RESUMEN
The poly-arginine peptides R18D and R18 represent novel potential neuroprotective treatments for acute ischaemic stroke. Here we examined whether R18D and R18 had any significant effects on the thrombolytic activity of alteplase (tPA) and tenecteplase (TNK) on clots formed from whole blood in an in vitro thrombolysis plate assay. R18D and R18 were examined at concentrations of 0.25, 0.5, 1, 2, 4, 8 and 16 µM during the 1-h thrombolytic assay. We also included the well-characterised neuroprotective NA-1 peptide as a control. R18D, R18 and NA-1 all reduced tPA or TNK percentage clot lysis by 0-9.35%, 0-3.44% and 0-4.8%, respectively. R18D, R18 and NA-1 had a modest and variable effect on the lag time, increasing the time to the commencement of thrombolysis by 0-9.9 min, 0-5.53 min and 0-7.16 min, respectively. Lastly, R18 and NA-1 appeared to increase the maximal activity of the thrombolysis reaction. In addition, the in vitro anti-excitotoxic neuroprotective efficacy of R18D and R18 was not affected by pre-incubation for 1-2 h or overnight with tPA or TNK, whereas only R18D retained high anti-excitotoxic neuroprotective efficacy when pre-incubated in a synthetic trypsin (TrypLE Express). The present in vitro findings suggest that neither R18D or R18 when co-administered with the thrombolytic inducing agents tPA or TNK are likely to have a significant impact when used clinically during clot thrombolysis and confirm the superior proteolytic stability of the R18D peptide.
Asunto(s)
Isquemia Encefálica , Accidente Cerebrovascular , Trombosis , Arginina , Fibrinolíticos/farmacología , Fibrinolíticos/uso terapéutico , Humanos , Péptidos/farmacología , Proteolisis , Accidente Cerebrovascular/tratamiento farmacológico , Tenecteplasa/farmacología , Tenecteplasa/uso terapéutico , Terapia Trombolítica , Activador de Tejido Plasminógeno/farmacología , Activador de Tejido Plasminógeno/uso terapéuticoRESUMEN
Poly-arginine peptides R18 and R18D have previously been demonstrated to be neuroprotective in ischaemic stroke models. Here we examined the proteolytic stability and efficacy of R18 and R18D in reducing infarct core growth and preserving the ischaemic penumbra following middle cerebral artery occlusion (MCAO) in the Sprague Dawley rat. R18 (300 or 1000 nmol/kg), R18D (300 nmol/kg) or saline were administered intravenously 10 min after MCAO induced using a filament. Serial perfusion and diffusion-weighted MRI imaging was performed to measure changes in the infarct core and penumbra from time points between 45- and 225-min post-occlusion. Repeated measures analyses of infarct growth and penumbral tissue size were evaluated using generalised linear mixed models (GLMMs). R18D (300 nmol/kg) was most effective in slowing infarct core growth (46.8 mm3 reduction; p < 0.001) and preserving penumbral tissue (21.6% increase; p < 0.001), followed by R18 at the 300 nmol/kg dose (core: 29.5 mm3 reduction; p < 0.001, penumbra: 12.5% increase; p < 0.001). R18 at the 1000 nmol/kg dose had a significant impact in slowing core growth (19.5 mm3 reduction; p = 0.026), but only a modest impact on penumbral preservation (6.9% increase; p = 0.062). The in vitro anti-excitotoxic neuroprotective efficacy of R18D was also demonstrated to be unaffected when preincubated for 1-3 h or overnight, in a cell lysate prepared from dying neurons or with the proteolytic enzyme, plasmin, whereas the neuroprotective efficacy of R18 was significantly reduced after a 2-h incubation. These findings highlight the capacity of poly-arginine peptides to reduce infarct growth and preserve the ischaemic penumbra, and confirm the superior efficacy and proteolytic stability of R18D, which indicates that this peptide is likely to retain its neuroprotective properties when co-administered with alteplase during thrombolysis for acute ischaemic stroke.
