RESUMO
Patients harboring CRLF2-rearranged B-lineage acute lymphocytic leukemia (B-ALL) face a 5-year survival rate as low as 20%. While significant gains have been made to position targeted therapies for B-ALL treatment, continued efforts are needed to develop therapeutic options with improved duration of response. Here, first we have demonstrated that patients with CRLF2-rearranged Ph-like ALL harbor elevated thymic stromal lymphopoietin receptor (TSLPR) expression, which is comparable with CD19. Then we present and evaluate the anti-tumor characteristics of 1B7/CD3, a novel CD3-redirecting bispecific antibody (BsAb) that co-targets TSLPR. In vitro, 1B7/CD3 exhibits optimal binding to both human and cynomolgus CD3 and TSLPR. Further, 1B7/CD3 was shown to induce potent T cell activation and tumor lytic activity in both cell lines and primary B-ALL patient samples. Using humanized cell- or patient-derived xenograft models, 1B7/CD3 treatment was shown to trigger dose-dependent tumor remission or growth inhibition across donors as well as induce T cell activation and expansion. Pharmacokinetic studies in murine models revealed 1B7/CD3 to exhibit a prolonged half-life. Finally, toxicology studies using cynomolgus monkeys found that the maximum tolerated dose of 1B7/CD3 was ≤1 mg/kg. Overall, our preclinical data provide the framework for the clinical evaluation of 1B7/CD3 in patients with CRLF2-rearranged B-ALL.
Assuntos
Anticorpos Biespecíficos , Linfoma de Células B , Leucemia-Linfoma Linfoblástico de Células Precursoras B , Humanos , Animais , Camundongos , Complexo CD3 , Anticorpos Biespecíficos/farmacologia , Anticorpos Biespecíficos/uso terapêutico , Linfoma de Células B/tratamento farmacológico , Antígenos CD19 , Linhagem Celular , Leucemia-Linfoma Linfoblástico de Células Precursoras B/patologia , Receptores de CitocinasRESUMO
Macrophages play an important role in immune responses including allograft rejection and they are one of the potential targets of anti-rejection therapies in organ transplantation. Macrophage alloreactivity relies on their phenotype/polarity, motility, phagocytosis and matrix degradation, which in turn depend on proper functioning of actin cytoskeleton and its regulators, the small GTPase RhoA and its downstream effector the Rho-associated protein kinase (ROCK). Several laboratories showed that administration of ROCK inhibitor Y-27632 to the graft recipient inhibits chronic rejection or rodent cardiac allografts. Here we studied the effect of Y-27632 on mouse peritoneal macrophage structure, polarity and functions in in vitro assays. We show that Y-27632 inhibitor affects macrophage phenotype/polarity, phagocytosis, migration, and matrix degradation. These novel findings suggest that the impediment of macrophage structure and function via interference with the RhoA/ROCK pathway has a potential to be therapeutically effective in organ transplantation.
Assuntos
Polaridade Celular/fisiologia , Rejeição de Enxerto/imunologia , Ativação de Macrófagos/imunologia , Macrófagos Peritoneais/citologia , Macrófagos Peritoneais/imunologia , Quinases Associadas a rho/antagonistas & inibidores , Aloenxertos , Amidas/farmacologia , Animais , Polaridade Celular/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Transplante de Coração , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Microscopia Eletrônica , Modelos Animais , Piridinas/farmacologiaRESUMO
Drug delivery is essential to achieve effective therapy. Herein we report on the only implantable nanochannel membrane with geometrically defined channels as small as 2.5 nm that achieves constant drug delivery in vivo. Nanochannels passively control the release of molecules by physico-electrostatic confinement, thereby leading to constant drug diffusion. We utilize a novel design algorithm to select the optimal nanochannel size for each therapeutic agent. Using nanochannels as small as 3.6 and 20 nm, we achieve sustained and constant plasma levels of leuprolide, interferon α-2b, letrozole, Y-27632, octreotide, and human growth hormone, all delivered at clinically-relevant doses. The device was demonstrated in dogs, rats, and mice and was capable of sustaining target doses for up to 70 days. To provide evidence of therapeutic efficacy, we successfully combined nanochannel delivery with a RhoA pathway inhibitor to prevent chronic rejection of cardiac allografts in a rat model. Our results provide evidence that the nanochannel platform has the potential to dramatically improve long-term therapies for chronic conditions.
