Antitussive drugs--past, present, and future.

Cough remains a serious unmet clinical problem, both as a symptom of a range of other conditions such as asthma, chronic obstructive pulmonary disease, gastroesophageal reflux, and as a problem in its own right in patients with chronic cough of unknown origin. This article reviews our current understanding of the pathogenesis of cough and the hypertussive state characterizing a number of diseases as well as reviewing the evidence for the different classes of antitussive drug currently in clinical use. For completeness, the review also discusses a number of major drug classes often clinically used to treat cough but that are not generally classified as antitussive drugs. We also reviewed a number of drug classes in various stages of development as antitussive drugs. Perhaps surprising for drugs used to treat such a common symptom, there is a paucity of well-controlled clinical studies documenting evidence for the use of many of the drug classes in use today, particularly those available over the counter. Nonetheless, there has been a considerable increase in our understanding of the cough reflex over the last decade that has led to a number of promising new targets for antitussive drugs being identified and thus giving some hope of new drugs being available in the not too distant future for the treatment of this often debilitating symptom.

The role of trigeminal nasal TRPM8-expressing afferent neurons in the antitussive effects of menthol.

The cold-sensitive cation channel TRPM8 is a target for menthol, which is used routinely as a cough suppressant and as an additive to tobacco and food products. Given that cold temperatures and menthol activate neurons through gating of TRPM8, it is unclear how menthol actively suppresses cough. In this study we describe the antitussive effects of (-)-menthol in conscious and anesthetized guinea pigs. In anesthetized guinea pigs, cough evoked by citric acid applied topically to the tracheal mucosa was suppressed by menthol only when it was selectively administered as vapors to the upper airways. Menthol applied topically to the tracheal mucosa prior to and during citric acid application or administered continuously as vapors or as an aerosol to the lower airways was without effect on cough. These actions of upper airway menthol treatment were mimicked by cold air delivered to the upper airways but not by (+)-menthol, the inactive isomer of menthol, or by the TRPM8/TRPA1 agonist icilin administered directly to the trachea. Subsequent molecular analyses confirmed the expression of TRPM8 in a subset of nasal trigeminal afferent neurons that do not coincidently express TRPA1 or TRPV1. We conclude that menthol suppresses cough evoked in the lower airways primarily through a reflex initiated from the nose.

Cholinergic brush cells in the trachea mediate respiratory responses to quorum sensing molecules.

AIM: The airway epithelial surface is constantly exposed to inhaled environmental factors and pathogens. Bitter "tasting" bacterial products such as quorum sensing molecules (QSM) can be detected by solitary chemosensory cells of the upper respiratory tract. Recently, we have shown that tracheal brush cells are cholinergic chemosensory cells affecting the respiration upon stimulation with bitter substances. Here, we explore the hypothesis that tracheal brush cells are capable of detection of bacterial products such as QSM resulting in changes in respiration and in induction of local effects, e.g. regulation of mucociliary clearance. MAIN METHODS: Functional analyses of respiration were performed in the trachea using a newly established model for investigation of respiration in spontaneously breathing anesthetized mice upon isolated tracheal stimulation. Influence of N-3-oxododecanoyl-homoserine lactone (3-OxoC(12)-HSL) on cilia-driven particle transport speed (PTS) in the airways was investigated in acutely excised and submerged mouse tracheae. KEY FINDINGS: 3-OxoC(12)-HSL, a Pseudomonas aeruginosa quorum sensing autoinducer, caused a drop in the respiratory rate 2 min after the application at the mucosal surface. The 3-OxoC(12)-HSL-induced effect on respiration was abolished by inhibition of nicotinic receptors with mecamylamine and by removal of the respiratory epithelium. At the same concentration, 3-OxoC(12)-HSL enhanced significantly PTS on the mucosal surface. SIGNIFICANCE: We conclude that cholinergic airway epithelial cells sense bacterial QSM in the airway lining fluid and communicate this to the CNS via ACh release and nicotinic stimulation of sensory neurons. In addition, QSM enhance PTS.

