Mapping the Orthosteric Binding Site of the Human 5-HT3 Receptor Using Photo-cross-linking Antagonists.

The serotonin-gated 5-HT3 receptor is a ligand-gated ion channel. Its location at the synapse in the central and peripheral nervous system has rendered it a prime pharmacological target, for example, for antiemetic drugs that bind with high affinity to the neurotransmitter binding site and prevent the opening of the channel. Advances in structural biology techniques have led to a surge of disclosed three-dimensional receptor structures; however, solving ligand-bound high-resolution 5-HT3 receptor structures has not been achieved to date. Ligand binding poses in the orthosteric binding site have been largely predicted from mutagenesis and docking studies. We report the synthesis of a series of photo-cross-linking compounds whose structures are based on the clinically used antiemetic drug granisetron (Kytril). These displaced [3H]granisetron from the orthosteric binding site with low nanomolar affinities and showed specific photo-cross-linking with the human 5-HT3 receptor. Detailed analysis by protein-MS/MS identified a residue (Met-228) near the tip of binding loop C as the covalent modification site.

Intestinal Microbiota Mediates the Susceptibility to Polymicrobial Sepsis-Induced Liver Injury by Granisetron Generation in Mice.

Sepsis-induced liver injury is recognized as a key problem in intensive care units. The gut microbiota has been touted as an important mediator of liver disease development; however, the precise roles of gut microbiota in regulating sepsis-induced liver injury are unknown. Here, we aimed to investigate the role of the gut microbiota in sepsis-induced liver injury and the underlying mechanism. Cecal ligation and puncture (CLP) was used to induce polymicrobial sepsis and related liver injury. Fecal microbiota transplantation (FMT) was used to validate the roles of gut microbiota in these pathologies. Metabolomics analysis was performed to characterize the metabolic profile differences between sepsis-resistant (Res; survived to 7 days after CLP) and sepsis-sensitive (Sen; moribund before or approximately 24 hours after CLP) mice. Mice gavaged with feces from Sen mice displayed more-severe liver damage than did mice gavaged with feces from Res mice. The gut microbial metabolic profile between Sen and Res mice was different. In particular, the microbiota from Res mice generated more granisetron, a 5-hydroxytryptamine 3 (5-HT3 ) receptor antagonist, than the microbiota from Sen mice. Granisetron protected mice against CLP-induced death and liver injury. Moreover, proinflammatory cytokine expression by macrophages after lipopolysaccharide (LPS) challenge was markedly reduced in the presence of granisetron. Both treatment with granisetron and genetic knockdown of the 5-HT3A receptor in cells suppressed nuclear factor kappa B (NF-кB) transactivation and phosphorylated p38 (p-p38) accumulation in macrophages. Gut microbial granisetron levels showed a significantly negative correlation with plasma alanine aminotransferase (ALT)/aspartate aminotransferase (AST) levels in septic patients. Conclusion: Our study indicated that gut microbiota plays a key role in the sensitization of sepsis-induced liver injury and associates granisetron as a hepatoprotective compound during sepsis development.

Evaluation of the maternal-fetal transfer of granisetron in an ex vivo placenta perfusion model.

The objective of this study was to estimate maternal-fetal transplacental passage of granisetron in an ex vivo placental perfusion model. Term human placentas (N=8) were collected immediately after delivery. A single cotyledon from each placenta was perfused granisetron concentration to mimic systemic maternal peak plasma concentrations following either IV (50ng/mL) or transdermal administration (5ng/mL). To assess drug transfer and accumulation, samples were collected from maternal and fetal compartments. In the 50ng/mL open model, the mean transport fraction was 0.21±0.08 with clearance index of 0.53±0.66. Fetal peak concentrations achieved was 5.6±6.6ng/mL with mean accumulation of 5.35±6.4ng/mL. No drug was detected in the fetal compartment with the 5ng/mL models. Transplacental passage of granisetron was inconsistent at the 50ng/mL concentration that achieved with IV dosing. However, there consistently was no detectable passage in all the placentas evaluated of the granisetron at 5ng/mL concentration that would be achieved after transdermal patch administration.

A single channel mutation alters agonist efficacy at 5-HT3A and 5-HT3AB receptors.

