Kevin B. Johnson

Pharmacological Properties of N-(3,5-Diamino-6-chloropyrazine-2-carbonyl)-N′-4-[4-(2,3-dihydroxypropoxy)phenyl]butyl-guanidine Methanesulfonate (552-02), a Novel Epithelial Sodium Channel Blocker with Potential Clinical Efficacy for Cystic Fibrosis Lung Disease

Amiloride improves mucociliary clearance (MC) by blocking airway epithelial sodium channels (ENaC) and expanding airway surface liquid (ASL). However, the low potency and rapid absorption of amiloride by airway epithelia translated into a short duration of efficacy as an aerosolized therapy for cystic fibrosis (CF) patients. To improve ENaC blocker CF pharmacotherapy, a more potent and durable ENaC blocker tailored for aerosol delivery was synthesized. Parion compound N-(3,5-diamino-6-chloropyrazine-2-carbonyl)-N′-4-[4-(2,3-dihydroxypropoxy)phenyl]butyl-guanidine methanesulfonate (552-02) was tested for potency and reversibility of ENaC block, epithelial absorption and biotransformation, selectivity, durability of ASL expansion under isotonic and hypertonic conditions in canine and human CF bronchial epithelial cells, and drug dissociation on ENaC in Xenopus oocytes. Short-circuit current assessed compound potency and reversibility, patch-clamp recordings of ENaC current assessed drug off-rate (koff), a gravimetric method and confocal microscopy measured mucosal water retention and ASL height, and drug absorption and biotransformation were assessed using liquid chromatography-mass spectrometry. Amiloride and 552-02 were tested in vivo for MC activity in sheep immediately and 4 to 6 h after aerosol dosing. Compared with amiloride, compound 552-02 was 60 to 100-fold more potent, it was 2 to 5-fold less reversible, it was slower at crossing the epithelium, and it exhibited a 170-fold slower koff value. 552-02 exhibited greater ASL expansion over 8 h in vitro, and it was more effective than amiloride at increasing MC immediately and 4 to 6 h after dosing. When combining hypertonic saline and 552-02, a synergistic effect on ASL expansion was measured in canine or CF bronchial epithelia. In summary, the preclinical data support the clinical use of 552-02 +/– hypertonic saline for CF lung disease.

Metabolic and functional mapping of the neural network subserving inferior collicular seizure generalization

The sensory-motor portion of the inferior collicular cortex is capable of seizure genesis that is characterized initially by coincident wild running behaviors and localized electrographic afterdischarge. With repeated stimulations, this seizure activity spreads into the forebrain, producing generalized tonic-clonic or myoclonic seizure activity. In order to characterize the neural network subserving this caudal-rostral seizure generalization, three mapping techniques were used: 2-deoxyglucose (2-DG) utilization, c-fos expression and local anesthetic microinjection. Kindled seizure generalization from the inferior collicular cortex produced a global increase in 2-DG accumulation, while relative 2-DG increases were found in the inferior collicular cortex, dorsal lateral lemniscus, dorsal central gray, peripeduncular nucleus, medial geniculate nucleus, substantia nigra, entopeduncular nucleus, ventroposterior and centromedian thalamus and tenia tectum, as well as the perirhinal, somatosensory and frontal cortices. Kindled seizure generalization also increased c-fos-like immunoreactivity (FLI) in the inferior collicular cortex, cuneiform nucleus, dorsal lateral nucleus of the lateral lemniscus, peripeduncular nucleus, caudal central gray, dentate gyrus of the hippocampus, rhinal fissure area of the perirhinal cortex and the frontal cortex. Microinjections of procaine into the amygdala, perirhinal cortex, entopeduncular nucleus, substantia nigra, peripeduncular nucleus, dorsal central gray, and pontine reticular nucleus all prevented generalized seizure behaviors, but had no effect on the wild running seizures. Conversely, procaine microinjection into the area of the cuneiform nucleus/pedunculopontine tegmental nucleus prevented the wild running seizure but did not block the generalized seizure activity. Neither wild running, nor generalized seizures were altered following procaine microinjections into the anterior thalamus, sub-thalamus, lateral hypothalamus, hippocampus or deep superior colliculus. Thus, specific forebrain sites form a widespread neural network that mediates the generalization of seizure activity from the inferior collicular cortex into the forebrain.

D1 Dopamine Receptor Binding and mRNA Levels Are Not Altered After Neonatal 6‐Hydroxydopamine Treatment: Evidence Against Dopamine‐Mediated Induction of D1 Dopamine Receptors During Postnatal Development

Abstract: The role of dopaminergic innervation on the postnatal developmental expression of D1 dopamine receptors was investigated. Bilateral destruction of dopa‐mine‐containing neurons was achieved by treating rats intracisternally with 6‐hydroxydopamine (6‐OHDA) on postnatal day 3, and rats were killed on day 21. To ensure effective reduction of D1 receptor activation by residual dopamine, a group of 6‐OHDA‐lesioned rats was given twice daily injections of the D1 receptor antagonist SCH‐23390, from day 4 to 20. D1 dopamine receptor binding was assessed in the caudate—putamen, nucleus accumbens, and olfactory tubercle by quantitative autoradiographic analysis of [3H]SCH‐23390 binding. In addition, the relative amount of D1A receptor mRNA was assessed by in situ hybridization of a 35S‐labeled riboprobe. In the developing rats, neither the amount of [3H]SCH‐23390 binding nor the amount of D1A receptor mRNA was altered by 6‐OHDA lesioning followed by chronic treatment with SCH‐23390. Thus, bilateral destruction of dopamine‐containing neurons and treatment with SCH‐23390 in neonatal rats did not interfere with the developmental expression of D1 receptors or alter the levels of mRNA that code for this receptor protein. Treatment of intact rats with SCH‐23390 from postnatal day 4 to 20 also did not alter [3H]SCH‐23390 binding or levels of D1 receptor mRNA. However, adult rats treated chronically with SCH‐23390 exhibited increased [3H]SCH‐23390 binding but did not show a significant change in D1 receptor mRNA levels.