Sensory nerves and airway irritability.

The lung, like many other organs, is innervated by a variety of sensory nerves and by nerves of the parasympathetic and sympathetic nervous systems that regulate the function of cells within the respiratory tract. Activation of sensory nerves by both mechanical and chemical stimuli elicits a number of defensive reflexes, including cough, altered breathing pattern, and altered autonomic drive, which are important for normal lung homeostasis. However, diseases that afflict the lung are associated with altered reflexes, resulting in a variety of symptoms, including increased cough, dyspnea, airways obstruction, and bronchial hyperresponsiveness. This review summarizes the current knowledge concerning the physiological role of different sensory nerve subtypes that innervate the lung, the factors which lead to their activation, and pharmacological approaches that have been used to interrogate the function of these nerves. This information may potentially facilitate the identification of novel drug targets for the treatment of respiratory disorders such as cough, asthma, and chronic obstructive pulmonary disease.

Cough sensors. I. Physiological and pharmacological properties of the afferent nerves regulating cough.

The afferent nerves regulating cough have been reasonably well defined. The selective effects of general anesthesia on C-fiber-dependent cough and the opposing effects of C-fiber subtypes in cough have led to some uncertainty about their regulation of this defensive reflex. But a role for C-fibers in cough seems almost certain, given the unique pharmacological properties of these unmyelinated vagal afferent nerves and the ability of many C-fiber-selective stimulants to evoke cough. The role of myelinated laryngeal, tracheal, and bronchial afferent nerve subtypes that can be activated by punctate mechanical stimuli, inhaled particulates, accumulated secretions, and acid has also been demonstrated. These "cough receptors" are distinct from the slowly and rapidly adapting intrapulmonary stretch receptors responding to lung inflation. Indeed, intrapulmonary rapidly and slowly adapting receptors and pulmonary C-fibers may play no role or a nonessential role in cough, or might even actively inhibit cough upon activation. A critical review of the studies of the afferent nerve subtypes most often implicated in cough is provided.

Role of nerves in asthmatic inflammation and potential influence of gastroesophageal reflux disease.

Inflammation of the lower airways is a defining characteristic of asthma. Microaspiration of refluxate may initiate an inflammatory response in the airways of patients with gastroesophageal reflux disease (GERD), thereby precipitating asthma. Airway nerves are likely to play a role in the pathogenesis of asthma and could potentially mediate airway inflammation initiated by GERD through axonal reflexes. Alternatively, refluxate may initiate airway reflexes from the esophagus that are markedly exaggerated by inflammation-induced enhancement of airway neuronal excitability. The characteristic features of inflammation in asthma are defined, and the potential role of nerves in inflammation is discussed. Mechanisms by which GERD may initiate airway inflammation or act synergistically with airway inflammation to produce asthma also are discussed.

Multiple mechanisms of reflex bronchospasm in guinea pigs.

The mechanisms of histamine- and bradykinin-induced reflex bronchospasm were determined in anesthetized guinea pigs. With intravenous administration, both autacoids evoked dose-dependent increases in tracheal cholinergic tone. Vagotomy or atropine prevented these tracheal reflexes. When delivered as an aerosol, bradykinin readily increased tracheal cholinergic tone, whereas histamine aerosols were much less effective at inducing tracheal reflexes. Also, unlike histamine, bradykinin could evoke profound increases in cholinergic tone without directly or indirectly (e.g., prostanoid dependent) inducing measurable airway smooth muscle contraction resulting in bronchospasm. Neither autacoid required de novo synthesis of prostanoids or nitric oxide to induce reflex tracheal contractions. Combined cyclooxygenase inhibition and tachykinin-receptor antagonism did, however, abolish all effects of bradykinin in the airways, whereas responses to histamine were unaffected by these pretreatments. The data indicate that histamine and bradykinin initiate reflex bronchospasm by differential activation of vagal afferent nerve subtypes. We speculate that selective activation of either airway C fibers or airway rapid adapting receptors can initiate reflex bronchospasm.