BACKGROUND AND PURPOSE: 5-HT3 receptors are composed of 5-HT3A subunits (homomeric receptors), or combinations of 5-HT3A and other 5-HT3 receptor subunits (heteromeric receptors, the best studied of which are 5-HT3AB receptors). Here we explore the effects of partial agonists at 5-HT3A and 5-HT3AB receptors, and the importance of a channel-lining residue in determining the efficacy of activation. EXPERIMENTAL APPROACH: Wild type and mutant 5-HT3A and 5-HT3AB receptors were expressed in Xenopus oocytes and examined using two-electrode voltage-clamp, or expressed in HEK293 cells and examined using [(3)H]granisetron binding. KEY RESULTS: Dopamine, quipazine and VUF10166 were partial agonists at wild type 5-HT3A and 5-HT3AB receptors, with quipazine and VUF10166 causing a long-lived (>20 min) inhibition of subsequent agonist responses. At 5-HT3A receptors, mCPBG was a partial agonist, but was a superagonist at 5-HT3AB receptors, as it produced a response 2.6× greater than that of 5-HT. A T6'S substitution in the 5-HT3A subunit decreased EC50 and increased Rmax of dopamine and quipazine at both homomeric and heteromeric receptors. The greatest changes were seen with VUF10166 at 5-HT3AT6'SB receptors, where it became a full agonist (EC50 = 7 nM) with an EC50 58-fold less than 5-HT (EC50 = 0.4 µM) and no longer caused inhibition of subsequent agonist responses. CONCLUSIONS AND IMPLICATIONS: These results indicate that a mutation in the pore lining domain in both 5-HT3A and 5-HT3AB receptors alters the relative efficacy of a series of agonists, changing some (e.g. quipazine) from apparent antagonists to potent and efficacious agonists.

Prolonged inhibition of 5-HT3 receptors by palonosetron results from surface receptor inhibition rather than inducing receptor internalization.

BACKGROUND AND PURPOSE: The 5-HT3 receptor antagonist palonosetron is an important treatment for emesis and nausea during cancer therapy. Its clinical efficacy may result from its unique binding and clearance characteristics and receptor down-regulation mechanisms. We investigated the mechanisms by which palonosetron exerts its long-term inhibition of 5-HT3 receptors for a better understanding of its clinical efficacy. EXPERIMENTAL APPROACH: Cell surface receptors (recombinantly expressed 5HT3A or 5HT3AB in COS-7 cells) were monitored using [³H]granisetron binding and ELISA after exposure to palonosetron. Receptor endocytosis was investigated using immunofluorescence microscopy. KEY RESULTS: Chronic exposure to palonosetron reduced the number of available cell surface [³H]granisetron binding sites. This down-regulation was not sensitive to either low temperature or pharmacological inhibitors of endocytosis (dynasore or nystatin) suggesting that internalization did not play a role. This was corroborated by our observation that there was no change in cell surface 5-HT3 receptor levels or increase in endocytic rate. Palonosetron exhibited slow dissociation from the receptor over many hours, with a significant proportion of binding sites being occupied for at least 4 days. Furthermore, our observations suggest that chronic receptor down-regulation involved interactions with an allosteric binding site. CONCLUSIONS AND IMPLICATIONS: Palonosetron acts as a pseudo-irreversible antagonist causing prolonged inhibition of 5-HT3 receptors due to its very slow dissociation. In addition, an irreversible binding mode persists for at least 4 days. Allosteric receptor interactions appear to play a role in this phenomenon.

Structural basis of ligand recognition in 5-HT3 receptors.

The 5-HT(3) receptor is a pentameric serotonin-gated ion channel, which mediates rapid excitatory neurotransmission and is the target of a therapeutically important class of anti-emetic drugs, such as granisetron. We report crystal structures of a binding protein engineered to recognize the agonist serotonin and the antagonist granisetron with affinities comparable to the 5-HT(3) receptor. In the serotonin-bound structure, we observe hydrophilic interactions with loop E-binding site residues, which might enable transitions to channel opening. In the granisetron-bound structure, we observe a critical cation-π interaction between the indazole moiety of the ligand and a cationic centre in loop D, which is uniquely present in the 5-HT(3) receptor. We use a series of chemically tuned granisetron analogues to demonstrate the energetic contribution of this electrostatic interaction to high-affinity ligand binding in the human 5-HT(3) receptor. Our study offers the first structural perspective on recognition of serotonin and antagonism by anti-emetics in the 5-HT(3) receptor.

