Empagliflozin, a novel selective sodium glucose cotransporter-2 (SGLT-2) inhibitor: characterisation and comparison with other SGLT-2 inhibitors.

AIMS: Empagliflozin is a selective sodium glucose cotransporter-2 (SGLT-2) inhibitor in clinical development for the treatment of type 2 diabetes mellitus. This study assessed pharmacological properties of empagliflozin in vitro and pharmacokinetic properties in vivo and compared its potency and selectivity with other SGLT-2 inhibitors. METHODS: [(14)C]-alpha-methyl glucopyranoside (AMG) uptake experiments were performed with stable cell lines over-expressing human (h) SGLT-1, 2 and 4. Two new cell lines over-expressing hSGLT-5 and hSGLT-6 were established and [(14)C]-mannose and [(14)C]-myo-inositol uptake assays developed. Binding kinetics were analysed using a radioligand binding assay with [(3)H]-labelled empagliflozin and HEK293-hSGLT-2 cell membranes. Acute in vivo assessment of pharmacokinetics was performed with normoglycaemic beagle dogs and Zucker diabetic fatty (ZDF) rats. RESULTS: Empagliflozin has an IC(50) of 3.1 nM for hSGLT-2. Its binding to SGLT-2 is competitive with glucose (half-life approximately 1 h). Compared with other SGLT-2 inhibitors, empagliflozin has a high degree of selectivity over SGLT-1, 4, 5 and 6. Species differences in SGLT-1 selectivity were identified. Empagliflozin pharmacokinetics in ZDF rats were characterised by moderate total plasma clearance (CL) and bioavailability (BA), while in beagle dogs CL was low and BA was high. CONCLUSIONS: Empagliflozin is a potent and competitive SGLT-2 inhibitor with an excellent selectivity profile and the highest selectivity window of the tested SGLT-2 inhibitors over hSGLT-1. Empagliflozin represents an innovative therapeutic approach to treat diabetes.

In vitro pharmacology of clinically used central nervous system-active drugs as inverse H(1) receptor agonists.

The human histamine H(1) receptor (H(1)R) is a prototypical G protein-coupled receptor and an important, well characterized target for the development of antagonists to treat allergic conditions. Many neuropsychiatric drugs are also known to potently antagonize this receptor, underlying aspects of their side effect profiles. We have used the cell-based receptor selection and amplification technology assay to further define the clinical pharmacology of the human H(1)R by evaluating >130 therapeutic and reference drugs for functional receptor activity. Based on this screen, we have reported on the identification of 8R-lisuride as a potent stereospecific partial H(1)R agonist (Mol Pharmacol 65:538-549, 2004). In contrast, herein we report on a large number of varied clinical and chemical classes of drugs that are active in the central nervous system that display potent H(1)R inverse agonist activity. Absolute and rank order of functional potency of these clinically relevant brain-penetrating drugs may possibly be used to predict aspects of their clinical profiles, including propensity for sedation.

Histamine H3-receptor isoforms.

Increasing evidence supports a role for HA as a neurotransmitter and neuromodulator in various brain functions, including emotion, cognition, and feeding. The recent cloning of the histamine H3 receptor allowed for the subsequent cloning of a variety of H3 receptor isoforms from different species as well as the H4 receptor. As a result a wide variety of H3-receptor isoforms are now known that display differential brain expression patterns and signalling properties. These recent discoveries are discussed in view of the growing interest of the H3 receptor as a target for the development of potential therapeutics.

8R-lisuride is a potent stereospecific histamine H1-receptor partial agonist.

The human histamine H1 receptor (H1R) is an important, well characterized target for the development of antagonists to treat allergic conditions. Many neuropsychiatric drugs are known to potently antagonize the H1R, thereby producing some of their side effects. In contrast, the tolerability and potential therapeutic utility of H1R agonism is currently unclear. We have used a cell-based functional assay to evaluate known therapeutics and reference drugs for H1R agonist activity. Our initial functional screen identified three ergot-based compounds possessing heretofore-unknown H1R agonist activity. 8R-lisuride demonstrated potent agonist activity in various assays including receptor selection and amplification technology, inositol phosphate accumulation, and activation of nuclear factor-kappaB with pEC50 values of 8.1, 7.9, and 7.9, respectively, and with varying degrees of efficacy. Based on these assays, 8R-lisuride is the most potent stereospecific partial agonist for the human H1R yet reported. Investigation of the residues involved in histamine and lisuride binding, using H1R mutants and molecular modeling, have revealed that although these ligands are structurally different, the lisuride-binding pocket in the H1R closely corresponds to the histamine-binding pocket. The discovery of a potent stereospecific partial H1R agonist provides a valuable tool to further characterize this important therapeutic target in vitro.

Histamine H(1)-receptor activation of nuclear factor-kappa B: roles for G beta gamma- and G alpha(q/11)-subunits in constitutive and agonist-mediated signaling.

Nuclear factor kappa B (NF-kappa B) is an important transcription factor in inflammation that has obtained a great interest as a drug target for the treatment of various allergic conditions. In this study, we show that the histamine H(1) receptor, which is also an important player in allergic and inflammatory conditions, activates NF-kappa B in both a constitutive and agonist-dependent manner. Moreover, the observed constitutive NF-kappa B activation is inhibited by various H(1)-receptor antagonists, suggesting that inverse agonism may account, at least in part, for their ascribed antiallergic properties. Investigation of the H(1) receptor-mediated NF-kappa B activation in transfected COS-7 cells indicates that the level of the observed constitutive activity of the H(1) receptor can be modulated by the expression levels of either G alpha-proteins or G beta gamma-heterodimers. Members of the G alpha(q/11)-family of G alpha-proteins are most effective in increasing H(1) constitutive activity. Also, coexpression of G beta(2) in combination with either G gamma(1) or G gamma(2) results in an increased constitutive activity of the H(1) receptor, whereas scavenging of G beta gamma-subunits by coexpression of G alpha(t) completely neutralizes the constitutive, but not the agonist-induced, NF-kappa B activity. Our data suggest that both G alpha(q/11)- and G beta gamma-subunits play a role in the agonist-induced, H(1) receptor-mediated NF-kappa B activation, but that constitutive NF-kappa B activation by the H(1) receptor is primarily mediated through G beta gamma-subunits.