Asunto(s)
Infarto de la Arteria Cerebral Media/tratamiento farmacológico , Fármacos Neuroprotectores/uso terapéutico , Péptidos/uso terapéutico , Animales , Encéfalo/efectos de los fármacos , Células Cultivadas , Fibrinolisina/metabolismo , Masculino , Fármacos Neuroprotectores/química , Fármacos Neuroprotectores/metabolismo , Péptidos/química , Péptidos/metabolismo , Estabilidad Proteica , Ratas Sprague-Dawley , EstereoisomerismoRESUMEN
Thrombolytic therapy with recombinant tissue plasminogen activator (rtPA) in ischaemic stroke has been associated with neurotoxicity, blood brain barrier (BBB) disruption and intra-cerebral hemorrhage. To examine rtPA cellular toxicity we investigated the effects of rtPA on cell viability in neuronal, astrocyte and brain endothelial cell (bEnd.3) cultures with and without prior exposure to oxygen-glucose deprivation (OGD). In addition, the neuroprotective peptide poly-arginine-18 (R18D; 18-mer of D-arginine) was examined for its ability to reduce rtPA toxicity. Studies demonstrated that a 4- or 24-h exposure of rtPA was toxic, affecting neuronal cell viability at ≥ 2 µM, and astrocyte and bEnd.3 cells viability at ≥ 5 µM. In addition, a 4-h exposure to rtPA after a period of OGD (OGD/rtPA) exacerbated toxicity, affecting neuronal, astrocyte and bEnd.3 cell viability at rtPA concentrations as low as 0.1 µM. Treatment of cells with low concentrations of R18D (0.5 and 1 µM) reduced the toxic effects of rtPA and OGD/rtPA, while on some occasions a higher 2 µM R18D concentrations exacerbated neuronal and bEnd.3 cell toxicity in OGD/rtPA exposed cultures. In exploratory studies we also demonstrated that OGD activates matrix metalloproteinase-9 (MMP-9) release into the supernatant of astrocyte and bEnd.3 cell cultures, but not neuronal cultures, and that OGD/rtPA increases MMP-9 activation. Furthermore, R18D decreased MMP-9 activation in OGD/rtPA treated astrocyte and bEnd.3 cell cultures. In summary, the findings show that rtPA can be toxic to neural cells and that OGD exacerbates toxicity, while R18D has the capacity to reduce rtPA neural cellular toxicity and reduce MMP-9 activation in astrocytes and bEnd.3. Poly-arginine-18 peptides, which are being developed as neuroprotective therapeutics for ischaemic stroke, therefore have the additional potential of reducing cytotoxic effects associated with rtPA thrombolysis in the treatment of ischaemic stroke.
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Péptidos y Proteínas de Señalización Intracelular/farmacología , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Activador de Tejido Plasminógeno/toxicidad , Animales , Animales Recién Nacidos , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/fisiología , Células Cultivadas , Relación Dosis-Respuesta a Droga , Ratones , Neuronas/patología , Ratas , Ratas Sprague-Dawley , Proteínas Recombinantes/toxicidadRESUMEN
We have previously demonstrated that Cationic Arginine-Rich Peptides (CARPs) and in particular poly-arginine-18 (R18; 18-mer of arginine) exhibit potent neuroprotective properties in both in vitro and in vivo neuronal injury models. Based on the current literature, there is a consensus that arginine residues by virtue of their positive charge and guanidinium head group is the critical element for imparting CARP neuroprotective properties and their ability to traverse cell membranes. This study examined the importance of guanidinium head groups in R18 for peptide cellular uptake, localization, and neuroprotection. This was achieved by using poly-ornithine-18 (O18; 18-mer of ornithine) as a control, which is structurally identical to R18, but possesses amino head groups rather than guanidino head groups. Epifluorescence and confocal fluorescence microscopy was used to examine the cellular uptake and localization of the FITC-conjugated R18 and O18 in primary rat cortical neurons and SH-SY5Y human neuroblastoma cell cultures. An in vitro cortical neuronal glutamic acid excitotoxicity model was used to compare the effectiveness of R18 and O18 to inhibit cell death and intracellular calcium influx, as well as caspase and calpain activation. Fluorescence imaging studies revealed cellular uptake of both FITC-R18 and FITC-O18 in neuronal and SH-SY5Y cells; however, intracellular localization of the peptides differed in neurons. Following glutamic acid excitotoxicity, only R18 was neuroprotective, prevented caspases and calpain activation, and was more effective at reducing neuronal intracellular calcium influx. Overall, this study demonstrated that for long chain cationic poly-arginine peptides, the guanidinium head groups provided by arginine residues are an essential requirement for neuroprotection but are not required for entry into neurons.
