In Vitro and In Vivo Antiviral Activity of Nylidrin by Targeting the Hemagglutinin 2-Mediated Membrane Fusion of Influenza A Virus.

Influenza A virus, one of the major human respiratory pathogens, is responsible for annual seasonal endemics and unpredictable periodic pandemics. Despite the clinical availability of vaccines and antivirals, the antigenic diversity and drug resistance of this virus makes it a persistent threat to public health, underlying the need for the development of novel antivirals. In a cell culture-based high-throughput screen, a ß2-adrenergic receptor agonist, nylidrin, was identified as an antiviral compound against influenza A virus. The molecule was effective against multiple isolates of subtype H1N1, but had limited activity against subtype H3N2, depending on the strain. By examining the antiviral activity of its chemical analogues, we found that ifenprodil and clenbuterol also had reliable inhibitory effects against A/H1N1 strains. Field-based pharmacophore modeling with comparisons of active and inactive compounds revealed the importance of positive and negative electrostatic patterns of phenyl aminoethanol derivatives. Time-of-addition experiments and visualization of the intracellular localization of nucleoprotein NP demonstrated that an early step of the virus life cycle was suppressed by nylidrin. Ultimately, we discovered that nylidrin targets hemagglutinin 2 (HA2)-mediated membrane fusion by blocking conformational change of HA at acidic pH. In a mouse model, preincubation of a mouse-adapted influenza A virus (H1N1) with nylidrin completely blocked intranasal viral infection. The present study suggests that nylidrin could provide a core chemical skeleton for the development of a direct-acting inhibitor of influenza A virus entry.

Structure-activity relationships for a series of bis(phenylalkyl)amines: potent subtype-selective inhibitors of N-methyl-D-aspartate receptors.

A series of bis(phenylalkyl)amines, structural analogues of ifenprodil and nylidrin, were synthesized and tested for antagonism of N-methyl-D-aspartate (NMDA) receptors. Potency and subunit selectivity were assayed by electrical recordings in Xenopus oocytes expressing three binary combinations of cloned rat NMDA receptor subunits: NR1A expressed in combination with either NR2A, NR2B, or NR2C. The bis(phenylalkyl)amines were selective antagonists of NR1A/2B receptors. Assayed under steady-state conditions, the most potent of these, N-[2-(4-hydroxyphenyl)ethyl]-5-phenylpentylamine hydrochloride (20), has an IC50 value of 8 nM and >1000-fold selectivity with respect to NR1A/2A and NR1A/2C receptors. The structure-activity relationship of the bis(phenylalkyl)amine series indicates that the piperidine ring and alkyl chain substitutions common to NR2B-selective antagonists such as ifenprodil, CP 101,606, and Ro 25-6981 are not necessary to generate potent and selective ligands. The primary determinants of potency are the phenolic OH group, acting as a hydrogen bond donor, the distance between the two rings, and an electrostatic interaction between the receptor and the basic nitrogen atom. This study provides a framework for designing structurally novel NR2B-selective antagonists which may be useful for treatment of a variety of neurological disorders.

Influence of extracellular pH on inhibition by ifenprodil at N-methyl-D-aspartate receptors in Xenopus oocytes.

Ifenprodil is an atypical N-methyl-D-aspartate (NMDA) receptor antagonist that selectively blocks receptors containing the NR2B subunit. It has been proposed that ifenprodil may act at a stimulatory polyamine site on NMDA receptors, although interactions between ifenprodil and polyamines are non-competitive. NMDA receptors are also inhibited by extracellular protons, and an interaction between protons and polyamine stimulation has been described. Using voltage-clamp recording of recombinant NR1/NR2B receptors expressed in oocytes, ifenprodil inhibition was found to be pH sensitive with a smaller inhibition at alkaline pH. Similar effects of pH were seen on inhibition by nylidrin, eliprodil, and haloperidol, which are thought to act at the ifenprodil binding site. The pH sensitivity of ifenprodil block occurs at NR1B/NR2B as well as NR1A/NR2B receptors, suggesting that it is not influenced by the exon-5 insert that is present in NR1B but absent in NR1A. Protons may directly affect the ifenprodil binding site or may alter the coupling of ifenprodil binding to inhibition of channel gating.

