d-jnki-1 and Inflammation

We aimed to investigate the safety, tolerability, and systemic diffusion of a single escalating dose of XG-102 (a 31-D-amino-acid peptide inhibiting JNK pathway activation), administered subconjunctivally in the treatment of post-surgery or post-trauma intraocular inflammation.. This is a dose-escalating, tolerance Phase Ib study. Twenty patients with post-surgery or post-traumatic intraocular inflammation were assigned to 1 of the 4 dose escalating (45, 90, 450, or 900 μg XG-102) groups of 5 patients each. Patients were evaluated at 24, 48 h, 8, and 28 days following the administration of XG-102, including laboratory tests, standard eye examinations, vital signs, and occurrence of adverse events. A single plasma quantification of XG-102 was performed 30 min after administration, according to previous pharmacokinetics studies performed on volunteers.. A total of 17 non-serious adverse events, considered unrelated to the study treatment, were reported for 10 patients. The adverse event incidence was not related to the drug dose. All patients experienced a decrease in intraocular inflammation as of 24 h post-administration and this decrease was sustained up to 28 days thereafter. No patient required local injection or systemic administration of corticoids following the administration of XG-102. XG-102 was undetectable in the first 3 dose groups. In the fourth-dose group (900 μg) the XG-102 plasma levels were above the limit of detection for 3 patients and above the limit of quantification for 1 patient.. In this first clinical trial using XG-102, administered as a single subconjunctival injection as adjunct therapy, in patients with recent post-surgery or post-trauma intraocular inflammation is safe and well tolerated. Further studies are required to evaluate its efficacy.

Topics: Adolescent; Aged; Dose-Response Relationship, Drug; Eye Diseases; Female; Humans; Inflammation; Injections, Intraocular; Male; MAP Kinase Kinase 4; MAP Kinase Signaling System; Middle Aged; Peptides; Postoperative Complications

Neonatal encephalopathy is associated with high mortality and life-long developmental consequences. Therapeutic options are very limited. We assessed the effects of D-JNKi, a small peptide c-Jun N-terminal kinase (JNK) MAP kinase inhibitor, on neuroinflammation, mitochondrial integrity and neuronal damage in a neonatal rat model of ischemic brain damage. Hypoxic-ischemic (HI) brain injury was induced in postnatal-day 7 rats by unilateral carotid artery occlusion and hypoxia, and was followed by intraperitoneal D-JNKi treatment. We demonstrate here for the first time that a single intraperitoneal injection with D-JNKi directly after HI strongly reduces neonatal brain damage by >85% with a therapeutic window of at least 6h. D-JNKi treatment also restored cognitive and motor function as analyzed at 9weeks post-insult. Neuroprotective D-JNKi treatment inhibited phosphorylation of nuclear c-Jun (P-c-Jun), and consequently reduced activity of the AP-1 transcription factor and production of cerebral cytokines/chemokines as determined at 3 and 24h post-HI. Inhibition of P-c-Jun by D-JNKi is thought to be mediated via inhibition of the upstream phosphorylation of cytosolic and nuclear JNK and/or by preventing the direct interaction of phosphorylated (P-)JNK with c-Jun. Surprisingly, however, HI did not induce a detectable increase in P-JNK in cytosol or nucleus. Notably, we show here for the first time that HI induces P-JNK only in the mitochondrial fraction, which was completely prevented by D-JNKi treatment. The hypothesis that mitochondrial JNK activation is key to HI brain injury was supported by data showing that treatment of rat pups with SabKIM1 peptide, a specific mitochondrial JNK inhibitor, is also neuroprotective. Inhibition of HI-induced mitochondrial JNK activation was associated with preservation of mitochondrial integrity as evidenced by prevention of ATP loss and inhibition of lipid peroxidation. The HI-induced increase in apoptotic markers (cytochrome c release and caspase 3 activation) as analyzed at 24h post-HI were also strongly reduced by D-JNKi and the mitochondrial anti-apoptotic proteins Bcl-2 and Bcl-xL were upregulated. Neuroprotection was lost after repeated 0+3h D-JNKi treatment which was associated with complete inhibition of the second peak of AP-1 activity and disability to upregulate mitochondrial Bcl-2 and Bcl-xL. We show here for the first time that D-JNKi treatment efficiently protects the neonatal brain against ischemic brain da

