RESUMO
ETHNOPHARMACOLOGICAL RELEVANCE: Ayurvedic medicine has been used in the treatment of diabetes mellitus for centuries. In Arabia and some areas of Africa, Commiphora myrrha (CM) has been extensively used as a plant-based remedy. We have previously shown that an aqueous CM resin solution directly stimulates insulin secretion from MIN6 cells, a mouse ß-cell line, and isolated mouse and human islets. However, the signaling pathways involved in CM-induced insulin secretion are completely unknown. Insulin secretion is normally triggered by elevations in intracellular Ca2+ ([Ca2+]i) through voltage gated Ca2+ channels (VGCC) and activation of protein kinases. Protein and lipid kinases such as protein kinase A (PKA), Ca2+-calmodulin dependent protein kinase II (CaMKII), phosphoinositide 3-kinases (PI3Ks), protein kinase C (PKC) and mitogen-activated protein kinase (MAPK), specifically extracellular signal-regulated kinases (ERK1/2), may be involved in receptor-operated insulin secretion. Therefore, we hypothesized that CM may induce insulin secretion by modulating the activity of VGCC and/or one or more of the above kinases. AIM OF THE STUDY: To investigate the possible molecular mechanism of action of CM-induced insulin secretion. The effects of aqueous CM resin extract on [Ca2+]i and protein kinase activation from ß-cells were examined. METHODS: The effect of aqueous CM resin solution on [Ca2+]i was assessed using Ca2+ microfluorimetry. The involvement of VGCC in CM-induced insulin secretion was investigated using static and perifusion insulin secretion experiments in the presence of either EGTA, a Ca2+ chelator, or nifedipine, a blocker of VGCC. The involvement of kinase activation in the stimulatory effect of CM on insulin secretion was examined by using static and perifusion insulin secretion experiments in the presence of known pharmacological inhibitors and/or downregulation of specific kinases. The effects of CM on phosphorylation of PKCζ and ERK1/2 were also assessed using the Wes™ capillary-based protein electrophoresis. RESULTS: Ca2+ microfluorimetry measurements showed that exposing MIN6 cells to CM (0.5-2 mg/mL) was not associated with changes in [Ca2+]i. Similarly, incubating MIN6 cells and mouse islets with EGTA and nifedipine, respectively, did not attenuate the insulin secretion induced by CM. However, incubating mouse and human islets with CM in the presence of staurosporine, a non-selective protein kinase inhibitor, completely blocked the effect of CM on insulin secretion. Exposing mouse islets to CM in the presence of H89, KN62 and LY294002, inhibitors of PKA, CaMKII and PI3K, respectively, did not reduce CM-induced insulin secretion. However, incubating mouse and human islets with CM in the presence of Ro 31-8220, a pan-PKC inhibitor, diminished insulin secretion stimulated by CM, whereas inhibiting the action of typical PKC (with Go6976) and PLCß (with U73122) did not affect CM-stimulated insulin secretion. Similarly, downregulating typical and novel PKC by chronic exposure of mouse islets to phorbol 12-myristate 13-acetate (PMA) was also not associated with a decrease in the stimulatory effect of CM on insulin secretion. Interestingly, CM-induced insulin secretion from mouse islets was inhibited in the presence of the PKCζ inhibitor ZIP and a MAPK inhibitor PD 98059. In addition, Wes™ capillary-based protein electrophoresis indicated that expression of the phosphorylated forms of PKCζ and ERK1/2, a MAPK, was significantly increased following exposure of INS-1832/13 cells, a rat insulinoma cell line, to CM. CONCLUSIONS: Our data indicate that CM directly stimulates insulin secretion through activating known downstream effectors of insulin-stimulus secretion coupling. Indeed, the increase in insulin secretion seen with CM is independent of changes in [Ca2+]i and does not involve activation of VGCC. Instead, the CM stimulatory effect on insulin secretion is completely dependent on protein kinase activation. Our findings indicate that CM could induce insulin exocytosis by stimulating the phosphorylation and activation of PKCζ, which in turn phosphorylates and activates ERK1/2.
