácido Araquidônico/metabolismo , Fosfolipases A2/metabolismo , Proteína bcl-X/metabolismo , Ácido 8,11,14-Eicosatrienoico/farmacologia , Animais , Linhagem Celular Tumoral , Feminino , Humanos , Camundongos , Camundongos Nus , Carga Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
A series of substituted styryl-acrylonitriles was designed and synthesized. The new compounds, called tyrenes, were tested for the ability to inhibit acute lymphocytic leukemia (ALL) cancer cell growth, as well as on their toxicity to normal bone marrow (NBM) cells. The results showed that 3,4-dihydroxystyryl-acrylonitriles, in particular CR-4, revealed great potency as antitumor agents, and also exhibited low toxicity to normal cells. The effectiveness of these compounds with extended conjugation may be due to their possible functioning as reactive Michael acceptors.
Assuntos
Acrilonitrila/análogos & derivados , Acrilonitrila/farmacologia , Antineoplásicos/síntese química , Antineoplásicos/farmacologia , Estirenos/química , Estirenos/farmacologia , Acrilonitrila/metabolismo , Antineoplásicos/química , Linhagem Celular Tumoral , Humanos , Estirenos/síntese químicaRESUMO
The hepoxilin analog PBT-3 [10(S)-hydroxy-11,12-cyclopropyleicosa-5Z,8Z,14Z-trienoic acid methyl ester] was previously shown to inhibit the aggregation of human platelets and to antagonize the binding of the thromboxane receptor agonist I-BOP [[1S-[1alpha,2alpha (Z),3beta(1E,3S*),4alpha]]-7-[3-[3-hydroxy-4-(4-iodophenoxy)-1-butenyl]-7-oxabicyclo[2.2.1]hept-2-yl]-5-heptenoic acid] in human platelets (Pace-Asciak et al., 2002). We show herein that PBT-3 inhibits, to different degrees, binding of the TP receptor antagonist [3H]SQ 29,548 [[1S-[1alpha,2alpha (Z),3alpha,4alpha]]-7-[3-[[2-[(phenylamino)carbonyl]hydrazino]methyl]-7-oxabicyclo[2.2. 1]hept-2-yl]-5-heptenoic acid], to the TP receptor isoforms in TPalpha- and TPbeta-transfected COS-7 cells. These isoforms possess a different tail length, the alpha being shorter than the beta isoform. In contrast, SQ 29,548 shows no selection for the two TP isoforms. The IC50 value for PBT-3 = 2.0 +/- 0.3 x 10-7 M was observed for TPalpha, whereas this was one-sixth less active on the TPbeta isoform (IC50 = 1.2 +/- 0.2 x 10-6 M), suggesting selectivity for the TPalpha isoform. To investigate whether the tail contributes to the difference in competition binding by PBT-3, we investigated the tailless TP isoform expressed in transfected COS-7 cells. Its IC50 was similar to that of the TPalpha isoform. In additional studies, we investigated the effect of PBT-3 on the collagen and I-BOP evoked intracellular calcium release and on the collagen and I-BOP evoked phosphorylation of pleckstrin. PBT-3 blocked both pathways further demonstrating its TP receptor antagonist activity. These results demonstrate that the action of PBT-3 in inhibiting platelet aggregation is mediated via inhibition of the TPalpha isoform of the thromboxane receptor and that the tail may play an important role in recognition of this TP receptor antagonist.
Assuntos
Ácido 8,11,14-Eicosatrienoico/análogos & derivados , Ácido 8,11,14-Eicosatrienoico/farmacologia , Receptores de Tromboxanos/antagonistas & inibidores , Animais , Autorradiografia , Plaquetas/efeitos dos fármacos , Plaquetas/metabolismo , Proteínas Sanguíneas/metabolismo , Western Blotting , Compostos Bicíclicos Heterocíclicos com Pontes , Células COS , Cálcio/sangue , Células Cultivadas , Colágeno/metabolismo , Densitometria , Relação Dose-Resposta a Droga , Eletroforese em Gel de Poliacrilamida , Ácidos Graxos Insaturados , Humanos , Hidrazinas/metabolismo , Técnicas In Vitro , Isomerismo , Fosfoproteínas/metabolismo , Fosforilação , Ensaio Radioligante , Receptores de Tromboxanos/genética , Receptores de Tromboxanos/metabolismo , Estimulação Química , TransfecçãoRESUMO
In recent years, synthetic tyrosine kinase inhibitors have made a rapid transition from basic research to therapeutic application. These compounds represent a major clinical advance in the approach to cancer in their relative specificity of action and decreased toxicity. We report here the effects of a novel tyrosine kinase inhibitor CR4 that interferes with growth-promoting pathways to markedly inhibit the growth and survival of both Philadelphia-positive and -negative acute lymphoblastic leukemia (ALL) as well as acute myeloid leukemia (AML). While efficiently ablating leukemic cell growth, normal cell growth and differentiation remain unaffected by CR4. CR4 demonstrates an ability to inhibit the function of multiple growth-critical kinases and yet exhibits a low level of cytotoxicity. These findings suggest that CR4 may prove to be highly effective as a therapeutic agent.