Topics: Animals; Animals, Newborn; Apoptosis; Blotting, Western; Brain Ischemia; Disease Models, Animal; Electrophoretic Mobility Shift Assay; Enzyme Inhibitors; Inflammation; MAP Kinase Kinase 4; Mitochondria; Neuroprotective Agents; Oxidative Stress; Peptides; Phosphorylation; Rats; Rats, Wistar; Real-Time Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction

The c-Jun-N-terminal kinase signaling pathway (JNK) is highly activated during ischemia and plays an important role in apoptosis and inflammation. We have previously demonstrated that D-JNKI1, a specific JNK inhibitor, is strongly neuroprotective in animal models of stroke. We presently evaluated if D-JNKI1 modulates post-ischemic inflammation such as the activation and accumulation of microglial cells. Outbred CD1 mice were subjected to 45 min middle cerebral artery occlusion (MCAo). D-JNKI1 (0.1 mg/kg) or vehicle (saline) was administered intravenously 3 h after MCAo onset. Lesion size at 48 h was significantly reduced, from 28.2+/-8.5 mm(3) (n=7) to 13.9+/-6.2 mm(3) in the treated group (n=6). Activation of the JNK pathway (phosphorylation of c-Jun) was observed in neurons as well as in Isolectin B4 positive microglia. We quantified activated microglia (CD11b) by measuring the average intensity of CD11b labelling (infra-red emission) within the ischemic tissue. No significant difference was found between groups. Cerebral ischemia was modelled in vitro by subjecting rat organotypic hippocampal slice cultures to oxygen (5%) and glucose deprivation for 30 min. In vitro, D-JNKI1 was found predominantly in NeuN positive neurons of the CA1 region and in few Isolectin B4 positive microglia. Furthermore, 48 h after OGD, microglia were activated whereas resting microglia were found in controls and in D-JNKI1-treated slices. Our study shows that D-JNKI1 reduces the infarct volume 48 h after transient MCAo and does not act on the activation and accumulation of microglia at this time point. In contrast, in vitro data show an indirect effect of D-JNKI1 on the modulation of microglial activation.

Topics: Animals; Brain Ischemia; Hippocampus; Immunohistochemistry; Inflammation; JNK Mitogen-Activated Protein Kinases; Male; Mice; Microglia; Neurons; Organ Culture Techniques; Peptides; Phosphorylation; Rats; Rats, Sprague-Dawley

Activation of extracellular signal-regulated kinase (ERK) in spinal cord neurons could serve as a marker for sensitization of dorsal horn neurons in persistent pain. ERK is normally activated by high-threshold noxious stimuli. We investigated how low-threshold mechanical stimuli could activate ERK after complete Freund's adjuvant (CFA)-induced inflammation. Unilateral injection of CFA induced ipsilateral heat hyperalgesia and bilateral mechanical allodynia. CFA-induced ERK activation in ipsilateral dorsal horn neurons declined after 2 days. Interestingly, low-threshold mechanical stimulation given by light touch either on the inflamed paw or the contralateral non-inflamed paw dramatically increased ERK phosphorylation in the dorsal horn ipsilateral to touch stimulation. Notably, light touch induced ERK phosphorylation mainly in superficial neurons in laminae I-IIo. Intrathecal administration of the astroglial toxin L-α-aminoadipate on post-CFA day 2 reversed CFA-induced bilateral mechanical allodynia but not heat hyperalgesia. Furthermore, L-α-aminoadipate, the glial inhibitor fluorocitrate, and a peptide inhibitor of c-Jun N-terminal Kinase all reduced light touch-evoked ERK activation ipsilateral to touch. Collectively, these data suggest that (i) ERK can be activated in superficial dorsal horn neurons by low-threshold mechanical stimulation under pathological condition and (ii) ERK activation by light touch is associated with mechanical allodynia and requires an astrocyte network.

Topics: 2-Aminoadipic Acid; Analysis of Variance; Animals; Astrocytes; Citrates; Disease Models, Animal; Edema; Excitatory Amino Acid Antagonists; Extracellular Signal-Regulated MAP Kinases; Freund's Adjuvant; Functional Laterality; Hyperalgesia; Inflammation; Male; Mitogen-Activated Protein Kinase Kinases; Peptides; Physical Stimulation; Posterior Horn Cells; Rats; Rats, Sprague-Dawley; Signal Transduction; Spinal Cord; Touch

