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Despite sensible guidelines for the use of opioid analgesics, respiratory depression remains a significant risk with a possibility of fatal outcomes. Clinicians need to find a balance of analgesia with manageable respiratory effects. The ampakine CX717 (Cortex Pharmaceuticals, Irvine, CA, USA), an allosteric enhancer of glutamate-stimulated AMPA receptor activation, has been shown to counteract opioid-induced respiratory depression in rats while preserving opioid-induced analgesia. Adopting a translational approach, we orally administered 1500 mg of CX717 to 16 male healthy volunteers in a placebo controlled double-blind study. Starting 100 min after CX717 or placebo intake, alfentanil was administered by computerized intravenous infusion targeting a plateau of effective alfentanil plasma concentrations of 100 ng/ml. One hour after start of opioid infusion, its effects were antagonized by intravenous injection of 1.6 mg of the classical opioid antidote naloxone. Respiration was quantified prior to drug administration (baseline), during alfentanil infusion and after naloxone administration by (i) counting the spontaneous respiratory frequency at rest and (ii) by employing hypercapnic challenge with CO2 rebreathing that assessed the expiratory volume at a carbon dioxide concentration in the breathable air of 55% (VE55). Pain was quantified at the same time points, immediately after assessment of respiratory parameters, by (i) measuring the tolerance to electrical stimuli (5 Hz sine increased by 0.2 mA/s from 0 to 20 mA and applied via two gold electrodes placed on the medial and lateral side of the mid-phalanx of the right middle finger) and (ii) by measuring the tolerance to heat (increased by 0.3°C/s from 32 to 52.5°C applied to a 3 x 3 cm2 skin area of the left volar forearm, after sensitization with 0.15 g capsaicin cream 0.1%). CX717 was tolerated by all subjects without side effects that would have required medical intervention. We observed that CX717 was approximately as effective as naloxone in reversing the opioid induced reduction of the respiratory frequency. Despite the presence of high plasma alfentanil concentrations, the respiratory frequency decreased only by 8.9 ± 22.4% when CX717 was pre-administered, which was comparable to the 7.0 ± 19.3% decrease observed after administration of naloxone. In contrast, after placebo pre-administration the respiratory rate decreased by 30.0 ± 21.3% (p=0.0054 for CX717 versus placebo). In agreement with this, periods of a very low respiratory frequency of <= 4 min-1 under alfentanil alone were shortened by ampakine pre-dosing by 52.9% (p=0.0182 for CX717 versus placebo). Furthermore, VE55 was decreased during alfentanil infusion by 55.9 ± 16.7% under placebo preadministration but only by 46.0 ± 18.1% under CX717 pre-administration (p=0.017 for CX717 versus placebo). Most importantly, in contrast to naloxone, CX717 had no effect on opioid induced analgesia. Alfentanil increased the pain tolerance to electrical stimuli by 68.7 ± 59.5% with placebo pre-administration. With CX717 pre-administration, the increase of the electrical pain tolerance was similar (54.6 ± 56.7%, p=0.1 for CX717 versus placebo). Similarly, alfentanil increased the heat pain tolerance threshold by 24.6 ± 10.0% with placebo pre-administration. Ampakine co-administration had also no effect on the increase of the heat pain tolerance of the capsaicin-sensitized skin (23.1 ± 8.3%, p=0.46 for CX717 versus placebo). The results of this study allow us to draw the conclusion, that opioid induced ventilatory depression can be selectively antagonized in humans by co-administering an ampakine. This is the first successful translation of a selective antagonism of opioidinduced respiratory depression from animal research into application in humans. Ampakines, namely CX717, thus are the first selective antidote for opioid-induced respiratory depression without loss of analgesia, available for the use in humans.