Neural regulation of airway smooth muscle tone.

Airway smooth muscle is innervated by sympathetic and parasympathetic nerves. When activated, airway nerves can markedly constrict bronchi either in vivo or in vitro, or can completely dilate a precontracted airway. The nervous system therefore plays a primary role in regulating airway caliber and its dysfunction is likely to contribute to the pathogenesis of airways diseases. The predominant contractile innervation of airway smooth muscle is parasympathetic and cholinergic in nature, while the primary relaxant innervation of the airways is comprised of noncholinergic (nitric oxide synthase- and vasoactive intestinal peptide-containing) parasympathetic nerves. These parasympathetic nerves are anatomically and physiologically distinct from one another and differentially regulated by reflexes. Sympathetic-adrenergic nerves play little if any role in directly regulating smooth muscle tone in the human airways. Activation of airway afferent nerves (rapidly adapting receptors, C-fibers) can evoke increases in airway smooth muscle parasympathetic nerve activity, or decreases in parasympathetic nerve activity (through activation of slowly adapting receptors). Extrapulmonary afferents can also modulate nerve mediated regulation of airway smooth muscle tone. In guinea pigs and rats, peripheral activation of tachykinin-containing airway afferent nerves evokes bronchospasm via release of substance P and neurokinin A. This effect of airway afferent nerve activation appears to be unique to guinea pigs and rats. The actions and interactions between the components of airway innervation are discussed.

Nicotinic acetylcholine receptor assays.

Nicotinic acetylcholine receptors (nAChRs) in peripheral tissues are localized almost exclusively to autonomic nerves and the motor end plates of striated musculature. Pharmacologic analyses of nicotinic receptor antagonist potencies can be conducted by assessing the ability of these compounds to inhibit responses elicited by preganglionic autonomic nerve stimulation or stimulation of the motor nerves innervating striated muscle in isolated tissue preparations. In addition, in some isolated tissues innervated by autonomic nerves, nicotinic receptor mediated responses can be elicited by exogenously administered agonists, and the effects of antagonists on these responses can be assessed using pharmacologic analyses. This unit describes the guinea pig trachea/esophagus preparation, in which nicotinic receptor pharmacology can be studied at synapses of the parasympathetic and sympathetic nervous system and the striated musculature of the esophagus. In addition, a preparation whereby the nicotinic receptors of the striated musculature of the diaphragm can be studied is described as are techniques for studying exogenous nicotinic agonist mediated effects in two smooth muscle preparations.Nicotinic acetylcholine receptors (nAChRs) in peripheral tissues are localized almost exclusively to autonomic nerves.

Regulation of baseline cholinergic tone in guinea-pig airway smooth muscle.