Assuntos
Inibidores Enzimáticos/farmacologia , Leucemia Mieloide/tratamento farmacológico , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológico , Proteínas Tirosina Quinases/antagonistas & inibidores , Doença Aguda , Animais , Antineoplásicos/farmacologia , Antineoplásicos/toxicidade , Divisão Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/toxicidade , Humanos , Leucemia Mieloide/patologia , Leucemia Mieloide/prevenção & controle , Infiltração Leucêmica/tratamento farmacológico , Infiltração Leucêmica/prevenção & controle , Camundongos , Camundongos SCID , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , Leucemia-Linfoma Linfoblástico de Células Precursoras/prevenção & controle , Transdução de Sinais/efeitos dos fármacos , Células Tumorais CultivadasRESUMO
Bleomycin has been suggested to incite plasma extravasation and influx of inflammatory cells leading to pulmonary fibrosis. We hypothesized that stable analogs of the 12-lipoxygenase product, hepoxilin, may attenuate these effects. We initially investigated the effects of the four hepoxilin analogs (PBT-1 to -4) coadministered intradermally with bleomycin and found that PBT-1 and -2 significantly opposed the vascular permeability effects of bleomycin in rat skin. We subsequently tested the hepoxilin analogs for their actions in opposing the intratracheal bleomycin-evoked acute inflammatory phase of lung fibrosis in the mouse, characterized by a marked accumulation of macrophages and an increase in the rate of collagen synthesis and deposition. We found that the bleomycin-evoked effects on macrophage influx were inhibited by all the hepoxilin analogs (PBT-1, -3, and -4 > PBT-2) administered i.p. for 8 days. Increased total lung collagen was completely abrogated by PBT-1 and -2, whereas PBT-3 and -4 had little effect. A dose-response study with PBT-1 indicated that the effective dose for inhibition of bleomycin-induced inflammatory and histological changes was below 10 microg/day. These studies demonstrate an in vivo action of stable analogs of hepoxilin and support an effect on inflammation and vascular permeability from these novel compounds, especially for PBT-1.
Assuntos
Ácido 8,11,14-Eicosatrienoico/uso terapêutico , Fibrose Pulmonar/tratamento farmacológico , Ácido 8,11,14-Eicosatrienoico/análogos & derivados , Animais , Bleomicina , Modelos Animais de Doenças , Masculino , Camundongos , Camundongos Endogâmicos CBA , Fibrose Pulmonar/induzido quimicamente , Ratos , Ratos WistarRESUMO
We report herein a novel class of thromboxane receptor (TP receptor) antagonists modeled on unstable natural lipids that we identified several years ago, the hepoxilins. These antagonists have been rendered chemically and biologically more stable than the natural compounds through structural modification by chemical synthesis. We demonstrate that the analogs inhibit the aggregation of human platelets in vitro evoked by the thromboxane receptor agonists, I-BOP ([1S-[1alpha,2alpha(Z),3beta(1E,3S*),4alpha]]-7-[3-[3-hydroxy-4-(4-iodophenoxy)-1-butenyl]-7-oxabi-cyclo[2.2.1]hept-2-yl]5-heptenoic acid) and U46619 (9,11-dideoxy-9alpha,11alpha-methanoepoxy-prosta-5Z,13E-dien-1-oic acid). The most potent of the analogs described, PBT-3 [10(S)-hydroxy-11,12-cyclopropyl-eicosa-5Z,8Z,14Z-trienoic acid methyl ester], has an IC(50) versus aggregation by I-BOP = 0.6 x 10(-7) M and versus U46619 = 7 x 10(-7) M, representing one of the most potent anti-aggregating substances so far described. PBT-3 also inhibits thromboxane formation and aggregation evoked by collagen with an IC(50) = 8 x 10(-7) M. Other PBT (hepoxilin cyclopropane) analogs so far tested were 5- to 10-fold less active, and the native hepoxilins were about 500-fold less active. Neither PBT-3 nor the other analogs inhibited 12-lipoxygenase, phospholipase A(2), or cyclooxygenase 1 or 2, and weakly stimulated adenyl cyclase (threshold stimulation at 10(-7) M and little selectivity for each of the PBT compounds). TP antagonism by PBT-3 was further demonstrated in receptor binding studies through use of (125)I-BOP, where the IC(50) for PBT-3 was 8 x 10(-9) M, approximately 16-fold less than for I-BOP itself. These findings identify a new mode of action of PBT-3 and other related analogs as primarily TP antagonists. These studies identify a new family of compounds useful in further development as novel therapeutics for thromboxane-mediated diseases.