Distinct binding and signaling activity of Acthar Gel compared to other melanocortin receptor agonists.

PURPOSE: To compare the binding and agonistic activity of Acthar® Gel and synthetic melanocortin receptor (MCR) agonists and examine how the activity of select agonists affects the in vivo production of corticosterone. MATERIALS AND METHODS: In vitro binding was determined using concentration-dependent displacement of the ligand [125I]Nle4, D-Phe7-α-melanocyte-stimulating hormone (α-MSH) on cells expressing MC1R, MC3R, MC4R, or MC5R. Functional activity was determined using a time-resolved fluorescence cyclic adenosine monophosphate (cAMP) assay in cells expressing MC1R, MC2R, MC3R, MC4R, or MC5R. In vivo corticosterone analyses were performed by measuring plasma corticosterone levels in Sprague Dawley rats. RESULTS: Acthar Gel and synthetic MCR agonists exhibited the highest binding at MC1R, lowest binding at MC5R, and moderate binding at MC3R and MC4R. Acthar Gel stimulated the production of cAMP in all 5 MCR-expressing cell lines, with MC2R displaying the lowest level of full agonist activity, 3-, 6.6-, and 10-fold lower than MC1R, MC3R, and MC4R, respectively. Acthar Gel was a partial agonist at MC5R. The synthetic MCR agonists induced full activity at all 5 MCRs, with the exception of α-MSH having no activity at MC2R. Acthar Gel treatment had less of an impact on in vivo production of corticosterone compared with synthetic ACTH1-24 depot. CONCLUSIONS: Acthar Gel bound to and activated each MCR tested in this study, with partial agonist activity at MC5R and the lowest level of full agonist activity at MC2R, which distinguished it from synthetic MCR agonists. The minimal activity of Acthar Gel at MC2R corresponded to lower endogenous corticosteroid production.

A novel small-molecule enantiomeric analogue of traditional (-)-morphinans has specific TLR9 antagonist properties and reduces sterile inflammation-induced organ damage.

TLR9 is a key determinant of the innate immune responses in both infectious and sterile injury. Specific antagonism of TLR9 is of great clinical interest to reduce tissue damage in a wide range of pathologies, and has been approached by modification of nucleic acids, the recognized ligand for TLR9. Such oligonucleotide-derived pharmacotherapeutics have limitations in specificity for nucleic acid receptors, significant potential for immunologic recognition with generation of innate and adaptive immune responses, and limited bioavailability. We have identified enantiomeric analogues of traditional (-)-morphinans as having TLR9 antagonist properties on reporter cell lines. One of these analogues (COV08-0064) is demonstrated to be a novel small-molecule antagonist of TLR9 with greater specificity for TLR9 than oligo-based antagonists. COV08-0064 has wide bioavailability, including the s.c. and oral routes. It specifically inhibits the action of TLR9 antagonists on reporter cells lines and the production of cytokines by TLR9 agonists from primary cells. It also has efficacy in limiting TLR9-mediated sterile inflammation in in vivo models of acute liver injury and acute pancreatitis. The identification of a morphinan-based novel small-molecule structure with TLR9 antagonism is a significant step in expanding therapeutic strategies in the field of sterile inflammatory injury.

Exogenous fluorescent tracer agents based on pegylated pyrazine dyes for real-time point-of-care measurement of glomerular filtration rate.

Novel pyrazine carboxamides bearing hydrophilic poly(ethylene glycol) (PEG) moieties were designed, synthesized, and evaluated for use as fluorescent glomerular filtration rate (GFR) tracer agents. Among these, compounds 4d and 5c that contain about 48 ethylene oxide units in the PEG chain exhibited the most favorable physicochemical and renal clearance properties. In vitro studies show that these two compounds have low plasma protein binding, a necessary condition for renal excretion. In vivo animal model results show that 4d and 5c have a higher urine recovery of the injected dose than iothalamate (a commonly considered gold standard GFR agent). Pharmacokinetic studies show that these two compounds exhibit a plasma clearance equivalent to iothalamate, but with a faster (i.e. lower) terminal half-life than iothalamate (possibly from restricted distribution into the extracellular space due to large molecular size and hydrodynamic volume). Furthermore, the plasma clearance of 4d and 5c remained unchanged upon blockage of the tubular secretion pathway with probenecid, a necessary condition for establishment of clearance via glomerular filtration exclusively. Finally, noninvasive real-time monitoring of this class of compounds was demonstrated by pharmacokinetic clearance of 5c by optical measurements in rat model, which correlates strongly with plasma concentration of the tracer. Hence, 4d and 5c are promising candidates for translation to the clinic as exogenous fluorescent tracer agents in real-time point-of-care monitoring of GFR.