Asunto(s)
Señalización del Calcio/efectos de los fármacos , Neuronas/metabolismo , Fármacos Neuroprotectores , Péptidos , Animales , Línea Celular Tumoral , Neuronas/patología , Fármacos Neuroprotectores/química , Fármacos Neuroprotectores/farmacocinética , Fármacos Neuroprotectores/farmacología , Péptidos/química , Péptidos/farmacocinética , Péptidos/farmacología , Ratas , Ratas Sprague-DawleyRESUMEN
Recent studies have highlighted that a novel class of neuroprotective peptide, known as cationic arginine-rich peptides (CARPs), have intrinsic neuroprotective properties and are particularly effective anti-excitotoxic agents. As such, the present study investigated the mechanisms underlying the anti-excitotoxic properties of CARPs, using poly-arginine-18 (R18; 18-mer of arginine) as a representative peptide. Cortical neuronal cultures subjected to glutamic acid excitotoxicity were used to assess the effects of R18 on ionotropic glutamate receptor (iGluR)-mediated intracellular calcium influx, and its ability to reduce neuronal injury from raised intracellular calcium levels after inhibition of endoplasmic reticulum calcium uptake by thapsigargin. The results indicate that R18 significantly reduces calcium influx by suppressing iGluR overactivation, and results in preservation of mitochondrial membrane potential (ΔΨm) and ATP production, and reduced ROS generation. R18 also protected cortical neurons against thapsigargin-induced neurotoxicity, which indicates that the peptide helps maintain neuronal survival when intracellular calcium levels are elevated. Taken together, these findings provide important insight into the mechanisms of action of R18, supporting its potential application as a neuroprotective therapeutic for acute and chronic neurological disorders.
Asunto(s)
Neuronas/metabolismo , Neuroprotección/efectos de los fármacos , Péptidos/farmacología , Receptores de Glutamato/genética , Animales , Calcio/metabolismo , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Corteza Cerebral/metabolismo , Corteza Cerebral/patología , Retículo Endoplásmico/efectos de los fármacos , Retículo Endoplásmico/metabolismo , Ácido Glutámico/química , Humanos , Mitocondrias/efectos de los fármacos , Mitocondrias/genética , Neuroprotección/genética , Fármacos Neuroprotectores/química , Fármacos Neuroprotectores/farmacología , Péptidos/química , Ratas , Receptores de Glutamato/químicaRESUMEN
BACKGROUND: Despite extensive studies, there are still no clinically available neuroprotective treatments for traumatic brain injury. OBJECTIVES: In previous studies we demonstrated beneficial treatment effects of polyarginine peptides R18 (18-mer of arginine; 300 nmol/kg) and R18D (18-mer of D-arginine; 1000 nmol/kg) in a rat model of impact-acceleration closed-head injury. METHODS: We examined the efficacy of R18D when intravenously administered at a low (100 nmol/kg) and high (1000 nmol/kg) dose, 30 minutes after a closed-head injury in male Sprague-Dawley rats. RESULTS: At postinjury day 3, treatment with R18D at the high dose significantly reduced axonal injury (Pâ¯=â¯0.044), whereas the low-dose treatment of R18D showed a trend for reduced axonal injury. Following assessment in the Barnes maze, both doses of R18D treatment appeared to improve learning and memory recovery compared with vehicle treatment at postinjury days 1 and 3, albeit not to a statistically significant level. Rotarod assessment of vestibulomotor recovery did not differ between R18D and the vehicle treatment groups. CONCLUSIONS: R18D modestly decreased axonal injury only at the highest dose used but had no significant effect on functional recovery. These findings warrant further studies with additional doses to better understand peptide pharmacodynamics and provide information to guide optimal dosing.
RESUMEN
Stroke is the second leading cause of death globally and represents a major cause of devastating long-term disability. Despite sustained efforts to develop clinically effective neuroprotective therapies, presently there is no clinically available neuroprotective agent for stroke. As a central mediator of neurodamaging events in stroke, mitochondria are recognised as a critical neuroprotective target, and as such, provide a focus for developing mitochondrial-targeted therapeutics. In recent years, cationic arginine-rich peptides (CARPs) have been identified as a novel class of neuroprotective agent with several demonstrated mechanisms of action, including their ability to target mitochondria and exert positive effects on the organelle. This review provides an overview on neuronal mitochondrial dysfunction in ischaemic stroke pathophysiology and highlights the potential beneficial effects of CARPs on mitochondria in the ischaemic brain following stroke.