Assuntos
Sistemas de Liberação de Medicamentos/instrumentação , Nanoestruturas/ultraestrutura , Preparações Farmacêuticas/administração & dosagem , Animais , Cães , Desenho de Equipamento , Feminino , Humanos , Masculino , Membranas Artificiais , Camundongos , Nanoestruturas/química , Próteses e Implantes , Ratos , Ratos Sprague-DawleyRESUMO
BACKGROUND: Current immunosuppressive regimens fail to avert chronic rejection (CR) of transplanted organs; however, selective targeting of actin-cytoskeletal regulators decreases T-cell motility and abrogates CR in rat model system. Administration of mutated class I major histocompatibility complex molecules or selective targeting of the RhoA pathway, which controls T-cell cytoskeletal activity, using Y27632 (a selective Rock1 inhibitor) resulted in reduced T-cell infiltration and abrogation of CR as judged from the neointimal index (13.9±19.7 vs. 45±37.5; P<0.001) and the number of affected vessels (30% vs. 60%; P<0.01). Here, we examined the role of mammalian target of rapamycin (mTOR) pathway in inhibition of CR. METHODS: A mutated class I major histocompatibility complex molecule that eliminates CR was delivered into ACI recipients of Wistar-Furth hearts at the time of transplantation with subtherapeutic cyclosporine (10 mg/kg on days 0-2). Controls included untreated and cyclosporine A-treated (10 mg/kg on days 0-2) heart allograft recipients. RESULTS: Western blotting and immunostaining showed that rat heart allografts with abolished CR exhibited down-regulation of the RAPA-sensitive mTORC1 components such as mTOR and Raptor and down-regulation of the RAPA-insensitive mTORC2 elements Rictor and Sin1. The mTOR regulator Deptor and its downstream target Rac1 were also inhibited. CONCLUSIONS: Abrogation of CR in rat model system involves modulation of two mTOR pathways: a RAPA-sensitive mTORC1 pathway regulating cellular proliferation and a RAPA-insensitive mTORC2 pathway regulating T-cell motility. Selective targeting of T-cell actin cytoskeletal pathways shows potential for pathway-targeted immunosuppression therapies.
Assuntos
Rejeição de Enxerto/prevenção & controle , Transplante de Coração/efeitos adversos , Antígenos de Histocompatibilidade/farmacologia , Imunossupressores/farmacologia , Complexos Multiproteicos/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Proteínas de Transporte/metabolismo , Doença Crônica , Ciclosporina/farmacologia , Rejeição de Enxerto/imunologia , Rejeição de Enxerto/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Masculino , Alvo Mecanístico do Complexo 1 de Rapamicina , Alvo Mecanístico do Complexo 2 de Rapamicina , Fosfoproteínas/metabolismo , Proteína Companheira de mTOR Insensível à Rapamicina , Ratos , Ratos Endogâmicos ACI , Ratos Wistar , Proteína Regulatória Associada a mTOR , Transdução de Sinais/efeitos dos fármacos , Sirolimo/farmacologia , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia , Fatores de Tempo , Proteínas rac1 de Ligação ao GTP/metabolismoRESUMO
Dilations to endothelium-derived hyperpolarizing factor (EDHF) are significantly attenuated in the middle cerebral artery (MCA) isolated from female compared to male rats. Since gap junctions appear to be involved in the EDHF pathway and cAMP has been shown to enhance gap junction permeability, we tested the hypothesis that elevation of cAMP would enhance EDHF-mediated dilations in female rat MCA. Vascular diameter was measured in perfused MCA segments using videomicroscopy in the presence and absence of IBMX, an inhibitor of cAMP phosphodiesterase. In the presence of L-NAME and indomethacin, dilation to 10(-4) M ATP was significantly reduced in females (48+/-12%) compared to males (92+/-2%). IBMX, an inhibitor of cAMP phosphodiesterase, had no significant effect on ATP-mediated dilations in both males and females. Basal cAMP levels were comparable in male and female MCAs (1.7 pmol/mg protein). Incubation with IBMX (2 x 10(-4) M) significantly elevated cAMP in both male (12.8 pmol/mg protein) and female (11.2 pmol/mg protein) MCAs. Our results demonstrate that reduced EDHF dilations in female rat MCA cannot be solely attributed to impaired cAMP signaling. Future studies will target other potential sites along the EDHF pathway in order to identify why EDHF dilations are reduced in the female compared to the male rat MCA.