Ondansetron and granisetron binding orientation in the 5-HT(3) receptor determined by unnatural amino acid mutagenesis.

The serotonin type 3 receptor (5-HT(3)R) is a ligand-gated ion channel found in the central and peripheral nervous systems. The 5-HT(3)R is a therapeutic target, and the clinically available drugs ondansetron and granisetron inhibit receptor activity. Their inhibitory action is through competitive binding to the native ligand binding site, although the binding orientation of the drugs at the receptor has been a matter of debate. Here we heterologously express mouse 5-HT(3)A receptors in Xenopus oocytes and use unnatural amino acid mutagenesis to establish a cation-π interaction for both ondansetron and granisetron to tryptophan 183 in the ligand binding pocket. This cation-π interaction establishes a binding orientation for both ondansetron and granisetron within the binding pocket.

Varenicline is a potent agonist of the human 5-hydroxytryptamine3 receptor.

Varenicline, a widely used and successful smoking cessation agent, acts as a partial agonist at nicotinic acetylcholine receptors. Here, we explore the effects of varenicline at human and mouse 5-Hydroxytryptamine(3) (5-HT(3)) receptors. Application of varenicline to human 5-HT(3) receptors expressed in Xenopus laevis oocytes reveal it is almost a full agonist (R(max) = 80%) with an EC(50) (5.9 µM) 3-fold higher than 5-HT. At mouse 5-HT(3) receptors varenicline is a partial agonist (R(max) = 35%) with an EC(50) (18 µM) 20-fold higher than 5-HT. Displacement of the competitive 5-HT(3) receptor antagonist [(3)H]granisetron reveals similar IC(50) values for varenicline at mouse and human receptors expressed in human embryonic kidney 293 cells, although studies in these cells using a membrane potential-sensitive dye show that again varenicline is a 4- or 35-fold less potent agonist than 5-HT in human and mouse receptors, respectively. Thus the data suggest that the efficacy, but not the affinity, of varenicline is greater at human 5-HT(3) receptors compared with mouse. Docking studies provide a possible explanation for this difference, because they suggest distinct orientations of the ligand in the mouse versus human 5-HT(3) agonist binding sites. Additional binding selectivity studies in a broad panel of recombinant receptors and enzymes confirmed an interaction with 5-HT(3) receptors but revealed no additional interactions of varenicline. Therefore, activation of human 5-HT(3) receptors may be responsible for some of the side effects that preclude use of higher doses during varenicline treatment.

An efficient and information-rich biochemical method design for fragment library screening on ion channels.

Drug discovery requires a simple, rapid, and cost-effective method for the early identification of novel leads and elimination of poor candidates. Here we present an experimental design that fulfils these criteria, using a ligand-gated ion channel expressed in a mammalian cell line, whose function can be probed using a voltage-sensitive dye. The experimental design is novel, as it uses the same screen to identify hit fragments and to characterize them as agonists or antagonists. The results were independently validated using radioligand binding, although the new technique has several advantages over radioligand methods. A number of novel high-affinity ligands were found. The method is broadly applicable to a wide range of receptor types including ligand-gated ion channels (LGICs), voltage-gated ion channels (VGICs), and G protein-coupled receptors (GPCRs), all of which are important drug targets.

Computational analysis of ligand recognition sites of homo- and heteropentameric 5-HT3 receptors.