Assuntos
Commiphora , Neoplasias Pancreáticas , Humanos , Ratos , Animais , Camundongos , Secreção de Insulina , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina , Ácido Egtázico , Nifedipino , Proteína Quinase C , Proteínas Quinases Dependentes de AMP Cíclico , Insulina , MAP Quinases Reguladas por Sinal Extracelular , Acetato de Tetradecanoilforbol , Fosfatidilinositol 3-QuinasesRESUMO
BACKGROUND: Folk medicines are attractive therapeutic agents for treating type 2 diabetes mellitus (T2DM). Most plant extracts that have been suggested to restore ß-cells function were tested in vivo. Some only have been tested in vitro to determine whether they have a direct effect on ß-cells islets of Langerhans. Currently, there are no defined criteria for screening of ß-cell-directed plant-based remedies as potential antidiabetic agents. SUMMARY: In this review, we have identified certain criteria/characteristics that can be used to generate a "screening portfolio" to identify plant extracts as potential ß-cell-directed agents for the treatment of T2DM. To validate our screening method, we studied the potential therapeutic efficacy of a Gymnema sylvestre (GS) extract using the screening criteria detailed in the review. Six criteria have been identified and validated using OSA®, a GS extract. By using this screening method, we show that OSA® fulfilled most of the criteria identified for an effective ß-cell-directed antidiabetic therapy, being an effective insulin-releasing agent at nontoxic concentrations; maintaining ß-cell insulin content by stimulating a concomitant increase in insulin gene transcription; maintaining ß-cell mass by protecting against apoptosis; and being effective at maintaining normoglycemia in vivo in a mouse model and a human cohort with T2DM. KEY MESSAGES: The present review has highlighted the importance of having a screening portfolio for plant extracts that have potential antidiabetic effects in the treatment of T2DM. We propose that this screening method should be adopted for future studies to identify new ß-cell-directed antidiabetic plant derived agents.
Assuntos
Diabetes Mellitus Tipo 2 , Gymnema sylvestre , Magnoliopsida , Animais , Camundongos , Humanos , Diabetes Mellitus Tipo 2/tratamento farmacológico , Insulina , Hipoglicemiantes/farmacologia , Hipoglicemiantes/uso terapêutico , Extratos Vegetais/farmacologia , Extratos Vegetais/uso terapêuticoRESUMO
Nerve growth factor (NGF) is an endogenously produced polypeptide that promotes the differentiation, survival, and repair of neurons in the central and peripheral nervous systems. While trophic proteins hold promise for the treatment of neuronal injury and disease, use of NGF is limited by its large molecular weight, lack of permeability through the blood-brain barrier, and peripheral side effects. Previously, we found that an extract of the Momordica cochinchinensis seed stimulated PC-12 neurite outgrowth. Bioactivity-guided fractioning of the seed extract suggested that the NGF mimetic agent was one of few defined proteins from this plant: one group being the defense Knottins and the other group of the lowest mass is the potent trypsin inhibitor MCoTI-II. Here, the NGF mimetic potential of this latter protein was investigated using two concurrent but different approaches. A biological study used recombinant purified MCoTI-II, which when tested in rat PC-12 cells grown on collagen, failed to initiate outgrowth relative to the positive control 7S NGF. In a separate computational study, the possibility was investigated such that MCoTI-II could exert an effect through binding to the serine protease γ-NGF subunit of the 7S NGF complex, analogous to its binding to its native receptor trypsin. Molecular dynamics simulations showed that MCoTI-II can bind stably to γ-NGF for >350 ns. Modeling indicated that this interaction could sterically inhibit 7S NGF complex formation, potentially altering the equilibrium between inactive complexed and free active NFG protein. In conclusion, the biological study now excludes the MCoTI-II protein as the NGF mimetic factor in the Momordica extract, an important and required step to identify the active component in this seed. On the other hand, the theoretical study has revealed a novel observation that may be of use in the development of strategies to affect NGF activity.