Atherosclerosis is accompanied by infiltration of macrophages to the intima of blood vessels. There they engulf oxLDL (oxidized low-density lipoproteins) and differentiate to foam cells. These cells are known as major promoters of atherosclerosis progression. In initial experiments I could demonstrate that foam cell formation caused a severe loss in the ability to produce IFNA (interferon A) in response to stimulation with the bacterial cell wall component LPS (lipopolysaccharide). Since IFNA is discussed to have anti-atherosclerotic potential and has the capability to induce immune tolerance, its inhibition in foam cells might promote the atherosclerotic process. For this reason the aim of my PhD project was to clarify the underlying molecular mechanisms that attenuate LPS-induced IFNA expression in foam cells. LPS activates TLR4 (Toll-like receptor 4) in macrophages. Downstream this receptor two distinct signaling pathways are activated, namely a MyD88 (myeloid differentiation primary response gene 88)-dependent and a TRIF (TIR-domain-containing adapter-inducing IFNA)-dependent one. Foam cell formation targeted the TRIF-dependent TLR4 signaling pathway, as seen by loss of IRF3 activation and IFNA expression inhibition, whereas MyD88-initiated NFBB (nuclear factor 'B-light-chain-enhancer' of activated B-cells) activation and subsequent TNF@ (tumor necrosis factor @) expression remained unaltered. The TRIF signaling cascade results in transactivation of the transcription factor IRF3 (interferon regulatory factor 3), the main activator of IFNA expression. This event demands IRF3 phosphorylation by TBK1 (TANK-binding kinase 1), whereas TBK1 needs to be recruited to TRAF3 (TNF receptor associated factor 3) by the scaffold protein TANK (TRAF family member-associated NFBB activator) for its activation. This work allowed to propose the following scheme: OxLDL utilizes SR-A1 (scavenger receptor A1) to activate IRAK4 (interleukin-1 receptor-associated kinase 4), IRAK1 and Pellino3. Active IRAK1 and Pellino3 associate with TRAF3 and Pellino3 promotes mono-ubiquitination of the adaptor molecule TANK. Mono-ubiquitination of TANK interrupts TBK1 recruitment to TRAF3 and thereby abrogates phosphorylation and transactivation of IRF3 as well as subsequent expression of IFNA. In this study I provide evidence for a negative regulatory role of Pellino3 for TRIF-dependent TLR4 signaling. This expands the current knowledge of the interplay between pathways downstream scavenger and Toll-like receptors. Due to the multifaceted roles of TLR4 signaling in pathology, the new TRIF-signaling inhibitor Pellino3 might be of importance as therapeutical target for disease intervention.
Die Präkonditionierung mit den bakteriellen Zellwandbestandteilen Lipopolysaccharid (LPS) oder Lipoteichonsäure (LTA) führt in vivo zu einer Reduktion der myokardialen Infarktgröße nach Ischämie und Reperfusion (I/R). Hierbei wird durch die Präkonditionierung u.a. die Akkumulation neutrophiler Granulozyten im Ischämiegebiet während der Reperfusionsphase reduziert und somit einer der wichtigsten Mechanismen bei der Entstehung des Reperfusionsschadens am Herzen vermindert. In dieser Studie bedienten wir uns eines ex vivo Modells nach Langendorff mit regionaler I/R und zellfreier Perfusion. Wir konnten erstmalig eine LTA-Präkonditionierung in einem leukozytenfreien System zeigen und somit demonstrieren, dass die LTA-Präkonditionierung Mechanismen involviert, die unabhängig sind von einer Akkumulation neutrophiler Granulozyten. 24 Stunden nach einer Vorbehandlung der Ratten mit LPS, LTA, Kochsalz und/oder Dexamethason wurden die Herzen entfernt und retrograd mit oxygenierter Krebs-Henseleit-Lösung perfundiert. Die Herzen wurden einer 20-minütigen Ischämie, gefolgt von einer 2-stündigen Reperfusionsphase, unterzogen. Das Infarktrisikogebiet (Evans-Blue-Färbung) und das Infarktgebiet (pNBT-Färbung) wurden planimetrisch bestimmt. Die ischämische Präkonditionierung (IPC) wurde als Positivkontrolle unseres Modells verwendet. LTA- und LPS-Präkonditionierung führten - ebenso wie IPC - bei gleicher Dosierung in vergleichbarem Umfang zu einer signifikanten Reduktion der Infarktgröße. Dieser Effekt konnte durch Vorbehandlung mit Dexamethason vollständig aufgehoben werden, so dass gefolgert werden kann, dass die Protektion durch LPS bzw. LTA. von der Modulation der inflammatorischen Vorgänge im Endothel und Myokard mit abhängt.