Inhibition of the 5-hydroxytryptamine receptor (5-HT(3)R), a member of the Cys-loop superfamily of Ligand-Gated Ion Channels (LGICs), has been recognized to have important antiemetic effects. With respect to the many other drugs already in use, such as the first generation 5-HT(3)R antagonist granisetron, palonosetron, a second generation antagonist, clearly demonstrates superior inhibition potency towards the 5-HT(3)Rs. Five different receptor monomers, the 5-HT(3)R A-E, have been identified although the A and B subunits are the only known to build functional receptors, the homopentameric 5-HT(3A)R and the heteropentameric 5-HT(3B-A)R (with BBABA subunit arrangement). At present, however, no three-dimensional structure has been reported for any of the 5-HT(3)R subunits. To understand the binding properties of agonists and antagonists, models of the extracellular portion of the 5-HT(3)R A and B subunits are built and assembled into the receptor (homo- and hetero-) pentameric structure on the basis of the known three-dimensional structure of the nicotinic-acetylcholine receptor (nACh-R). The results of docking studies of the natural agonist serotonin and the antagonists palonosetron and granisetron into the modelled homomeric and heteromeric 5-HT(3)R binding interfaces, provide a possible rationalization both of the higher potency of palonosetron with respect to other antagonists, and of its previously reported allosteric binding and positive cooperativity properties.

Agonists and antagonists bind to an A-A interface in the heteromeric 5-HT3AB receptor.

The 5-HT3 receptor is a member of the Cys-loop family of transmitter receptors. It can function as a homopentamer (5-HT3A-only subunits) or as a heteropentamer. The 5-HT3AB receptor is the best characterized heteropentamer. This receptor differs from a homopentamer in its kinetics, voltage dependence, and single-channel conductance, but its pharmacology is similar. To understand the contribution of the 5-HT3B subunit to the binding site, we created homology models of 5-HT3AB receptors and docked 5-HT and granisetron into AB, BA, and BB interfaces. To test whether ligands bind in any or all of these interfaces, we mutated amino acids that are important for agonist and antagonist binding in the 5-HT3A subunit to their corresponding residues in the 5-HT3B subunit and vice versa. Changes in [3H]granisetron binding affinity (Kd) and 5-HT EC50 were determined using receptors expressed in HEK-293 cells and Xenopus oocytes, respectively. For all A-to-B mutant receptors, except T181N, antagonist binding was altered or eliminated. Functional studies revealed that either the receptors were nonfunctional or the EC50 values were increased. In B-to-A mutant receptors there were no changes in Kd, although EC50 values and Hill slopes, except for N170T mutant receptors, were similar to those for 5-HT3A receptors. Thus, the experimental data do not support a contribution of the 5-HT3B subunit to the binding pocket, and we conclude that both 5-HT and granisetron bind to an AA binding site in the heteromeric 5-HT3AB receptor.

The antimalarial drugs quinine, chloroquine and mefloquine are antagonists at 5-HT3 receptors.

BACKGROUND AND PURPOSE: The antimalarial compounds quinine, chloroquine and mefloquine affect the electrophysiological properties of Cys-loop receptors and have structural similarities to 5-HT(3) receptor antagonists. They may therefore act at 5-HT(3) receptors. EXPERIMENTAL APPROACH: The effects of quinine, chloroquine and mefloquine on electrophysiological and ligand binding properties of 5-HT(3A) receptors expressed in HEK 293 cells and Xenopus oocytes were examined. The compounds were also docked into models of the binding site. KEY RESULTS: 5-HT(3) responses were blocked with IC (50) values of 13.4 microM, 11.8 microM and 9.36 microM for quinine, chloroquine and mefloquine. Schild plots indicated quinine and chloroquine behaved competitively with pA (2) values of 4.92 (K (B)=12.0 microM) and 4.97 (K (B)=16.4 microM). Mefloquine displayed weakly voltage-dependent, non-competitive inhibition consistent with channel block. On and off rates for quinine and chloroquine indicated a simple bimolecular reaction scheme. Quinine, chloroquine and mefloquine displaced [(3)H]granisetron with K (i) values of 15.0, 24.2 and 35.7 microM. Docking of quinine into a homology model of the 5-HT(3) receptor binding site located the tertiary ammonium between W183 and Y234, and the quinoline ring towards the membrane, stabilised by a hydrogen bond with E129. For chloroquine, the quinoline ring was positioned between W183 and Y234 and the tertiary ammonium stabilised by interactions with F226. CONCLUSIONS AND IMPLICATIONS: This study shows that quinine and chloroquine competitively inhibit 5-HT(3) receptors, while mefloquine inhibits predominantly non-competitively. Both quinine and chloroquine can be docked into a receptor binding site model, consistent with their structural homology to 5-HT(3) receptor antagonists.