Asunto(s)
Arginina/administración & dosificación , Isquemia Encefálica/tratamiento farmacológico , Mitocondrias/efectos de los fármacos , Fármacos Neuroprotectores/administración & dosificación , Péptidos/administración & dosificación , Accidente Cerebrovascular/tratamiento farmacológico , Animales , Isquemia Encefálica/complicaciones , Isquemia Encefálica/metabolismo , Humanos , Mitocondrias/metabolismo , Accidente Cerebrovascular/complicaciones , Accidente Cerebrovascular/metabolismo , Resultado del TratamientoRESUMEN
Hypoxic-ischaemic encephalopathy (HIE) remains the leading cause of mortality and morbidity in neonates, with no available neuroprotective therapeutic agent. In the development of a therapeutic for HIE, we examined the neuroprotective efficacy of the poly-arginine peptide R18D (arginine 18 mer synthesised with D-arginine) in a perinatal model of hypoxia-ischaemia (HI; common carotid and external carotid occlusion + 8%O2 /92%N2 for 2.5 hr) in the P7 Sprague-Dawley rat. R18D was administered intraperitoneally 30 min (doses 10, 30, 100, 300 and 1,000 nmol/kg), 60 min (doses 30 and 300 nmol/kg) or 120 min (doses 30 and 300 nmol/kg) after HI. Infarct volumes and behavioural outcomes were measured 48 hr after HI. When administered 30 min after HI, R18D at varying doses reduced infarct volume by 23.7% to 35.6% (p = 0.009 to < 0.0001) and resulted in improvements in the negative geotactic response and wire-hang times, at a dose of 30 nmol/kg. When administered 60 min after HI, R18D at the 30 nmol/kg dose reduced total infarct volume by 34.2% (p = 0.002), whilst the 300 nmol/kg dose improved wire-hang time. When administered 120 min after HI, R18D at the 30 and 300 nmol/kg doses had no significant impact on infarct volume, but the 300 nmol/kg dose improved the negative geotactic response. This study further confirms the neuroprotective properties of poly-arginine peptides, demonstrating that R18D can reduce infarct volume and improve behavioural outcomes after HI if administered up to 60 min after HI and improve behavioural outcomes up to 2 hr after HI.
Asunto(s)
Hipoxia-Isquemia Encefálica/tratamiento farmacológico , Fármacos Neuroprotectores/farmacología , Péptidos/farmacología , Animales , Infarto Encefálico/tratamiento farmacológico , Femenino , Masculino , Neuroprotección , Ratas , Ratas Sprague-Dawley , Reflejo de Enderezamiento/efectos de los fármacosRESUMEN
In a recent study, we highlighted the importance of cationic charge and arginine residues for the neuroprotective properties of poly-arginine and arginine-rich peptides. In this study, using cortical neuronal cultures and an in vitro glutamic acid excitotoxicity model, we examined the neuroprotective efficacy of different modifications to the poly-arginine-9 peptide (R9). We compared an unmodified R9 peptide with R9 peptides containing the following modifications: (i) C-terminal amidation (R9-NH2); (ii) N-terminal acetylation (Ac-R9); (iii) C-terminal amidation with N-terminal acetylation (Ac-R9-NH2); and (iv) C-terminal amidation with D-amino acids (R9D-NH2). The three C-terminal amidated peptides (R9-NH2, Ac-R9-NH2, and R9D-NH2) displayed neuroprotective effects greater than the unmodified R9 peptide, while the N-terminal acetylated peptide (Ac-R9) had reduced efficacy. Using the R9-NH2 peptide, neuroprotection could be induced with a 10 min peptide pre-treatment, 1-6 h before glutamic acid insult, or when added to neuronal cultures up to 45 min post-insult. In addition, all peptides were capable of reducing glutamic acid-mediated neuronal intracellular calcium influx, in a manner that reflected their neuroprotective efficacy. This study further highlights the neuroprotective properties of poly-arginine peptides and provides insight into peptide modifications that affect efficacy.