Hydrophilic pyrazine dyes as exogenous fluorescent tracer agents for real-time point-of-care measurement of glomerular filtration rate.

Various hydrophilic pyrazine-bis(carboxamides) derived from 3,5-diamino-pyrazine-2,5-dicarboxylic acid bearing neutral and anionic groups were prepared and evaluated for use as fluorescent glomerular filtration rate (GFR) tracer agents. Among these, the dianionic d-serine pyrazine derivatives 2d and 2j, and the neutral dihydroxypropyl 2h, exhibited favorable physicochemical and clearance properties. In vitro studies show that 2d, 2h, and 2j have low plasma protein binding, a necessary condition for renal excretion. In vivo animal model results show that these three compounds exhibit a plasma clearance equivalent to iothalamate (a commonly considered gold standard GFR agent). In addition, these compounds have a higher urine recovery compared to iothalamate. Finally, the plasma clearance of 2d, 2h, and 2j remained unchanged upon blockage of the tubular secretion pathway with probenecid, a necessary condition for establishment of clearance via glomerular filtration only. Hence, 2d, 2h, and 2j are promising candidates for translation to the clinic as exogenous fluorescent tracer agents in real-time point-of-care monitoring of GFR.

Fluorescence-enhanced europium-diethylenetriaminepentaacetic (DTPA)-monoamide complexes for the assessment of renal function.

Real-time, noninvasive assessment of glomerular filtration rate (GFR) is essential not only for monitoring critically ill patients at the bedside, but also for staging and monitoring patients with chronic kidney disease. In our pursuit to develop exogenous luminescent probes for dynamic optical monitoring of GFR, we have prepared and evaluated Eu(3+) complexes of several diethylenetriamine pentaacetate (DTPA)-monoamide ligands bearing molecular "antennae" to enhance metal fluorescence via intramolecular ligand-metal fluorescence resonance energy transfer process. The results show that Eu-DTPA-monoamide complex 18b, which contains a quinoxanlinyl antenna, exhibits large (ca. 2700-fold) Eu(3+) fluorescence enhancement. Indeed, complex 18b exhibits the highest fluorescent enhancement observed thus far in the DTPA-type metal complexes. The renal clearance property was assessed using the corresponding radioactive (111)In complex 18a, and the data suggest that this complex clears via a complex mechanism that includes glomerular filtration.

Detection of individual microbubbles of ultrasound contrast agents: imaging of free-floating and targeted bubbles.

RATIONALE AND OBJECTIVES: During echo examinations with microbubble contrast, individual "dots" of ultrasound reflection can be visualized. To address the question whether these signals represent individual microbubbles, very dilute suspensions of ultrasound contrast agents or individual microbubbles attached to Petri dishes were prepared and studied by ultrasound imaging. METHODS: Microbubble suspensions were diluted in saline and evaluated by a clinical ultrasound imaging system. Microbubble concentration was verified by Coulter counter. Single microbubble preparation on a Petri dish was established by streptavidin-biotin interaction under microscopy control and subjected to ultrasound imaging. RESULTS: Ultrasound of dilute microbubble dispersions demonstrated distinct white foci; concentration of these sites was consistent with signals from individual microbubbles as determined by Coulter. Individual microbubbles immobilized on polystyrene were also visualized by ultrasound. CONCLUSION: Ultrasound medical systems can resolve backscatter signals from individual microbubbles of ultrasound contrast, both in solution and in the targeted immobilized state, implying picogram sensitivity.