Interactions of granisetron with an agonist-free 5-HT3A receptor model.

A new homology model of type-3A serotonin receptors (5-HT(3A)Rs) was built on the basis of the electron microscopic structure of the nicotinic acetylcholine receptor and with an agonist-free binding cavity. The new model was used to re-evaluate the interactions of granisetron, a 5-HT(3A)R antagonist. Docking of granisetron identified two possible binding modes, including a newly identified region for antagonists formed by loop B, C, and E residues. Amino acid residues L184-D189 in loop B were mutated to alanine, while Y143 and Y153 in loop E were mutated to phenylalanine. Mutation H185A resulted in no detectable granisetron binding, while D189A resulted in a 22-fold reduction in affinity. Y143F and Y153F decreased granisetron affinity to the same extent as Y143A and Y153A mutations, supporting the role of the OH groups of these tyrosines in loop E. Modeling and mutation studies suggest that granisetron plays its antagonist role by hindering the closure of the back wall of the binding cavity.

CYP1A1 is a major enzyme responsible for the metabolism of granisetron in human liver microsomes.

Granisetron, a potent 5-HT3 receptor antagonist, has been reported to be mainly metabolized to 7-hydroxygranisetron and a lesser extent to 9'-desmethylgranisetron in humans. A previous study indicated that cytochrome P450 (CYP)3A4 is a major catalyst of 9'-demethylation, although the major CYP isoform(s) responsible for 7-hydroxylation are unknown. To clarify granisetron 7-hydroxylase, the in vitro metabolism of granisetron using expressed human CYPs and human liver microsomes was investigated. 7-Hydroxygranisetron was produced almost exclusively by CYP1A1, while, apparently, 9'-desmethylgranisetron was preferentially produced by CYP3A4. Marked inter-individual differences in the ratio of the formation of 7-hydroxygranisetron and 9'-desmethylgranisetron in human liver microsomes was observed. Granisetron 7-hydroxylase activity was strongly correlated with benzo[a]pyrene 3-hydroxylase activity (p<0.0001), but not with testosterone 6beta-hydroxylase activity in human liver microsomes. Furthermore, an anti-human CYP1A1 antibody completely inhibited 7-hydroxylation in human liver microsomes, however, the reaction was not inhibited at all by an anti-CYP3A4 antibody. On the other hand, granisetron 9'-demethylase activity correlated significantly not only with testosterone 6beta-hydroxylase activity (p<0.0001) but also with benzo[a]pyrene 3-hydroxylase activity (p<0.01). Consistent with this, both the anti-CYP1A1 and anti-human CYP3A4 antibodies inhibited the 9'-demethylase activity. These data indicate that CYP1A1 is a major enzyme responsible for the metabolism of granisetron via a main 7-hydroxylation pathway and an alternative 9'-demethylation route. This is the first report demonstrating the substantial contribution of CYP1A1 to the metabolism of a drug, although its role in the metabolism of environmental compounds is well established.

Allosteric modulation of 5-HT3 serotonin receptors.

[(3)H]Granisetron binding to 5-HT(3) type serotonin receptors was examined in homogenates of rat forebrain and NG 108-15 cells. We have applied an allosteric model to 5-HT(3) receptor binding for the first time. Slope factors of displacement improved the modelling. Serotonin displaced [(3)H]granisetron binding with micromolar potency in forebrain and with nanomolar potency in NG 108-15 cells. Racemic and (+)verapamil, ifenprodil and GYKI-46903 were used as representative allosteric inhibitors of 5-HT(3) receptors. They displaced [(3)H]granisetron binding with great negative cooperativity (alpha>10) and exerted great negative cooperativity with serotonin binding (beta>10). Great negative cooperativity of these agents with serotonin and [(3)H]granisetron binding cannot be distinguished from dual competitive displacement. Trichloroethanol (data from literature) had no cooperativity with [(3)H]granisetron binding (alpha~1) and exhibit positive cooperativity with serotonin (beta<1) in displacement. The allosteric model can lead to a more quantitative method in vitro to develop allosteric agents for 5-HT(3) receptors.

Spatial orientation of the antagonist granisetron in the ligand-binding site of the 5-HT3 receptor.