Subtype-selective antagonism of NMDA receptors by nylidrin.

The 1,4-di-substituted piperidines ifenprodil, eliprodil, CP 101,606 ((1S,2S)-1-(4-hydroxyphenyl)-2-(4-hydroxy-4-phenylpiperidino)-1-propanol ) and Ro 25-6981 ((R-(R*,S*))-alpha-(4-hydroxyphenyl)-beta-methyl-4-(phenyl-methyl)-1- piperidinepropanol) are allosteric antagonists of NMDA receptors. Inhibition of diheteromeric NMDA receptors by this class of antagonist is characterized by pronounced selectivity for NR1/2B subunit combinations. In the current study, we assayed effects of nylidrin, a structurally-related non-piperidine, on recombinant and neuronal NMDA receptors. Nylidrin was a potent (IC50 = 0.18 microM) antagonist of NR1A/2B receptors expressed in Xenopus oocytes and was at least 150-fold weaker against NR1A/2A and NR1A/2C receptors. The blockade of NR1A/2B responses by nylidrin was not surmounted by increasing the concentrations of glutamate or glycine and was not voltage-dependent. Potency of inhibition increased approximately 3-fold upon lowering extracellular pH from 8 to 6.8. Nylidrin inhibited NMDA responses in cultured rat cortical neurons with similar potency and apparent mechanism of action as the NR1A/2B receptors. Our results suggest that nylidrin interacts with the same allosteric inhibitory site previously described for the related piperidine antagonists, and should serve as a structural lead for designing novel subtype-selective inhibitors of NMDA receptors.

The hemodynamic effects of antenatal indomethacin and a beta-sympathomimetic agent on the fetus and the newborn: a randomized study.

To study the effect of antenatal indomethacin or nylidrin hydrochloride treatment on the fetal and neonatal ductus arteriosus and tricuspid valve function, 84 pregnant women with threatened premature birth between 22.9 and 34.0 wk gestation and 94 of their offspring born at 24.7 to 41.6 wk of gestation were studied by Doppler echocardiography. Forty-six women were treated with indomethacin and 38 with nylidrin. Both peak systolic and peak diastolic velocities in the ductus increased after administration of indomethacin and exceeded the corresponding velocities in the fetuses of the nylidrin group (p = 0.0001). Ductal constriction occurred in 42 of 49 fetuses treated with indomethacin (86%). Tricuspid valve regurgitation (TR) was evident in 11 of 49 fetuses treated with indomethacin (22%). The mean gestational age of the fetuses with TR (30.0 wk) tended to be higher than those without TR (28.3 wk, p = 0.056). In the nylidrin group, no fetus had ductal constriction or TR. A significant increase in peak systolic velocity (r = 0.54, p = 0.0001) and in peak diastolic velocity (r = 0.46, p = 0.0001) in the ductus with advancing gestational age was demonstrated in the indomethacin group; however, in the nylidrin group, there was a less remarkable increase in peak systolic velocity (r = 0.35, p = 0.04) and no increase in peak diastolic velocity (r = 0.02, p = 0.93). In infants born at or before 35 wk gestation, incidences of both spontaneous closure and indomethacin-induced closure of ductus were similar in both study groups (p > 0.1).(ABSTRACT TRUNCATED AT 250 WORDS)

Decreased numbers of lymphocyte beta 2-adrenoceptors in pregnant women receiving beta 2-adrenergic agonist therapy.

Intravenous infusion of buphenine hydrochloride was administered on 4 successive days to 8 pregnant women with imminent preterm labor. Serial blood samples taken before and throughout the study were assayed for lymphocyte beta 2-adrenoceptor density and cyclic adenosine-3',5'-monophosphate (cAMP). The lymphocyte beta 2-adrenoceptor density declined significantly (p less than 0.01) during the treatment. The plasma cAMP concentration was highest 4 h after commencement of infusion and decreased thereafter. Despite the decrease in lymphocyte beta 2-adrenoceptor density, the clinical response remained good and the parturients did not go into labor until several days after infusion was started.

[The effect of partusisten, dilatol, papaverine and magnesium sulfate on uterine smooth muscle in vitro]. / Die Wirkungen von Partusisten, Dilatol, Papaverin und Magnesiumsulfat auf die glatte Uterusmuskulatur in vitro.