Constitutive signaling of the human cytomegalovirus-encoded chemokine receptor US28.

Previously it was shown that the HHV-8-encoded chemokine receptor ORF74 shows considerable agonist-independent, constitutive activity giving rise to oncogenic transformation (Arvanitakis, L., Geras-Raaka, E., Varma, A., Gershengorn, M. C., and Cesarman, E. (1997) Nature 385, 347-350). In this study we report that a second viral-encoded chemokine receptor, the human cytomegalovirus-encoded US28, also efficiently signals in an agonist-independent manner. Transient expression of US28 in COS-7 cells leads to the constitutive activation of phospholipase C and NF-kappaB signaling via G(q/11) protein-dependent pathways. Whereas phospholipase C activation is mediated via Galpha(q/11) subunits, the activation of NF-kappaB strongly depends on betagamma subunits with a preference for the beta(2)gamma(1) dimer. The CC chemokines RANTES (regulated on activation, normal T cell expressed and secreted) and MCP-1 (monocyte chemotactic protein-1) act as neutral antagonists at US28, whereas the CX(3)C chemokine fractalkine acts as a partial inverse agonist with IC(50) values of 1-5 nm. Our data suggest that a high level of constitutive activity might be a more general characteristic of viral G protein-coupled receptors and that human cytomegalovirus might exploit this G protein-coupled receptor property to modulate the homeostasis of infected cells via the early gene product US28.

Constitutive activity of the histamine H(1) receptor reveals inverse agonism of histamine H(1) receptor antagonists.

Transient expression of the wild-type human histamine H(1) receptor in SV40-immortalised African green monkey kidney cells resulted in an agonist-independent elevation of the basal levels of the second messenger inositoltrisphospate. Several histamine H(1) receptor antagonists, including the therapeutically used anti-allergics cetirizine, loratadine and epinastine reduced this constitutive histamine H(1) receptor activity. Inverse agonism, i.e., stabilisation of an inactive conformation of the human histamine H(1) receptor, may therefore be a key component of the anti-allergic mechanism of action of clinically used antihistamines.

Cloning and expression of a complementary DNA encoding a molluscan octopamine receptor that couples to chloride channels in HEK293 cells.

A cDNA encoding a G-protein-coupled receptor was cloned from the central nervous system of the pond snail Lymnaea stagnalis. The predicted amino acid sequence of this cDNA most closely resembles the Drosophila tyramine/octopamine receptor, the Locusta tyramine receptor, and an octopamine receptor (Lym oa1) that we recently cloned from Lymnaea. After stable expression of the cDNA in HEK293 cells, we found that [3H]rauwolscine binds with high affinity to the receptor (KD = 6.2.10(-9) M). Octopamine appears to be the most potent naturally occurring agonist to displace the [3H]rauwolscine binding (Ki = 3.0.10(-7) M). Therefore, the receptor is considered to be an octopamine receptor and is consequently designated Lym oa2. The novel receptor shares little pharmacological resemblance with Lym oa1, indicating that the two receptors represent different octopamine receptor subfamilies. Octopaminergic stimulation of Lym oa2 does not induce changes in intracellular concentrations of cAMP or inositol phosphates. However, electrophysiological experiments indicate that octopamine is able to activate a voltage-independent Cl- current in HEK293 cells stably expressing Lym oa2. Although opening of this chloride channel most probably does not require the activation of either protein kinase A or C, it can be blocked by inhibition of protein phosphorylation.

Molecular cloning and pharmacological characterization of a molluscan octopamine receptor.

We describe the cloning and functional expression of a cDNA encoding a novel G protein-coupled receptor, which was isolated from the central nervous system of the pond snail Lymnaea stagnalis. The amino acid sequence predicted by this cDNA shows highest similarity with the sequence of the Locusta tyramine receptor, the Drosophila tyramine/octopamine receptor, and the mammalian alpha-adrenergic receptors. On expression in mammalian cells, [3H]rauwolscine, an alpha2-adrenergic receptor antagonist, binds with high affinity (K(D) = 2.9 x 10(-9) M) to the receptor. Of several tested neurotransmitters, octopamine (which is considered to be the invertebrate counterpart of norepinephrine) showed the highest affinity (1.9 x 10(-6) M) for the receptor. Therefore, we consider this receptor to be the first true octopamine receptor to be cloned. The ligand binding properties of the novel receptor, designated Lym oa1, seem to be distinct from any of the binding profiles described for octopamine receptors in tissue preparations. Although the pharmacological profile of Lym oa1 shows some similarity with that of Tyr/Oct-Dro and Tyr-Loc, there are also clear differences. In particular, phentolamine, chlorpromazine, and mianserine display markedly higher affinities for Lym oa1 than for the insect receptors. As far as the vertebrate adrenergic receptors are concerned, the ligand binding properties of Lym oa1 resemble alpha2-adrenergic receptors more than they do alpha- or beta-adrenergic receptors. Octopaminergic stimulation of Lym oa1 induces an increase in both inositol phosphates and cAMP (EC50 = 9.1 x 10(-7) M and 5.1 x 10(-6) M, respectively). This is in contrast to the signal transduction pathways described for the related tyramine- and alpha2-adrenergic receptors, which couple in an inhibitory way to adenylyl cyclase.