Asunto(s)
Corteza Cerebral/metabolismo , Ácido Glutámico/toxicidad , Neuronas/metabolismo , Fármacos Neuroprotectores/farmacología , Péptidos/farmacología , Animales , Señalización del Calcio/efectos de los fármacos , Corteza Cerebral/patología , Neuronas/patología , Fármacos Neuroprotectores/química , Péptidos/química , RatasRESUMEN
BACKGROUND: We recently reported that poly-arginine peptides have neuroprotective properties both in vitro and in vivo. In cultured cortical neurons exposed to glutamic acid excitotoxicity, we demonstrated that neuroprotective potency increases with polymer length plateauing at R15 to R18 (R = arginine resides). In an in vivo study in rats, we also demonstrated that R9D (R9 peptide synthesised with D-isoform amino acids) administered intravenously at a dose of 1000 nmol/kg 30 min after permanent middle cerebral artery occlusion (MCAO) reduces infarct volume. Based on these positive in vitro and in vivo findings, we decided to examine the neuroprotective efficacy of the L-isoform poly-arginine peptides, R12, R15 and R18 when administered at a dose of 1000 nmol/kg 30 min after permanent MCAO in the rat. RESULTS: At 24 h post-MCAO, there was reduced total infarct volume for R12 (12.8 % reduction) and R18 (20.5 % reduction), but this reduction only reached statistical significance for R18. Brain slice analysis revealed significantly reduced injury in coronal slices 4 and 5 for R18, and slice 5 for R12. The R15 peptide had no effect on infarct volume. Peptide treatment did not reveal any statistical significant improvement in functional outcomes. CONCLUSION: While these findings confirm the in vivo neuroprotective properties of poly-arginine peptides, additional dose studies are required particularly in less severe transient MCAO models so as to further assess the potential of these agents as a stroke therapy.
Asunto(s)
Encéfalo/efectos de los fármacos , Encéfalo/patología , Infarto de la Arteria Cerebral Media/tratamiento farmacológico , Infarto de la Arteria Cerebral Media/patología , Fármacos Neuroprotectores/farmacología , Péptidos/farmacología , Animales , Modelos Animales de Enfermedad , Evaluación Preclínica de Medicamentos , Masculino , Actividad Motora/efectos de los fármacos , Distribución Aleatoria , Ratas Sprague-Dawley , Recuperación de la Función/efectos de los fármacos , Prueba de Desempeño de Rotación con Aceleración Constante , Índice de Severidad de la Enfermedad , Pérdida de Peso/efectos de los fármacosRESUMEN
Using proteomics, we identified nucleoside diphosphate kinase A (NDPKA; also known as NME/NM23 nucleoside diphosphate kinase 1: NME1) to be up-regulated in primary cortical neuronal cultures by erythropoietin (EPO) preconditioning. To investigate a neuroprotective role of NDPKA in neurons, we used a RNAi construct to knock-down and an adenoviral vector to overexpress the protein in cortical neuronal cultures prior to exposure to three ischemia-related injury models; excitotoxicity (L-glutamic acid), oxidative stress (hydrogen peroxide), and in vitro ischemia (oxygen-glucose deprivation). NDPKA down-regulation had no effect on neuronal viability following injury. By contrast, NDPKA up-regulation increased neuronal survival in all three-injury models. Similarly, treatment with NDPKA recombinant protein increased neuronal survival, but only against in vitro ischemia and excitotoxicity. These findings indicate that the NDPKA protein may confer a neuroprotective advantage following injury. Furthermore, as exogenous NDPKA protein was neuroprotective, it suggests that a cell surface receptor may be activated by NDPKA leading to a protective cell-signaling response. Taken together both NDPKAs intracellular and extracellular neuroprotective actions suggest that the protein is a legitimate therapeutic target for the design of drugs to limit neuronal death following stroke and other forms of brain injury.