Inhibitory effects of Partusisten, Dilatol, Papaverin and Magnesium-sulfat on the activity of smooth muscle were tested in vitro on 18 isolated stripes of rats uteri, exhibiting both a high sensitivity against oxytocin and a distinct spontaneous activity. Minimal inhibitory concentrations of all drugs tested were determined, and their influence on the frequency and amplitude of contraction as well as on lag phase between inhibition and onset of spontaneous activity were registered. Basing on these experimental data conclusions were drawn concerning their clinical relevance.

The effects of beta-adrenergic agonists on cone systems in the cat eye.

The effects of the beta-adrenergic agonist nylidrin and the beta 2-adrenergic agonist clenbuterol on electroretinogram and optic nerve response were studied in the isolated and arterially perfused, light-adapted cat eye. Two cone mechanisms, short wavelength-sensitive and long wavelength-sensitive, were functionally separated by means of intense yellow adaptation. A reversible increase in b-wave amplitude in response to nylidrin or clenbuterol was observed for the cone systems. Both drugs also caused a reversible alteration in configuration of the optic nerve response, mainly a depression of the late components related in time to the changes in the electroretinogram. These observations suggest that beta-adrenergic mechanisms are involved in cone systems. The greater increase in b-wave amplitude on 558-nm stimulation and preliminary evidence for greater increase in sensitivity observed in the V-log I function compared with 439 nm further suggest that the short and long wavelength cone systems are affected differently by beta-adrenergic agonists.

Effects of beta-agonists on b- and c-waves implicit for adrenergic mechanisms in cat retina.

Nylidrin (buphenine) is a beta-adrenergic agonist known to dilate peripheral vessels and used therapeutically in retinal degeneration and glaucoma. We studied retinal function under beta-agonists in arterially perfused cat eyes and observed a dose-dependent, reversible increase in b-wave amplitude and a decrease in c-wave amplitude in concentrations from 4.5 to 120 microM. A half maximal response was obtained at 40 to 50 microM. The optic nerve response to light showed dose-dependent reversible changes under nylidrin. Standing potential, light peak, intraocular pressure, vascular resistance, and diameter of or retinal vessels showed no consistent changes under nylidrin. The effect were inhibited by each of the beta-blocking agents propranolol, ICI 118, and oxprenolol (in sequence of decreasing potency). Another potent beta 2-agonist, clenbuterol, was used to determine the extent to which the responses to nylidrin were due to beta-receptor-mediated action. Clenbuterol had similar effects on the b-wave and optic nerve response at slightly higher concentrations (30 200 microM) but more variable effects on the c-wave. The data are interpreted as functional evidence that beta-adrenergic mechanisms are involved in retinal signal processing. This concept is corroborated by identification of beta-adrenergic binding sites in cat retina (Bruinink et al., 1986).

Effects of buphenine (nylidrin) on the perfused mammalian eye.

Buphenine (nylidrin), a beta-adrenergic agonist, is used therapeutically for its vasodilating effect on the peripheral circulation and possibly on the cerebral circulation as well. In spite of its ophthalmic usage in degenerative retinal disease and glaucoma, buphenine's ocular effects and their mechanisms are not sufficiently established. Consequently, we studied the action of 4.5-120 microM buphenine in isolated, arterially perfused cat eyes, and then used light-evoked electrical signals, perfusion flow rates and the diameters of the retinal vessels as parameters for the drug's effect. Our findings showed that buphenine induced a marked, dose-related, reversible increase in the amplitude of the electroretinogram (ERG) b-wave, a decrease in the c-wave, but no significant changes in the standing potential and light peak of the DC-ERG. The compound action potential of the optic nerve revealed dose-dependent and reversible changes in configuration. However, the flow of perfusate was not affected by the drug, and the diameter of retinal vessels did not change significantly. Our studies suggest that the interaction of buphenine with retinal adrenergic receptors is not related to the vasculature present but to elements involved in information processing. It is likely that these receptors are linked to neurons, since the beta-agonist affected the ERG b-waves as well as the compound action potential of the optic nerve.

Effects of isoxuprine and nylidrin on adrenoreceptors in rat vas deferens.