Assuntos
Fatores Biológicos/fisiologia , AMP Cíclico/metabolismo , Artéria Cerebral Média/fisiologia , Vasodilatação/fisiologia , Animais , Feminino , Junções Comunicantes/fisiologia , Masculino , Análise por Pareamento , Ratos , Ratos Long-Evans , Sistemas do Segundo Mensageiro/fisiologia , Fatores Sexuais , Transdução de Sinais/fisiologiaRESUMO
BACKGROUND: Unlike in peripheral vessels, the endothelium-derived hyperpolarizing factor (EDHF)-mediated component to P2Y(2) receptor-mediated dilations is significantly attenuated in the middle cerebral artery (MCA) of female rats compared to male rats. One aspect to the EDHF phenomenon is activation of the intermediate calcium-sensitive potassium (IK(Ca)) channels located on the endothelium. In an attempt to pinpoint the site along the EDHF pathway that is compromised in females, we tested the hypothesis that direct activation of IK(Ca) channels with DCEBIO would elicit attenuated hyperpolarization in the endothelium and smooth muscle of females compared to males. METHODS: Inhibitors of nitric oxide synthase and cyclooxygenase were present throughout all experiments. Vessel diameter changes were assessed in pressurized and luminally perfused MCAs. Membrane potential changes in the endothelium and smooth muscle were measured using the perforated patch clamp method and sharp electrodes, respectively. RESULTS AND CONCLUSIONS: The maximum vasodilation to 3 x 10(-4)M DCEBIO was significantly reduced in females (37 +/- 9%) compared to intact males (70 +/- 4%). Endothelial cell hyperpolarization to DCEBIO was similar in both males and females. Smooth muscle cell hyperpolarization was attenuated in females (2 +/- 1 mV) compared to males (15 +/- 3 mV). Taken together, our data suggest that the transfer of hyperpolarization from the endothelium to the smooth muscle is impeded in the female rat MCA.