Asunto(s)
Isquemia Encefálica/metabolismo , Corteza Cerebral/metabolismo , Eritropoyetina/uso terapéutico , Fármacos Neuroprotectores/uso terapéutico , Nucleósido-Difosfato Quinasa/biosíntesis , Regulación hacia Arriba/fisiología , Animales , Isquemia Encefálica/prevención & control , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/fisiología , Células Cultivadas , Corteza Cerebral/efectos de los fármacos , Eritropoyetina/farmacología , Regulación de la Expresión Génica , Células HEK293 , Humanos , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Fármacos Neuroprotectores/farmacología , Nucleósido-Difosfato Quinasa/farmacología , Nucleósido-Difosfato Quinasa/uso terapéutico , Ratas , Ratas Sprague-Dawley , Regulación hacia Arriba/efectos de los fármacosRESUMEN
Cell-penetrating peptides (CPPs) are small peptides (typically 5-25 amino acids), which are used to facilitate the delivery of normally non-permeable cargos such as other peptides, proteins, nucleic acids, or drugs into cells. However, several recent studies have demonstrated that the TAT CPP has neuroprotective properties. Therefore, in this study, we assessed the TAT and three other CPPs (penetratin, Arg-9, Pep-1) for their neuroprotective properties in cortical neuronal cultures following exposure to glutamic acid, kainic acid, or in vitro ischemia (oxygen-glucose deprivation). Arg-9, penetratin, and TAT-D displayed consistent and high level neuroprotective activity in both the glutamic acid (IC50: 0.78, 3.4, 13.9 µM) and kainic acid (IC50: 0.81, 2.0, 6.2 µM) injury models, while Pep-1 was ineffective. The TAT-D isoform displayed similar efficacy to the TAT-L isoform in the glutamic acid model. Interestingly, Arg-9 was the only CPP that displayed efficacy when washed-out prior to glutamic acid exposure. Neuroprotection following in vitro ischemia was more variable with all peptides providing some level of neuroprotection (IC50; Arg-9: 6.0 µM, TAT-D: 7.1 µM, penetratin/Pep-1: >10 µM). The positive control peptides JNKI-1D-TAT (JNK inhibitory peptide) and/or PYC36L-TAT (AP-1 inhibitory peptide) were neuroprotective in all models. Finally, in a post-glutamic acid treatment experiment, Arg-9 was highly effective when added immediately after, and mildly effective when added 15 min post-insult, while the JNKI-1D-TAT control peptide was ineffective when added post-insult. These findings demonstrate that different CPPs have the ability to inhibit neurodamaging events/pathways associated with excitotoxic and ischemic injuries. More importantly, they highlight the need to interpret neuroprotection studies when using CPPs as delivery agents with caution. On a positive note, the cytoprotective properties of CPPs suggests they are ideal carrier molecules to deliver neuroprotective drugs to the CNS following injury and/or potential neuroprotectants in their own right.
Asunto(s)
Péptidos de Penetración Celular/farmacología , Corteza Cerebral/patología , Ácido Glutámico/toxicidad , Isquemia/patología , Ácido Kaínico/toxicidad , Neuronas/patología , Fármacos Neuroprotectores/farmacología , Secuencia de Aminoácidos , Animales , Proteínas Portadoras/química , Proteínas Portadoras/farmacología , Supervivencia Celular/efectos de los fármacos , Péptidos de Penetración Celular/química , Células Cultivadas , Cisteamina/análogos & derivados , Cisteamina/química , Cisteamina/farmacología , Productos del Gen tat/química , Productos del Gen tat/farmacología , Concentración 50 Inhibidora , Modelos Biológicos , Datos de Secuencia Molecular , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Neurotoxinas/toxicidad , Oligopéptidos/química , Oligopéptidos/farmacología , Péptidos/química , Péptidos/farmacología , Ratas , Ratas Sprague-DawleyRESUMEN
[This corrects the article DOI: 10.1016/j.heliyon.2019.e02390.].
RESUMEN
Our laboratory focuses on the development of novel neuroprotective cationic peptides, such poly-arginine-18 (R18: 18-mer of l-arginine; net charge +18) and its d-enantiomer R18D in stroke and other brain injuries. In the clinical development of R18/R18D, their cationic property raises potential safety concerns on their non-specific effects to induce mast cell degranulation and hemolysis. To address this, we first utilised primary human cultured mast cells (HCMCs) to examine anaphylactoid effects. We also included as controls, the well-characterised neuroprotective TAT-NR2B9c peptide and the widely used heparin reversal peptide, protamine. Degranulation assay based on ß-hexosaminidase release demonstrated that R18 and R18D did not induce significant mast cell degranulation in both untreated (naïve) and IgE-sensitised HCMCs in a dose-response study to a maximum peptide concentration of 16 µM. Similarly, TAT-NR2B9c and protamine did not induce significant mast cell degranulation. To examine hemolytic effects, red blood cells (RBCs), were incubated with the peptides at a concentration range of 1-16 µM in the absence or presence of 2% plasma. Measurement of hemoglobin absorbance revealed that only R18 induced a modest, but significant degree of hemolysis at the 16 µM concentration, and only in the absence of plasma. This study addressed the potential safety concern of the application of the cationic neuroprotective peptides, especially, R18D, on anaphylactoid responses and hemolysis. The findings indicate that R18, R18D, TAT-NR2B9c and protamine are unlikely to induce histamine mediated anaphylactoid reactions or RBC hemolysis when administered intravenously to patients.