The interaction of isoxuprine and nylidrin with alpha 1- and beta 2-adrenoreceptors in rat vas deferens was examined using radioligand binding assays and physiological studies in vitro. Isoxuprine and nylidrin have a greater affinity for binding to alpha 1 (isoxuprine KD = 59 +/- 15 nM; nylidrin KD = 41 +/- 3 nM) than beta 2-(isoxuprine KD = 3,900 +/- 500 nM; nylidrin KD = 900 +/- 50 nM) adrenoreceptors in rat vas deferens. Vas deferens from rats pretreated for 16-24 h with reserpine (3 mg/kg i.p.) were exposed to 10 microM phenoxybenzamine for 15 min to inactivate alpha-adrenoreceptors. Under these conditions high concentrations of both isoxuprine and nylidrin relaxed vas deferens contracted with 55 mM K+, however the relaxation was not blocked by the beta-adrenoreceptor antagonist propranolol (10 microM). Both isoxuprine and nylidrin were potent competitive antagonists of alpha 1-adrenoreceptor mediated contraction of vas deferens. pA2 values for isoxuprine (6.9 +/- .05) and nylidrin (7.1 +/- .08) agreed well with KD values for binding to alpha 1-adrenoreceptors in vas deferens. The greater potency of isoxuprine and nylidrin in inhibiting alpha 1-adrenoreceptors than binding to beta 2-adrenoreceptors or causing nonspecific relaxation suggest that alpha-adrenoreceptor antagonist actions of these drugs may be important in their ability to inhibit smooth muscle tone.

Ocular hypotension in the rabbit. Receptor mechanisms of pirbuterol and nylidrin.

Pirbuterol and nylidrin, both purported sympathomimetic amines, reduced intraocular pressure (IOP) when given topically (50 microliter, 0.1%) to albino rabbits. Pirbuterol increased the cyclic-AMP concentration in aqueous humor by a factor of 3.25, while nylidrin had no effect on aqueous cyclic-AMP nor on adenylate cyclase activity of iris-ciliary body membranes assayed in vitro. Studies of the receptor affinity of pirbuterol, timolol and nylidrin were carried out on iris-ciliary body membranes by competition binding with radioactive ligands. Four ligands were used that appear to label separate subpopulations of adrenergic receptors; dihydroalprenolol (beta-receptors), WB-4101 (alpha 1-receptors) prazosin (alpha 1-receptor subpopulation) and yohimbine (alpha 2-receptors). Pirbuterol and timolol showed exclusive selectivity for beta-receptors with high affinities (Kd 12.6 and 0.48 nM, respectively) compared with other adrenergic receptor populations in iris-ciliary body. Nylidrin had high affinities for beta-receptors (Kd 22 nM) and for the subpopulation of alpha 1-receptors labelled by prazosin (Kd 6.5 nM), but showed 100-fold lower affinity and complex binding characteristics to the two other classes of alpha-adrenergic receptor sites labelled by WB-4101 and yohimbine, respectively. The results show that pirbuterol and timolol are highly beta-receptor selective and that hypotensive responses to these drugs are not mediated by the other classes of alpha-adrenergic receptor determined in this study. However, the hypotensive response to nylidrin may be related to its prazosin-like (alpha 1-receptor) antagonist properties with additional activity at beta-receptors.

Antiallergic activity of nylidrin hydrochloride (RHC 3432-A). I. Effect on release of histamine in vitro from rat peritoneal mast cells, guinea pig lung slices and human basophils.

Nylidrin (RHC 3432-A) has been investigated for its antiallergic activity in three in vitro models. Nylidrin was an effective inhibitor of IgE-mediated release of histamine from passively sensitized rat peritoneal mast cells and human basophils, and of IgG1-mediated release of histamine from passively sensitized guinea pig lung slices. The inhibition of the release of histamine by nylidrin in all three models was not antagonized by propranolol, indicating that nylidrin does not inhibit histamine release via stimulation of beta-adrenergic receptors. Isoproterenol and epinephrine were effective as inhibitors of the release of histamine only from guinea pig lung while salbutamol and terbutaline had no effect on immunologic release of histamine in all three models. Detailed comparative studies with disodium cromoglycate (DSCG) indicated that the mechanism of action of nylidrin in the rat mast cell model is different from that of DSCG.