Assuntos
Benzimidazóis/farmacologia , Ativação do Canal Iônico/efeitos dos fármacos , Artéria Cerebral Média/efeitos dos fármacos , Canais de Potássio Cálcio-Ativados/agonistas , Vasodilatação/efeitos dos fármacos , Vasodilatadores/farmacologia , Animais , Fatores Biológicos/metabolismo , Relação Dose-Resposta a Droga , Endotélio Vascular/efeitos dos fármacos , Feminino , Junções Comunicantes/metabolismo , Técnicas In Vitro , Masculino , Potenciais da Membrana/efeitos dos fármacos , Artéria Cerebral Média/metabolismo , Músculo Liso Vascular/efeitos dos fármacos , Técnicas de Patch-Clamp , Canais de Potássio Cálcio-Ativados/metabolismo , Ratos , Ratos Long-Evans , Fatores Sexuais , Fatores de TempoRESUMO
Little is known about the presence and function of two-pore domain K(+) (K(2P)) channels in vascular smooth muscle cells (VSMCs). Five members of the K(2P) channel family are known to be directly activated by arachidonic acid (AA). The purpose of this study was to determine 1) whether AA-sensitive K(2P) channels are expressed in cerebral VSMCs and 2) whether AA dilates the rat middle cerebral artery (MCA) by increasing K+ currents in VSMCs via an atypical K+ channel. RT-PCR revealed message for the following AA-sensitive K(2P) channels in rat MCA: tandem of P domains in weak inward rectifier K+ (TWIK-2), TWIK-related K+ (TREK-1 and TREK-2), TWIK-related AA-stimulated K+ (TRAAK), and TWIK-related halothane-inhibited K+ (THIK-1) channels. However, in isolated VSMCs, only message for TWIK-2 was found. Western blotting showed that TWIK-2 is present in MCA, and immunohistochemistry further demonstrated its presence in VSMCs. AA (10-100 microM) dilated MCAs through an endothelium-independent mechanism. AA-induced dilation was not affected by inhibition of cyclooxygenase, epoxygenase, or lipoxygenase or inhibition of classical K+ channels with 10 mM TEA, 3 mM 4-aminopyridine, 10 microM glibenclamide, or 100 microM Ba2+. AA-induced dilations were blocked by 50 mM K+, indicating involvement of a K+ channel. AA (10 microM) increased whole cell K+ currents in dispersed cerebral VSMCs. AA-induced currents were not affected by inhibitors of the AA metabolic pathways or blockade of classical K+ channels. We conclude that AA dilates the rat MCA and increases K+ currents in VSMCs via an atypical K+ channel that is likely a member of the K(2P) channel family.
Assuntos
Artérias Cerebrais/metabolismo , Canais de Potássio de Domínios Poros em Tandem/metabolismo , Actinas/metabolismo , Animais , Ácido Araquidônico/metabolismo , Western Blotting , Artérias Cerebrais/efeitos dos fármacos , Eletrofisiologia , Imuno-Histoquímica , Técnicas In Vitro , Masculino , Potenciais da Membrana/efeitos dos fármacos , Músculo Liso Vascular/citologia , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/metabolismo , Bloqueadores dos Canais de Potássio/farmacologia , Canais de Potássio/metabolismo , Canais de Potássio de Domínios Poros em Tandem/efeitos dos fármacos , Ratos , Ratos Long-Evans , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Vasodilatação/efeitos dos fármacosRESUMO
We tested the hypotheses that EDHF in rat middle cerebral arteries (MCAs) involves 1) metabolism of arachidonic acid through the epoxygenase pathway, 2) metabolism of arachidonic acid through the lipoxygenase pathway, or 3) reactive oxygen species. EDHF-mediated dilations were elicited in isolated and pressurized rat MCAs by activation of endothelial P2Y(2) receptors with either UTP or ATP. All studies were conducted after the inhibition of nitric oxide synthase and cyclooxygenase with N(omega)-nitro-l-arginine methyl ester (10 microM) and indomethacin (10 microM), respectively. The inhibition of epoxygenase with miconazole (30 microM) did not alter EDHF dilations to UTP, whereas the structurally different epoxygenase inhibitor N-methylsulfonyl-6-(2-propargyloxyphenyl)hexanoic acid (20 or 40 microM) only modestly inhibited EDHF at the highest concentration of UTP. An antagonist of epoxyeicosatrienoic acids, 14,15-epoxyeicosa-5(Z)-enoic acid, had no effect on EDHF dilations to UTP. Chronic inhibition of epoxygenase in the rat with 1-aminobenzotriazol (50 mg/kg twice daily for 5 days) did not alter EDHF dilations. The inhibition of the lipoxygenase pathway with either 10 microM baicalein or 10 microM nordihydroguaiaretic acid produced no major inhibitory effects on EDHF dilations. The combination of superoxide dismutase (200 U/ml) and catalase (140 U/ml) had no effect on EDHF dilations. Neither tiron (10 mM), a cell-permeable scavenger of reactive oxygen species, nor deferoxamine (1 or 10 mM), an iron chelator that blocks the formation of hydroxyl radicals, altered EDHF dilations in rat MCAs. We conclude that EDHF dilations in the rat MCA do not involve the epoxygenase pathway, lipoxygenase pathway, or reactive oxygen species including H(2)O(2).