Assuntos
Ciclotídeos/metabolismo , Miniproteínas Nó de Cistina/metabolismo , Fatores de Crescimento Neural/metabolismo , Crescimento Neuronal , Extratos Vegetais/metabolismo , Animais , Biomimética , Simulação por Computador , Ciclotídeos/química , Miniproteínas Nó de Cistina/química , Simulação de Dinâmica Molecular , Momordica/química , Fatores de Crescimento Neural/química , Células PC12 , Ligação Proteica , Conformação Proteica , Ratos , Serina Endopeptidases/metabolismoRESUMO
PURPOSE: Codrituzumab, a humanized antibody against glypican-3, is highly expressed in HCC. A phase I study evaluated the combination with sorafenib in HCC. PATIENTS AND METHODS: In a 3 + 3 design, codrituzumab was given intravenously in various doses with sorafenib 400 mg twice daily to patients with advanced HCC, age ≥18, ECOG 0-1, Child-Pugh A and B7, adequate organ functions, and no prior systemic therapy, with tumor assessment by RECIST 1.0 and safety by CTCAE 3.0. PK and pre, during, and post-therapy 124I radiolabeled codrituzumab PET scan imaging were performed. RESULTS: 41 patients were enrolled: 2.5 mg/kg weekly (qw) (12), 5 mg/kg qw (12), 10 mg/kg qw (3), 1600 mg every 2 weeks (q2w) (6), and 1600 mg qw (7). Two drug limiting toxicities occurred: grade 3 hyponatremia at 5 mg/kg and grade 3 hyponatremia and hyperglycemia at 1600 mg q2w. Adverse events occurred in 80% of patients, including at least one ≥grade 3: ten (25%) increased AST, three (7.5%) increased ALT, and ten (25%) increased lipase. There were no responses and nine (25.7%) had stable disease. PK C max and AUCt of codrituzumab and sorafenib were comparable to single-agent data. Thirteen out of 14 patients showed 124I radiolabeled codrituzumab uptake in tumor. In all three patients who underwent a post-progression PET, glypican-3 remained expressed. CONCLUSION: Codrituzumab plus sorafenib were tolerated at 1600 mg q2w and 400 mg bid, respectively, with no responses. Codrituzumab exerts selective distribution to HCC cells, and GPC3 does not show any down-regulation post-progression (NCT00976170).
Assuntos
Anticorpos Monoclonais Humanizados/administração & dosagem , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Carcinoma Hepatocelular/tratamento farmacológico , Glipicanas/antagonistas & inibidores , Neoplasias Hepáticas/tratamento farmacológico , Adulto , Idoso , Idoso de 80 Anos ou mais , Anticorpos Monoclonais Humanizados/efeitos adversos , Anticorpos Monoclonais Humanizados/farmacocinética , Feminino , Humanos , Radioisótopos do Iodo , Masculino , Pessoa de Meia-Idade , Niacinamida/administração & dosagem , Niacinamida/efeitos adversos , Niacinamida/análogos & derivados , Niacinamida/farmacocinética , Compostos de Fenilureia/administração & dosagem , Compostos de Fenilureia/efeitos adversos , Compostos de Fenilureia/farmacocinética , Tomografia por Emissão de Pósitrons , SorafenibeRESUMO
Only about 1 in 5,000 investigational agents in a preclinical stage acquires Food and Drug Administration approval. Among many reasons for this includes an inefficient transition from preclinical to clinical phases, which exponentially increase the cost and the delays the process of drug development. Positron emission tomography (PET) is a nuclear imaging technique that has been used for the diagnosis, risk stratification, and guidance of therapy. However, lately with the advance of radiochemistry and of molecular imaging technology, it became evident that PET could help novel drug development process. By using a PET radioligand to report on receptor occupancy during novel agent therapy, it may help assess the effectiveness, efficacy, and safety of such a new medication in an early preclinical stage and help design successful clinical trials even at a later phase. In this article, we explore the potential implications of PET in the development of new heart failure therapies and review PET's application in the respective pathophysiologic pathways such as myocardial perfusion, metabolism, innervation, inflammation, apoptosis, and cardiac remodeling.