RESUMEN
Neuronal cell death caused by glutamate excitotoxicity is prevalent in various neurological disorders and has been associated with the transcriptional activation of activator protein-1 (AP-1). In this study, we tested 19 recently isolated AP-1 inhibitory peptides, fused to the cell penetrating peptide TAT, for their efficacy in preventing cell death in cortical neuronal cultures following glutamate excitotoxicity. Five peptides (PYC19D-TAT, PYC35D-TAT, PYC36D-TAT, PYC38D-TAT, PYC41D-TAT) displayed neuroprotective activity in concentration responses in both l- and retro-inverso d-isoforms with increasing levels of neuroprotection peaking at 83%. Interestingly, the D-TAT peptide displayed a neuroprotective effect increasing neuronal survival to 25%. Using an AP-1 luciferase reporter assay, we confirmed that the AP-1 inhibitory peptides reduce AP-1 transcriptional activation, and that c-Jun and c-Fos mRNA following glutamate exposure is reduced. In addition, following glutamate exposure the AP-1 inhibitory peptides decreased calpain-mediated alpha-fodrin cleavage, but not neuronal calcium influx. Finally, as neuronal death following glutamate excitotoxicity was transcriptionally independent (actinomycin D insensitive), our data indicate that activation of AP-1 proteins can induce cell death via non-transcriptional pathways. Thus, these peptides have potential application as therapeutics directly or for the rational design of small molecule inhibitors in both apoptotic and necrotic neuronal death associated with AP-1 activation.
Asunto(s)
Corteza Cerebral/metabolismo , Agonistas de Aminoácidos Excitadores/toxicidad , Ácido Glutámico/toxicidad , Fármacos Neuroprotectores/farmacología , Fragmentos de Péptidos/fisiología , Factor de Transcripción AP-1/antagonistas & inhibidores , Secuencia de Aminoácidos , Animales , Línea Celular , Células Cultivadas , Corteza Cerebral/efectos de los fármacos , Corteza Cerebral/patología , Humanos , Datos de Secuencia Molecular , Fragmentos de Péptidos/genética , Ratas , Ratas Sprague-Dawley , Factores de Tiempo , Factor de Transcripción AP-1/fisiología , Transcripción Genética/fisiologíaRESUMEN
There are virtually no clinically available neuroprotective drugs for the treatment of acute and chronic neurological disorders, hence there is an urgent need for the development of new neuroprotective molecules. Cationic arginine-rich peptides (CARPs) are an expanding and relatively novel class of compounds, which possess intrinsic neuroprotective properties. Intriguingly, CARPs possess a combination of biological properties unprecedented for a neuroprotective agent including the ability to traverse cell membranes and enter the CNS, antagonize calcium influx, target mitochondria, stabilize proteins, inhibit proteolytic enzymes, induce pro-survival signaling, scavenge toxic molecules, and reduce oxidative stress as well as, having a range of anti-inflammatory, analgesic, anti-microbial, and anti-cancer actions. CARPs have also been used as carrier molecules for the delivery of other putative neuroprotective agents across the blood-brain barrier and blood-spinal cord barrier. However, there is increasing evidence that the neuroprotective efficacy of many, if not all these other agents delivered using a cationic arginine-rich cell-penetrating peptide (CCPPs) carrier (e.g., TAT) may actually be mediated largely by the properties of the carrier molecule, with overall efficacy further enhanced according to the amino acid composition of the cargo peptide, in particular its arginine content. Therefore, in reviewing the neuroprotective mechanisms of action of CARPs we also consider studies using CCPPs fused to a putative neuroprotective peptide. We review the history of CARPs in neuroprotection and discuss in detail the intrinsic biological properties that may contribute to their cytoprotective effects and their usefulness as a broad-acting class of neuroprotective drugs.