Antiallergic activity of nylidrin hydrochloride (RHC 3432-A). II. A lack of correlation between inhibition of mediator release and levels of cyclic AMP.

Nylidrin hydrochloride has weak beta-adrenergic agonist properties with rat mast cells, and shows synergy with 3-isobutyl-1-methylxanthine (IBMX) in raising intracellular cAMP in purified mast cells; both of these properties can be blocked by dl-propranolol. However, the action of nylidrin hydrochloride as an inhibitor of histamine secretion from purified rat mast cells is not antagonized by dl-propranolol, nor is it potentiated by IBMX. Nylidrin-induced elevation of cellular cAMP in purified rat mast cells shows no correlation with nylidrin-induced inhibition of histamine release. Thus, nylidrin hydrochloride inhibits release of histamine from mast cells by a novel, non-adrenergic mechanism, which is not dependent on increased levels of cAMP.

Central cardiovascular effects of nylidrin (buphenine).

The injection of small doses of nylidrin into the vertebral artery lowered blood pressure in chloralose-anesthetized cats. Cerebroventricular perfusion of nylidrin also decreased blood pressure and inhibited or modified cardiovascular reflexes. There was inhibition of the reflex bradycardia evoked by i.v. noradrenaline or angiotensin. Likewise the pressure increase due to bilateral carotid occlusion was diminished. The blood pressure decrease elicited by stimulation of the central stump of a cut vagus nerve was inhibited or reversed. The pattern of central cardiovascular effects of nylidrin seemed not compatible with its well known beta-sympathomimetic properties. Indirect evidence points to an interasction with central alpha-receptors.

[Paradoxical effect of buphenine on intraocular pressure in aphakic eyes (author's transl)]. / Die paradoxe Wirkung von Buphenin auf den intraokularen Druck in aphaken Augen.

The effect of topical administration of Buphenine was studied in a group of aphakic patients (53 eyes). The action of the drug was paradoxical: it induced an increase, often remarkable, in intraocular pressure in more than 70% of the eyes thus treated. The cause of the elevated IOP is discussed.

Effects of buphenin on the rat portal vein.

The vasodilator and tocolytic substance buphenin (10 mumol/l) stimulated the spontaneous phasic activity of some (8 out of 18) isolated rat portal vein preparations; 0,1-1 mmol/l buphenin diminished or abolished the activity in all preparations. The isotonic and isometric tonic contractions of portal vein in response to adrenaline, noradrenaline and phenylephrine (0,1-1 mumol/l) disappeared almost completely after addition of buphenin in equimolar concentrations, whereas acetylcholine contractions persisted. The beta-adrenergic blocking agents propranolol and dichloisoprenaline (10 mumol/l) only slightly antagonized the inhibitory effect of buphenin on the contractile responses to catecholamines. It is concluded that buphenin exerts dual action upon rat portal vein: the drug partially stimulates the beta-receptors and partially blocks the alpha-adrenergic receptors.

Nylidrin: a potent anti-hypertensive agent in hypertensive rats.

Nylidrin HCl lowered the blood pressure and increased the heart rate of conscious, spontaneously hypertensive rats (SHR), Goldblatt hypertensive rats, and DOCA hypertensive rates. In SHR, the minimum effective dose was 0.5 mg/kg, s.c. At that dose, the anti-hypertensive activity of nylidrin lasted more than 3 hours. Propranolol reversed both effects on blood pressure and heart rate by nylidrin, whereas atenolol, a specific cardiac beta 1 blocker, only blocked or reversed the tachycardia caused by nylidrin but did not block the anti-hypertensive effect of the compound. In normotensive rats of Wistar/Kyoto strain (WKY) and Holtzman strain, nylidrin at 5 or 10 mg/kg, s.c., produced a transient hypotensive effect which lasted less than an hour and tachycardia that persisted for several hours. In WKY, atenolol prolonged the hypotensive activity of the compound by partially reversing the tachycardia. These observations indicate that SHR is more sensitive than WKY to the anti-hypertensive activity of nylidrin, which is probably caused by vasodilatation mediated by beta 2 receptors.