Assuntos
Fármacos Cardiovasculares/farmacologia , Insuficiência Cardíaca/tratamento farmacológico , Tomografia por Emissão de Pósitrons/métodos , Desenho de Fármacos , Avaliação Pré-Clínica de Medicamentos/métodos , Distribuição TecidualRESUMO
UNLABELLED: Humanized A33 (huA33) is a promising monoclonal antibody that recognizes A33 antigen, which is present in more than 95% of colorectal cancers and in normal bowel. In this study, we took advantage of quantitative PET to evaluate (124)I huA33 targeting, biodistribution, and safety in patients with colorectal cancer. We also determined the biodistribution of (124)I-huA33 when a large dose of human intravenous IgG (IVIG) was administered to manipulate the Fc receptor or when (124)I-huA33 was given via hepatic arterial infusion (HAI). METHODS: We studied 25 patients with primary or metastatic colorectal cancer; 19 patients had surgical exploration or resection. Patients received a median of 343 MBq (44.4-396 MBq) and 10 mg of (124)I-huA33. Nineteen patients received the antibody intravenously and 6 patients via HAI, and 5 patients also received IVIG. RESULTS: Ten of 12 primary tumors were visualized in 11 patients. The median concentration in primary colon tumors was 0.016% injected dose per gram, compared with 0.004% in normal colon. The PET-based median ratio of hepatic tumor uptake to normal-liver uptake was 3.9 (range, 1.8-22.2). Quantitation using PET, compared with well counting of serum and tissue, showed little difference. Prominent uptake in bowel hindered tumor identification in some patients. Pharmacokinetics showed that patients receiving IVIG had a significantly shorter serum half-time (41.6 ± 14.0 h) than those without (65.2 ± 9.8 h). There were no differences in clearance rates among the intravenous group, IVIG group, and HAI group, nor was there any difference in serum area under the curve, maximum serum concentration, or volume of distribution. Weak titers of human-antihuman antibodies were observed in 6 of 25 patients. No acute side effects or significant toxicities were associated with huA33. CONCLUSION: Good localization of (124)I-huA33 in colorectal cancer with no significant toxicity has been observed. PET-derived (124)I concentrations agreed well with those obtained by well counting of surgically resected tissue and blood, confirming the quantitative accuracy of (124)I-huA33 PET. The HAI route had no advantage over the intravenous route. No clinically significant changes in blood clearance were induced by IVIG.
Assuntos
Neoplasias Colorretais/imunologia , Radioisótopos do Iodo/farmacologia , Glicoproteínas de Membrana/química , Tomografia por Emissão de Pósitrons/métodos , Idoso , Área Sob a Curva , Colo/diagnóstico por imagem , Neoplasias Colorretais/diagnóstico por imagem , Neoplasias Colorretais/terapia , Feminino , Humanos , Imunoglobulinas Intravenosas/metabolismo , Imunoglobulinas Intravenosas/farmacocinética , Masculino , Pessoa de Meia-Idade , Metástase Neoplásica , Estudos Prospectivos , Radioimunoterapia/métodos , Resultado do TratamentoRESUMO
Plant-derived extracts have been used as folk remedies for Type 2 diabetes mellitus (T2DM) for many centuries, and offer the potential of cheap and readily available alternatives to conventional pharmaceuticals in developing countries. Extracts of Costus pictus (CP), a plant belonging to the Costaceae family, are reported to have antidiabetic activity in vivo. The exact molecular mode of action(s) of CP is unclear but the antihyperglycemic effect seen in animal studies was associated with dramatic increases in insulin secretion so in our study we have measured the effect of aqueous CP extract on insulin secretion in vitro from the MIN6 ß-cell line and isolated mouse and human islets. Our data demonstrate that CP has a direct stimulatory effect on insulin secretion at basal but not stimulatory glucose concentrations which was not associated with compromised membrane integrity or decrease ß-cell viability. Single cell calcium microfluorimetry measurements showed that CP caused elevations in ß-cell intracellular Ca(2+) concentrations ([Ca(2+)](i)), an effect which was completely abolished by the removal of extracellular Ca(2+) or blockade of voltage-gated Ca(2+) channels (VGCC). These in vitro observations suggest that one mode of action of CP is through stimulating insulin secretion which may be mediated, in part, by the ability of CP to increase [Ca(2+)](i) levels through VGCC. CP extracts may provide an affordable and inexpensive alternative for treating patients with T2DM.