The serotonin type 3 receptor (5-HT(3)R) is a member of the cys-loop ligand-gated ion channel (LGIC) superfamily. Like almost all membrane proteins, high-resolution structural data are unavailable for this class of receptors. We have taken advantage of the high degree of homology between LGICs and the acetylcholine binding protein (AChBP) from the freshwater snail Lymnea stagnalis, for which high-resolution structural data are available, to create a structural model for the extracellular (i.e., ligand-binding) domain of the 5-HT(3)R and to perform a series of ligand docking experiments to delineate the architecture of the ligand-binding site. Structural models were created using homology modeling with the AChBP as a template. Docking of the antagonist granisetron was carried out using a Lamarckian genetic algorithm to produce models of ligand-receptor complexes. Two energetically similar conformations of granisetron in the binding site were obtained from the docking simulations. In one model, the indazole ring of granisetron is near Trp90 and the tropane ring is near Arg92; in the other, the orientation is reversed. We used double-mutant cycle analysis to determine which of the two orientations is consistent with experimental data and found that the data are consistent with the model in which the indazole ring of granisetron interacts with Arg92 and the tropane ring interacts with Trp90. The combination of molecular modeling with double-mutant cycle analysis offers a powerful approach for the delineation of the architecture of the ligand-binding site.

Locating an antagonist in the 5-HT3 receptor binding site using modeling and radioligand binding.

We have used a homology model of the extracellular domain of the 5-HT(3) receptor to dock granisetron, a 5-HT(3) receptor antagonist, into the binding site using AUTODOCK. This yielded 13 alternative energetically favorable models. The models fell into 3 groups. In model type A the aromatic rings of granisetron were between Trp-90 and Phe-226 and its azabicyclic ring was between Trp-183 and Tyr-234, in model type B this orientation was reversed, and in model type C the aromatic rings were between Asp-229 and Ser-200 and the azabicyclic ring was between Phe-226 and Asn-128. Residues located no more than 5 A from the docked granisetron were identified for each model; of 26 residues identified, 8 were found to be common to all models, with 18 others being represented in only a subset of the models. To identify which of the docking models best represents the ligand-receptor complex, we substituted each of these 26 residues with alanine and a residue with similar chemical properties. The mutant receptors were expressed in human embryonic kidney (HEK)293 cells and the affinity of granisetron determined using radioligand binding. Mutation of 2 residues (Trp-183 and Glu-129) ablated binding, whereas mutation of 14 other residues caused changes in the [(3)H]granisetron binding affinity in one or both mutant receptors. The data showed that residues both in and close to the binding pocket can affect antagonist binding and overall were found to best support model B.

Facilitation of cholinergic transmission by combined treatment of ondansetron with flumazenil after cortical cholinergic deafferentation.

We have studied the effects of concomitant blockade of 5-HT(3) and GABA(A) receptors on acetylcholine (ACh) release in the frontal cortex of rats with a selective cholinergic lesion. Lesions were performed by microinjection of the cholinergic toxin 192 IgG-saporin into the nucleus basalis magnocellularis. Single treatment with either the 5-HT(3) receptor antagonist ondansetron, 0.1 microg/kg, or the GABA(A) receptor benzodiazepine site antagonist flumazenil, 10 mg/kg, did not affect ACh release. However, the combined ondansetron + flumazenil administration significantly increased ACh release to a similar extent as a depolarising stimulus with K(+), 100 mM, at both 7 and 30 days post-lesion. Cortical perfusion with the combined ondansetron + flumazenil treatment also increased [(3)H]ACh efflux "in vitro" 30 days after lesion, suggesting that local events within the frontal cortex may participate in the interaction of ondansetron with GABAergic neurons, modulating ACh release in situations of cholinergic hypoactivity. No differences in the expression of 5-HT(3) and GABA(A) receptors in the frontal cortex were found after the cholinergic lesion. These results suggest that a combined ondansetron + flumazenil treatment would contribute to restoring a diminished cholinergic function and may provide a basis for using this treatment in the therapy of cognitive disorders associated with degeneration of the cholinergic system.

Effects of aprepitant on the pharmacokinetics of ondansetron and granisetron in healthy subjects.