Assuntos
Ácido Araquidônico/metabolismo , Fatores Biológicos/metabolismo , Artérias Cerebrais/metabolismo , Peróxido de Hidrogênio/metabolismo , Amidas/farmacologia , Animais , Artérias Cerebrais/efeitos dos fármacos , Inibidores de Ciclo-Oxigenase/farmacologia , Inibidores Enzimáticos/farmacologia , Indometacina/farmacologia , Lipoxigenase/metabolismo , Masculino , Miconazol/farmacologia , NG-Nitroarginina Metil Éster/farmacologia , Oxirredutases/metabolismo , Ratos , Ratos Long-EvansRESUMO
There is now strong evidence that an endothelial mechanism, other than nitric oxide or prostacyclin, exists for dilating arteries and arterioles. This third pathway has been named endothelium-derived hyperpolarizing factor (EDHF) and should not be confused with endothelium-derived relaxing factor, which is nitric oxide. Currently, there are several ideas for the mechanism of EDHF, which may vary among vessels of different organs and species. During some pathologic states, EDHF can be up-regulated. This up-regulation often occurs as the dilator effects of endothelium-derived nitric oxide are suppressed. The up-regulated EDHF may serve in a protective capacity to help maintain blood flow to organs and tissues during these stressful states. Many anesthetics attenuate the dilator actions of EDHF; however, the full clinical implications of this anesthetic-related attenuation are not known. Like its cousins, nitric oxide and prostacyclin, EDHF is an important regulator of blood flow and should prove to be an important clinical consideration as we gain more knowledge of its mechanisms of action.
Assuntos
Fatores Biológicos/fisiologia , Endotélio Vascular/fisiologia , Epoprostenol/fisiologia , Óxido Nítrico/fisiologia , Animais , Fatores Biológicos/química , Endotélio Vascular/química , Epoprostenol/química , Humanos , Músculo Liso Vascular/química , Músculo Liso Vascular/fisiologia , Óxido Nítrico/químicaRESUMO
Very little is known regarding the mechanism of action for the endothelium-derived hyperpolarizing factor (EDHF) response in cerebral vessels. The authors tested two hypotheses: (1) activation of the cytoplasmic form of phospholipase A (cPLA ) is involved with EDHF-mediated dilations in rat middle cerebral arteries; and (2) activation of the cPLA involves an increase in endothelial Ca through activation of phospholipase C. Middle cerebral arteries were isolated from the rat, pressurized to 85 mm Hg, and luminally perfused. The EDHF response was elicited by luminal application of uridine triphosphate (UTP) after NO synthase and cyclooxygenase inhibition (10 mol/L -nitro-l-arginine methyl ester and 10 mol/L indomethacin, respectively). AACOCF and PACOCF, inhibitors of cPLA (Ca -sensitive) and Ca -insensitive PLA (iPLA ), dose dependently attenuated the EDHF response. A selective inhibitor for iPLA2, haloenol lactone suicide substrate, had no effect on the EDHF response. The EDHF response elicited by UTP was accompanied by an increase in endothelial Ca (144 to 468 nmol/L), and the EDHF dilation was attenuated with U73122, a phospholipase C inhibitor. The authors conclude that the EDHF response elicited by luminal UTP in rat middle cerebral arteries involved activation of phospholipase C, an increase in endothelial Ca, and activation of cPLA.