1. We quantified baseline cholinergic tone in the trachealis of mechanically ventilated guinea-pigs and determined the influence of vagal afferent nerve activity on this parasympathetic tone. 2. There was a substantial amount of baseline cholinergic tone in the guinea-pig trachea, eliciting contractions of the trachealis that averaged 24.6 +/- 3.5 % (mean +/- s.e.m.) of the maximum attainable contraction. This tone was essentially abolished by vagotomy or ganglionic blockade, suggesting that it was dependent upon on-going pre-ganglionic input arising from the central nervous system. 3. Cholinergic tone in the trachealis could be markedly and rapidly altered (either increased or decreased) by changes in ventilation (e. g. cessation of ventilation; hyperpnoea; slow, deep breathing) and by lung distention (via positive end-expiratory pressure). These effects were not accompanied by marked alterations in blood gases and were abolished by vagotomy or atropine. By contrast, tachykinin receptor antagonists, which abolished capsaicin-induced bronchospasm, were without effect on baseline cholinergic tone. This and other evidence suggests that capsaicin-sensitive nerves have little if any influence on baseline parasympathetic tone. Likewise, while activation of afferent nerves innervating the larynx can alter airway parasympathetic nerve activity, transection of the superior laryngeal nerves was without effect on baseline cholinergic tone. 4. Cutting the vagus nerves caudal to the recurrent laryngeal nerves, thus leaving the preganglionic parasympathetic innervation of the trachealis intact but disrupting all afferent nerves innervating the lungs and intrapulmonary airways, abolished baseline cholinergic tone in the trachea. Sham vagotomy or cutting the vagi caudal to the lungs did not reduce baseline cholinergic tone. 5. The results indicate that baseline airway cholinergic nerve activity is necessarily dependent upon afferent nerve activity arising from the intrapulmonary airways and lungs. More specifically, the data are consistent with the hypothesis that on-going activity arising from the nerve terminals of intrapulmonary rapidly adapting receptors determines the level of baseline airway cholinergic tone.

Evidence for an esophageal origin of VIP-IR and NO synthase-IR nerves innervating the guinea pig trachealis: a retrograde neuronal tracing and immunohistochemical analysis.

Nonadrenergic noncholinergic (NANC) relaxations of the guinea pig trachea are thought to be mediated by vasoactive intestinal peptide (VIP) and nitric oxide (NO). Physiological studies have indicated that the parasympathetic ganglion neurons mediating NANC relaxations of the guinea pig trachea but not the ganglion neurons mediating cholinergic contractions are in some way associated with the adjacent esophagus. In the present study, we attempted to locate precisely the noncholinergic parasympathetic ganglia innervating the trachealis. Two days after injection of the retrograde neuronal tracer DiI into the trachealis of organotypic cultures of the guinea pig trachea and esophagus, neurons within the myenteric plexus of the esophagus or closely associated with the outer striated longitudinal muscle layers of the esophagus were labeled. Subsequent immunohistochemical analyses revealed that a majority of the retrogradely labeled neurons possessed VIP immunoreactivity (IR) or NO synthase (NOS)-IR or had VIP-IR nerve fibers associated with their cell bodies. By contrast, no labeling of esophageal neurons was seen when the tissue between the trachea and esophagus had been disrupted by blunt dissection prior to tracer injection or when the cultures were treated with the axonal transport inhibitor colchicine. The results of these experiments provide the first direct evidence that VIP-IR and NOS-IR neurons intrinsic to the guinea pig esophagus project axons to the adjacent trachealis. Based on their location and phenotype and the results of our previous studies, it is likely that these neurons are the postganglionic parasympathetic neurons mediating NANC relaxations of the trachealis.

Localization of heme oxygenase-2 immunoreactivity to parasympathetic ganglia of human and guinea-pig airways.

Carbon monoxide (CO), an activator of soluble guanylate cyclase and generated enzymatically by heme oxygenase-2 (HO-2), is thought to function as an intra- and intercellular neurotransmitter in the central and peripheral nervous system. In the present study, the distribution of HO-2 in airway nerves from both humans and guinea pigs was assessed. HO-2 was found in all neuronal perikarya of the intrinsic ganglia of guinea-pig airways and in all ganglion nerve cell bodies localized to the trachea and bronchi of humans. By contrast, nerve fibers innervating the smooth muscle, lamina propria, and epithelium of the airways in both species were devoid of HO-2 immunoreactivity. HO-1, the inducible isoform of heme oxygenase, was not found in airway nerves. The pattern of distribution of HO-2 observed suggests that CO might serve as a modulator of synaptic neurotransmission in the lung and airways rather than as a bona fide neurotransmitter in the smooth muscle, vasculature, or glands. Consistent with this hypothesis, 8-bromo-cyclic guanosine monophosphate (cGMP) (30 microM), a stable, pharmacologically active analog of cGMP, markedly inhibited vagally-mediated cholinergic contractions of the isolated guinea-pig trachea. In subsequent studies, however, neither inhibiting heme oxygenase with zinc protoporphyrin-IX (30 microM) nor inhibiting the soluble isoform of guanylate cyclase with ODQ (3 microM) had measurable effects on vagally-mediated cholinergic contractions of the trachea. These results indicate that CO could play a modulatory role in efferent (parasympathetic) synaptic neurotransmission in the airways, but under normal conditions may not be activated to an appreciable extent during periods of elevated vagal activity.