BACKGROUND: The neurokinin-1-receptor antagonist aprepitant, when given in combination with a corticosteroid and a 5-hydroxytryptamine type 3 (5-HT(3))-receptor antagonist, has been shown to be effective for the prevention of acute and delated chemotherapy-induced nausea and vomiting (CINV). OBJECTIVE: Two studies were conducted to determine whether concomitant administration of aprepitant altered the pharmacokinetic profiles of ondansetron and granisetron, two 5-HT(3)-receptor antagonists commonly used as antiemetic therapy for CINV. METHODS: The 2 studies were randomized, open-label, crossover trials conducted in healthy subjects aged between 18 and 46 years. Study 1 involved the following 2 treatment regimens: aprepitant 375 mg PO, dexamethasone 20 mg PO, and ondansetron 32 mg IV on day 1, followed by aprepitant 250 mg PO and dexamethasone 8 mg PO on days 2 through 5; and dexamethasone 20 mg PO and ondansetron 32 mg IV on day 1, followed by dexamethasone 8 mg PO on days 2 through 5. Study 2 involved the following 2 treatment regimens: aprepitant 125 mg PO with granisetron 2 mg PO on day 1, followed by aprepitant 80 mg PO on days 2 and 3; and granisetron 2 mg PO on day 1 only. Individual plasma samples were used to estimate area under the plasma concentration-time curve from time zero to infinity (AUC(0- infinity )), peak plasma concentration, and apparent terminal elimination half-life (t(12)) of both ondansetron and granisetron. RESULTS: Study 1 included 19 subjects (10 women, 9 men), and study 2 included 18 subjects (11 men, 7 women). Coadministration of aprepitant 375 mg produced a small but statistically significant increase in the AUC(0- infinity ) for intravenous ondansetron (from 1268.3 to 1456.5 ng.h/mL; P = 0.019), with no significant effect on peak concentration at the end of the infusion (360.8 ng/mL with aprepitant vs 408.4 ng/mL without) or t(12) (5.0 vs 4.5 hours, respectively). Coadministration of aprepitant 125 mg/80 mg did not alter the mean pharmacokinetic characteristics of oral granisetron (AUC(0- infinity ), 101.4 ng.h/mL with aprepitant vs 92.2 ng.h/mL without; maximum plasma concentration, 9.0 ng/mL with and without aprepitant; time to maximum plasma concentration, both 3.0 hours; t(12), 6.5 vs 6.9 hours, respectively). CONCLUSION: Concomitant administration of aprepitant had no clinically significant effect on the mean pharmacokinetic characteristics of either ondansetron or granisetron in these healthy subjects.

Pharmacological characterization of the 5-HT1A, 5-HT2 and 5-HT3 receptors in the bovine ciliary muscle.

We aimed to investigate the effects of serotonin (5-hydroxytryptamine, 5-HT) on the bovine ciliary muscle and subsequently to characterize and identify the subtypes of 5-HT receptors involved in the serotonin-evoked contractility muscle. The binding of [3H]ketanserin, [3H]granisetron and [3H]8-hydroxy-2-(di-n-propylamino)tetralin ([3H]8-OH-DPAT) was analyzed. All labelled compounds bound with high affinity to a single site in the membrane preparations studied. The affinity (K(d)) of the binding site was 7.5+/-1.2 nM for [3H]ketanserin, 6.9+/-0.8 nM for [3H]granisetron and 4.4+/-0.31 nM for [3H]8-OH-DPAT. The density of receptors (B(max)) was 1062+/-43.0 fmol/mg protein for [3H]ketanserin, 566+/-2.32 fmol/mg protein for [3H]granisetron and 205+/-4.63 fmol/mg protein for [3H]8-OH-DPAT. The serotonin-induced contraction appeared to be competitively antagonized by ketanserin (0.1, 1 and 10 microM) and ondansetron (0.1, 10 and 100 microM) which produced a pA(2) value of 8.5+/-0.12 and 8.0+/-0.19, respectively. 8-OH-DPAT and 5-carboxamidotryptamine (5-CT) proved to be completely ineffective. We conclude that serotonin induces bovine ciliary muscle contraction via 5-HT(2) and 5-HT(3) receptors while the 5-HT(1A) receptors, although present, do not mediate the contractile response.