Inhibition of c-Jun N-terminal kinase (JNK) by a cell-penetrating, protease-resistant JNK peptide (D-JNKI-1) before hemorrhage and resuscitation (H/R) ameliorated the H/R-induced hepatic injury and blunted the proinflammatory changes. Here we tested the hypothesis if JNK inhibition at a later time point-after hemorrhagic shock but before the onset of resuscitation-in a rat model of H/R also confers protection. Twenty-four male Sprague-Dawley rats (250 - 350 g) were randomly divided into 4 groups: 2 groups of shock animals were hemorrhaged to a MAP of 32 to 37 mmHg for 60 min and randomly received either D-JNKI-1 (11 mg/kg i.p.) or sterile saline as vehicle immediately before the onset of resuscitation. Two groups of sham-operated animals underwent surgical procedures without H/R and were either D-JNKI-1 or vehicle treated. Rats were killed 2 h later. Serum activity of alanine aminotransferase and serum lactate dehydrogenase after H/R increased 3.5-fold in vehicle-treated rats as compared with D-JNKI-1-treated rats. Histopathological analysis revealed that hepatic necrosis and apoptosis (hematoxylin-eosin, TUNEL, and M30, respectively) were significantly inhibited in D-JNKI-1-treated rats after H/R. Hepatic oxidative (4-hydroxynonenal) and nitrosative (3-nitrotyrosine) stress as well as markers of inflammation (hepatic and serum IL-6 levels and hepatic infiltration with polymorphonuclear leukocytes) were also reduced in D-JNKI-1-treated rats. LPS-stimulated TNF-alpha release from whole blood from hemorrhaged and resuscitated animals was higher in vehicle-treated rats as compared with D-JNKI-1-treated rats. c-Jun N-terminal kinase inhibition after hemorrhage before resuscitation resulted in a reduced activation of c-Jun. Taken together, these results indicate that D-JNKI-1 application after hemorrhagic shock before resuscitation blunts hepatic damage and proinflammatory changes during resuscitation. Hence, JNK inhibition is even protective when initiated after blood loss before resuscitation. These experimental results indicate that the JNK pathway may be a possible treatment option for the harmful consequences of H/R.

Topics: Alanine Transaminase; Animals; Apoptosis; In Situ Nick-End Labeling; Inflammation; Interleukin-6; JNK Mitogen-Activated Protein Kinases; Lactate Dehydrogenases; Liver; Male; Necrosis; Peptides; Random Allocation; Rats; Rats, Sprague-Dawley; Resuscitation; Shock, Hemorrhagic; Time Factors

Hemorrhage and resuscitation (H/R) leads to phosphorylation of mitogen-activated stress kinases, an event that is associated with organ damage. Recently, a specific, cell-penetrating, protease-resistant inhibitory peptide of the mitogen-activated protein kinase c-JUN N-terminal kinase (JNK) was developed (D-JNKI-1). Here, using this peptide, we tested if inhibition of JNK protects against organ damage after H/R. Male Sprague-Dawley rats were treated with D-JNKI-1 (11 mg/kg, i.p.) or vehicle. Thirty minutes later, rats were hemorrhaged for 1 h to a MAP of 30 to 35 mmHg and then resuscitated with 60% of the shed blood and twice the shed blood volume as Ringer lactate. Tissues were harvested 2 h later. ANOVA with Tukey post hoc analysis or Kruskal-Wallis ANOVA on ranks, P < 0.05, was considered significant. c-JUN N-terminal kinase inhibition decreased serum alanine aminotransferase activity as a marker of liver injury by 70%, serum creatine kinase activity by 67%, and serum lactate dehydrogenase activity by 60% as compared with vehicle treatment. The histological tissue damage observed was blunted after D-JNKI-1 pretreatment both for necrotic and apoptotic cell death. Hepatic leukocyte infiltration and serum IL-6 levels were largely diminished after D-JNKI-1 pretreatment. The extent of oxidative stress as evaluated by immunohistochemical detection of 4-hydroxynonenal was largely abrogated after JNK inhibition. After JNK inhibition, activation of cJUN after H/R was also reduced. Hemorrhage and resuscitation induces a systemic inflammatory response and leads to end-organ damage. These changes are mediated, at least in part, by JNK. Therefore, JNK inhibition deserves further evaluation as a potential treatment option in patients after resuscitated blood loss.

Topics: Animals; Blood Pressure; Humans; Inflammation; Inflammation Mediators; JNK Mitogen-Activated Protein Kinases; Liver; Male; Peptides; Random Allocation; Rats; Rats, Sprague-Dawley; Resuscitation; Shock, Hemorrhagic