RESUMEN
Cationic arginine-rich peptides represent a novel class of peptides being developed as neuroprotective agents for stroke and other acute and chronic neurological disorders. As a group, cationic arginine-rich peptides have a diverse range of other biological properties including the ability to traverse cell membranes, modulate immune responses, antagonise ion channel receptor function, as well as possessing cardioprotective, anti-nociceptive, anti-microbial and anti-cancer properties. A sound understanding of their safety profile is essential for the design of future clinical trials and for ensuring translational success with these compounds. At present, while many neuroprotective cationic arginine-rich peptides have been examined in preclinical animal neuroprotection studies, few have been assessed in human safety studies. Despite this, the safety of the prototypical cationic arginine-rich peptide, protamine, which has been in clinical use for over 70 years to reverse the anticoagulant effects of heparin and as an excipient in certain insulin preparations, is well established. In addition, the poly-arginine peptide R9 (ALX40-4C) was developed as an anti-human inmmunodeficiency virus therapeutic in the mid-1990s, and more recently, the neuroprotective cationic arginine-rich peptides TAT-NR2B9c (NA-1), CN-105 and RD2 are being evaluated for the treatment of ischaemic stroke, haemorrhagic stroke and Alzheimer's disease, respectively. Based on the available clinical data, cationic arginine-rich peptides as a group appear to be safe when administered at therapeutic doses by a slow intravenous infusion. While protamine, owing to its isolation from salmon milt and homology with human sperm protamine, can trigger anaphylactic and anaphylactoid reactions in a small proportion of patients previously exposed to the peptide (e.g. diabetic patients), who are allergic to fish or have undergone a vasectomy, such reactions are unlikely to be triggered in individuals exposed to non-protamine cationic arginine-rich peptides.
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Fármacos Neuroprotectores/uso terapéutico , Péptidos/uso terapéutico , Accidente Cerebrovascular/prevención & control , Humanos , Fármacos Neuroprotectores/administración & dosificación , Péptidos/administración & dosificaciónRESUMEN
Poly-arginine peptide-18 (R18) is neuroprotective in different rodent middle cerebral artery occlusion (MCAO) stroke models. In this study, we examined whether R18 treatment could reduce ischemic brain injury and improve functional outcome in a nonhuman primate (NHP) stroke model. A stroke was induced in male cynomolgus macaques by MCAO distal to the orbitofrontal branch of the MCA through a right pterional craniotomy, using a 5-mm titanium aneurysm clip for 90 min. R18 (1000 nmol/kg) or saline vehicle was administered intravenously 60 min after the onset of MCAO. Magnetic resonance imaging (MRI; perfusion-weighted imaging, diffusion-weighted imaging, or T2-weighted imaging) of the brain was performed 15 min, 24 h, and 28 days post-MCAO, and neurological outcome was assessed using the NHP stroke scale (NHPSS). Experimental endpoint was 28 days post-MCAO, treatments were randomized, and all procedures were performed blinded to treatment status. R18 treatment reduced infarct lesion volume by up to 65.2% and 69.7% at 24 h and 28 days poststroke, respectively. Based on NHPSS scores, R18-treated animals displayed reduced functional deficits. This study confirms the effectiveness of R18 in reducing the severity of ischemic brain injury and improving functional outcomes after stroke in a NHP model, and provides further support for its clinical development as a stroke neuroprotective therapeutic.
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Encéfalo/efectos de los fármacos , Péptidos y Proteínas de Señalización Intracelular/farmacología , Fármacos Neuroprotectores/farmacología , Recuperación de la Función/efectos de los fármacos , Accidente Cerebrovascular/patología , Animales , Encéfalo/patología , Modelos Animales de Enfermedad , Macaca fascicularis , MasculinoRESUMEN
BACKGROUND AND PURPOSE: Brain injury after stroke and other cerebral ischemic events is a leading cause of death and disability worldwide. Our purpose here is to argue in favor of combined mild hypothermia (35 degrees C) and magnesium as an acute neuroprotective treatment to minimize ischemic brain injury. METHODS: Drawing on our own experimental findings with mild hypothermia and magnesium, and in light of the moderate hypothermia trials in cardiac arrest/resuscitation and magnesium trials in ischemic stroke (IMAGES, FAST-Mag), we bring attention to the advantages of mild hypothermia compared with deeper levels of hypothermia, and highlight the existing evidence for its combination with magnesium to provide an effective, safe, economical, and widely applicable neuroprotective treatment after brain ischemia. With respect to effectiveness, our own laboratory has shown that combined mild hypothermia and magnesium treatment has synergistic neuroprotective effects and reduces brain injury when administered several hours after global and focal cerebral ischemia. CONCLUSIONS: Even when delayed, combined treatment with mild hypothermia and magnesium has broad therapeutic potential as a practical neuroprotective strategy. It warrants further experimental investigation and presents a good case for assessment in clinical trials in treating human patients after brain ischemia.