Pharmacological analysis of the tachykinin receptors that mediate activation of nonadrenergic, noncholinergic relaxant nerves that innervate guinea pig trachealis.

Previous studies indicated that antidromic stimulation of capsaicin-sensitive vagal afferent fibers activated, via peripheral release of tachykinins, nonadrenergic, noncholinergic parasympathetic ganglion neurons that mediate relaxations of guinea pig trachealis. On the basis of the effects of selective agonists and inhibition with a nonselective receptor antagonist (SR 48968), we speculated that tachykinin-mediated activation of neurokinin3 (NK3) receptors might be involved. Using the recently developed NK3-selective receptor antagonist SR 142801, we further assessed the role of NK3 receptors in these relaxant responses. Relaxations of the guinea pig trachea elicited by antidromic stimulation of capsaicin-sensitive vagal afferent nerves were markedly inhibited by 0.3 microM SR 142801 and were abolished by a combination of SR 142801 and either of the NK1-selective receptor antagonists SR 140333 and CP 99994 (0.3 microM each). The NK3 receptor antagonist had similar effects on the relaxant responses elicited by capsaicin and substance P, but it had no effect on relaxations of the trachealis elicited by electrical field stimulation of the postganglionic nerves that innervate the trachealis or by stimulation of the preganglionic parasympathetic vagal nerves that innervate the trachea. These results and the observation that the ganglion neurons that mediate these responses are densely innervated by substance P-containing nerve fibers lead us conclude that stimulation of capsaicin-sensitive visceral afferent fibers activates, upon peripheral release of tachykinins, nonadrenergic, noncholinergic inhibitory neurons innervating guinea pig trachealis via activation of both NK3 and NK1 receptors.

Localization of cholinergic nerves in lower airways of guinea pigs using antisera to choline acetyltransferase.

Primary antiserum to choline acetyltransferase (ChAT), a specific marker for cholinergic nerves, was used to characterize the distribution of cholinergic nerve fibers and nerve cell bodies in guinea pig airways. ChAT immunoreactive nerve fibers were localized to the smooth muscle throughout the conducting airways and in the lamina propria of the trachea and large bronchi. Likewise, all nerve cell bodies in the ganglia intrinsic to the trachea and bronchi displayed a cholinergic phenotype. By contrast, ChAT immunoreactive nerve fibers were infrequently seen in the lamina propria of the peripheral airways and were absent in the airway epithelium. No evidence for colocalization of ChAT and the enzyme synthesizing the putative relaxant neurotransmitter nitric oxide was observed. These results provide further evidence for the key role played by cholinergic nerves in regulating airway smooth muscle tone and bronchial blood flow and provide further evidence that acetylcholine is not coreleased with the neurotransmitter(s) mediating relaxations of airway smooth muscle.

Correlation of vasoactive intestinal peptide and nitric oxide synthase with choline acetyltransferase in the airway innervation.