Assuntos
Canais de Cálcio/química , Costus/química , Insulina/metabolismo , Ilhotas Pancreáticas/efeitos dos fármacos , Extratos Vegetais/farmacologia , Animais , Cálcio/metabolismo , Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio/metabolismo , Linhagem Celular , Diabetes Mellitus Tipo 2/tratamento farmacológico , Humanos , Secreção de Insulina , Ilhotas Pancreáticas/metabolismo , Camundongos , Extratos Vegetais/químicaRESUMO
Leaves of the Gymnema sylvestre (GS) plant have been used to treat diabetes mellitus for millennia, but the previously documented insulin secretagogue effects of GS extracts in vitro may be non-physiological through damage to the beta-cells. We have now examined the effects of a novel GS extract (termed OSA) on insulin secretion from the MIN6 beta-cell line and isolated human islets of Langerhans. Insulin secretion from MIN6 cells was stimulated by OSA in a concentration-dependent manner, with low concentrations (0.06-0.25 mg/ml) having no deleterious effects on MIN6 cell viability, while higher concentrations (> or = 0.5 mg/ml) caused increased Trypan blue uptake. OSA increased beta-cell Ca2+ levels, an effect that was mediated by Ca2+ influx through voltage-operated calcium channels. OSA also reversibly stimulated insulin secretion from isolated human islets and its insulin secretagogue effects in MIN6 cells and human islets were partially dependent on the presence of extracellular Ca2+. These data indicate that low concentrations of the GS isolate OSA stimulate insulin secretion in vitro, at least in part as a consequence of Ca2+ influx, without compromising beta-cell viability. Identification of the component of the OSA extract that stimulates regulated insulin exocytosis, and further investigation of its mode(s) of action, may provide promising lead targets for Type 2 diabetes therapy.
Assuntos
Gymnema sylvestre/química , Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Ilhotas Pancreáticas/efeitos dos fármacos , Ilhotas Pancreáticas/metabolismo , Extratos Vegetais/química , Extratos Vegetais/farmacologia , Animais , Cálcio/metabolismo , Linhagem Celular , Humanos , Secreção de Insulina , Células Secretoras de Insulina/efeitos dos fármacos , Camundongos , Microscopia , Água/químicaRESUMO
INTRODUCTION: The in vitro and in vivo behavior of the radiolabeled monoclonal antibody MORAb-003 was investigated as a prelude to a clinical trial. METHODS: The cellular retention of 111In- and 131I-labeled MORAb-003 was investigated using IGROV1 and SW620 cells. Biodistribution studies in tumor-bearing mice were performed with the more favorable agent. RESULTS: Five 1,4,7,10-tetraazacyclododecane-N,N',N",N'"-tetraacetic acid (DOTA) molecules were conjugated to MORAb-003 with no apparent loss of immunoreactivity. Radiolabeled MORAb-003 had a high affinity for the folate receptor alpha (FRA) expressed by both IGROV1 and SW620 cells and was found to bind to around 8 x 10(5) and 7 x 10(5) sites/cell, respectively. Both cancer cell lines were found to internalize both 131I- and 111In-labeled MORAb-003, but 111In was retained and 131I was released as iodide. In athymic mice, 111In-DOTA-MORAb-003 was cleared from the blood with a single exponential biological clearance rate of 110 h. The uptake in SW620 tumors was 32+/-5%ID/g after 4 days. The clearance rate of activity from normal organs such as liver, kidney and spleen was similar to the blood clearance and was 5.36%ID/g, 4.03%ID/g and 4.36%ID/g at 1 day postinjection and 2.14%ID/g, 1.65%ID/g and 3.74%ID/g after 8 days, respectively. In a pilot clinical study, the biodistribution and tumor targeting of 111In-MORAb-003 was assessed in three patients undergoing treatment with cold MORAb-003. CONCLUSION: MORAb-003 is an attractive antibody for radioimmunoscintigraphy and possibly radioimmunotherapy of FRA-expressing cancers in addition to its potential direct therapeutic effects.