A newly identified role for superoxide in inflammatory pain.

Novel classes of pain-relieving molecules are needed to fill the void between nonsteroidal anti-inflammatory agents and narcotics. Our studies have identified superoxide as a novel mediator of hyperalgesia (clinically defined as an augmented sensitivity to painful stimuli) and have exposed potential pathways through which this radical modulates the hyperalgesic response. The role of superoxide in pain was elucidated using a superoxide dismutase mimetic, M40403 [a manganese(II) complex with a bis(cyclo-hexylpyridine-substituted) macrocyclic ligand]. Intraplantar injection of carrageenan in rats led to time-dependent development of peripheral inflammation [measured parameters of inflammation included paw edema, cytokine release in the paw exudates, nitrotyrosine formation (a marker of peroxynitrite formation and oxidative stress), and poly-ADP-ribose-polymerase activation (the nuclear enzyme activated by superoxide/peroxynitrite)] and hyperalgesia. M40403 blocked all measured parameters of inflammation and hyperalgesia. Furthermore, when given therapeutically (2 h after the induction of hyperalgesia) either by intravenous or intrathecal administration, M40403 but not its inactive congener M40404 inhibited hyperalgesia with a rapid onset of action. Our results also show that, at the level of the spinal cord and time of peak hyperalgesia, endogenous manganese superoxide dismutase was nitrated and subsequently deactivated, losing its capacity to remove superoxide. The antihyperalgesic effects of M40403 were not reversed by naloxone excluding the potential involvement of an opiate pathway. Collectively, these studies have unraveled a critical role for superoxide in the nociceptive signaling cascade both peripherally and centrally. The discovery of this pathway opens a new therapeutic strategy for the development of novel nonnarcotic antihyperalgesic agents.

Albumin-binding MR blood pool agents as MRI contrast agents in an intracranial mouse glioma model.

Intravenous MRI contrast agents are commonly used to improve the detection of intracranial tumors and other central nervous system (CNS) lesions for diagnosis and treatment planning. Two small-molecule, albumin-binding blood pool contrast agents (MP-2269 and MS-325) of potential clinical significance were evaluated at 1.5 Tesla in a mouse glioma model and compared with an extracellular contrast agent (OptiMARK). Tumor image contrast was significantly enhanced and long-lived following administration of 30 micromole/kg of the blood pool agents: specifically, contrast enhancement peaked slowly at 25-30 min following administration, remained constant for >3 hr, and returned to baseline within 20 hr. Comparable but "transient" enhancement was achieved using 100 micromole/kg OptiMARK: specifically, contrast enhancement peaked rapidly at 2-5 min following administration and then declined over 40 min. The blood pool contrast agents demonstrated an approximately threefold increased dose-effectiveness and a lengthened window of tumor contrast enhancement in comparison to commonly available extracellular contrast agents. This demonstrates the potential of alternative contrast-enhanced (CE) MRI examination protocols for tumor detection.

Microbubbles induce renal hemorrhage when exposed to diagnostic ultrasound in anesthetized rats.

The generation of ultrasound (US) bioeffects using a clinical imaging system is controversial. We tested the hypothesis that the presence of microbubbles in the US field of a medical imager induces biologic effects. Both kidneys of anesthetized rats were insonified for 5 min using a medical imaging system after the administration of microbubbles. One kidney was insonified using a continuous mode (30 Hz) and the opposite kidney was insonified using an intermittent (1 Hz) technique. The microbubbles were exposed to three different transducer frequencies and four transducer output powers. After insonification, the animals were euthanized, the kidneys were removed and their gross appearance scored under "blinded" conditions using a defined scale. After the administration of microbubbles, US imaging of the kidney caused hemorrhage in the renal tissue. The severity and area of hemorrhage increased with an increase in the transducer power and a decrease in the transducer frequency. Intermittent insonification in the presence of microbubbles produced a greater degree of renal hemorrhage than continuous imaging techniques.