Vasoactive intestinal peptide (VIP) and nitric oxide (NO) are potent mediators of neural airway smooth muscle relaxation. The major contractile mediator released by airway nerves under physiological conditions is acetylcholine (ACh). In the present study, we have correlated the immunohistochemical distribution of the relaxant mediators using antisera to VIP, to the marker enzyme of catecholamine synthesis tyrosine hydroxylase (TH) and to the NO-generating enzyme NO-synthase (NOS) with the distribution of the ACh-synthesizing enzyme, choline acetyltransferase (ChAT), and of substance P (SP), a neuropeptide present in sensory nerve fibers. In guinea-pig airways, VIP- and NOS-immunoreactivity (IR) were present in numerous nerve fibers in the airway smooth muscle and around submucosal glands; some fibers were also seen in the lamina propria and around blood vessels. The neuronal cell bodies in the intrinsic ganglia were devoid of both VIP- and NOS-IR. In contrast, all neuronal cell bodies in the intrinsic ganglia were immunoreactive for ChAT. In human airways, immunoreactivity for VIP, NOS, and ChAT was found in airway intrinsic neuronal perikarya. Whereas ChAT-IR appeared to be most frequent in the cell bodies, VIP-IR was seen in the largest number of nerve fibers in the airways. Therefore, in guinea pigs, a clear neuroanatomical and neurochemical separation of relaxant and of constrictor pathways is seen, whereas in human airways, both separate pathways as well as coexpression of VIP-/NOS- and of ChAT-IR are found.

Effects of organotypic culture on parasympathetic innervation of guinea pig trachealis.

Nonadrenergic, noncholinergic (NANC) relaxations of airway smooth muscle are thought to be mediated by vasoactive intestinal peptide (VIP) and nitric oxide (NO). Previous studies of the parasympathetic innervation of guinea pig trachealis suggest that the ganglion neurons mediating NANC relaxations but not cholinergic contractions are associated with the esophagus. In this study, the location of the neurons mediating these responses and their neurochemical phenotype was further assessed. Guinea pig tracheas maintained in organotypic culture for 2 days with the adjacent esophagus intact displayed cholinergic contractions and NANC relaxations to electrical field stimulation (EFS) as well as VIP and NO synthase (NOS) nerve fiber densities that were similar to those of control tracheas. By contrast, in tracheas cultured without the esophagus, NANC relaxations to EFS were not observed, and VIP and NOS nerve fiber densities were reduced > 80%. EFS-induced cholinergic contractions were unaffected by esophagus removal. These results provide further evidence that NANC relaxations are mediated by VIP and NO coreleased from noncholinergic parasympathetic nerve endings derived from neurons intrinsic to the esophagus.

Inhibition by zinc protoporphyrin-IX of vasoactive intestinal peptide-induced relaxations of guinea pig isolated trachea.

Carbon monoxide, formed as a product of heme oxygenase activity, has been postulated to act as an intra- and intercellular messenger molecule. We addressed the hypothesis that heme oxygenase is involved in the relaxation of the guinea pig trachealis elicited by vasoactive intestinal peptide (VIP) or by electrical field stimulation. Immunohistochemical studies revealed the presence of heme oxygenase-II in airway smooth muscle and epithelium. Zinc protoporphyrin-IX (ZnPPn), an inhibitor of heme oxygenase, effectively inhibited VIP-induced relaxations of tracheal smooth muscle. Surprisingly, the potency of ZnPPn was increased if the drug was preincubated with the VIP solution before addition to the tissue bath. The relaxant responses to 3-morpholinosydnonimine were unaffected by ZnPPn. Zinc deuteroporphyrin-IX 2,4 bisglycol, a more potent inhibitor of heme oxygenase than ZnPPn, did not affect the VIP responses. ZnPPn (300 microM) had no effect on nonadrenergic, noncholinergic relaxations of the guinea pig trachea. These data indicate that although ZnPPn is an efficacous inhibitor of VIP-induced relaxations of the guinea pig trachealis, it is unlikely that heme oxygenase plays an important role in this response. Rather, the data are consistent with the hypothesis that ZnPPn inhibits the VIP response via an interaction with the VIP molecules themselves. Although the results demonstrate the existence of heme oxygenase-II in the guinea pig trachealis, they do not support the hypothesis that it plays a role in electrical field stimulation-induced nonadrenergic, noncholinergic relaxations.