Design and synthesis of long acting inhaled corticosteroids for the treatment of asthma.

In this Letter we present data for a novel series of ICS for the treatment of asthma. 'Inhalation by design' principles have been applied to a series of highly potent steroidal GR agonists, with a focus on optimising the potential therapeutic index in human. Pharmacokinetic properties were tuned with high intrinsic clearance and low oral bioavailability in mind, to minimise systemic exposure and reduce systemically driven adverse events. High CYP mediated clearance as well as glucuronidation were targeted to achieve high intrinsic clearance coupled with multiple routes of clearance to minimise drug-drug interactions. Furthermore, pharmaceutical properties such as stability, crystallinity and solubility were considered to ensure compatibility with a dry powder inhaler. This work culminated in the identification of the clinical candidate 15, which demonstrates preclinically the desired efficacy and safety profiles confirming its potential as an inhaled agent for the treatment of asthma.

Design and synthesis of a fluorescent muscarinic antagonist.

The design and concise synthesis of a fluorescent tolterodine-BODIPY (boron dipyrromethene) conjugate is described which possesses potent antimuscarinic activity. This derivative illustrates proof-of-concept for the preparation of other useful fluorophoric antimuscarinic agents which have potential utility in receptor occupancy studies and high throughput screens.

The molecular cloning and functional expression of the dog CCR5.

Activation of CCR5 by specific chemokines is involved in the regulation of the immunological response of leukocytes at sites of inflammation. In addition, CCR5 serves as a fusion co-factor for macrophage-tropic strains of human immunodeficiency virus type 1 (HIV-1). Consequently, several CCR5 antagonists are currently in development for the treatment of HIV-1 infection. The dog CCR5 gene was cloned in order to characterise the chemokine binding site of the dog receptor for comparison across species. The deduced amino acid sequence of the dog CCR5 has close homology to the human receptor (80% identity). A HEK-293 cell line expressing the dog recombinant receptor was generated and immunoblot analysis with an anti-human CCR5 antibody revealed a 58kDa band in the cell lysate. In functional calcium signalling assays, the CCR5 endogenous ligands MIP-1alpha, MIP-1beta and RANTES evoked a robust response in the dog recombinant CCR5 cells. In a CRE-Luc (cAMP response element-luciferase) reporter gene assay, MIP-1beta (0.01-30nM) produced concentration-dependent inhibition of forskolin induced elevation in cAMP levels, and was equipotent in dog, human and macaque recombinant CCR5 cells (EC(50) 0.4, 0.21 and 0.47nM, respectively). These data suggest that chemokine signalling is conserved in the dog CCR5.

Molecular cloning and radioligand binding characterization of the chemokine receptor CCR5 from rhesus macaque and human.

The aim of this study was to determine if macaque represents a suitable species for the pre-clinical evaluation of novel CCR5 antagonists, such as maraviroc (UK-427,857). To do this we cloned and expressed CCR5 from rhesus macaque and compared the binding properties of [125I]-MIP-1beta and [3H]-maraviroc with human recombinant CCR5. [125I]-MIP-1beta bound with similar high affinity to CCR5 from macaque (K(d) = 0.24 +/- 0.05 nM) and human (K(d) = 0.23 +/- 0.05 nM) and with similar kinetic properties. In competition binding studies the affinity of a range of human chemokines for macaque CCR5 was also similar to human CCR5. Maraviroc inhibited binding of [125I]-MIP-1beta to CCR5 from macaque and human with similar potency (IC50 = 17.50 +/- 1.24 nM and 7.18 +/- 0.93 nM, respectively) and antagonised MIP-1beta induced intracellular calcium release mediated through CCR5 from macaque and human with similar potency (IC50 = 17.50 +/- 3.30 nM and 12.07 +/- 1.89, respectively). [3H]-maraviroc bound with high affinity to CCR5 from macaque (K(d) = 1.36+/-0.07 nM) and human (K(d) = 0.86 +/- 0.08 nM), but was found to dissociate approximately 10-fold more quickly from macaque CCR5. However, as with the human receptor, maraviroc was shown to be a high affinity, potent functional antagonist of macaque CCR5 thereby indicating that the macaque should be a suitable species in which to evaluate the pharmacology, safety and potential mechanism-related toxicology of novel CCR5 antagonists.

Opioid receptor characterisation of neuronally stimulated isolated human vas deferens.

Rodent vas deferens is routinely used as a native tissue preparation to assess opioid pharmacology of new compounds. The aim of this study was to investigate the effects of a selected number of opioid compounds in the human vas deferens. Stable contractions to electrical field stimulation (EFS) were inhibited by guanethidine (1 µM) and tetrodotoxin (TTX, 1 µΜ), confirming neuronally induced contractions. Contractile responses to EFS were inhibited by the selective δ-opioid agonists (DPDPE ([D-Pen2,5]enkephalin), PF-391459 (3-{4-[(R)-[(2S,5R)-4-benzyl-2,5-dimethylpiperazin-1-yl](3-hydroxyphenyl)methyl]phenyl}propanoic acid) and SNC-80 ((+)-4-[(αR)-α-((2 S,5R)-4-allyl-2,5-dimethyl-1-piperazinyl)-3-methoxybenzyl]-N,N-diethylbenzamide)) (tested from 1 ρM to 10 µM or maximum), and the µ-opioid agonists DAMGO ([D-Ala(2), NMe-Phe(4), Gly-ol(5)]-enkephalin), loperamide and SC-50484 (N-{2-[(N-acetyl-L-phenylalanyl)amino]-1,1-dimethylethyl}-L-tyrosinamide). There was no effect using the selective κ agonist U50488 (trans-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)cyclohexyl]-benzeneacetamide) (tested from 1 ρM to 10 µM). The selective δ-opioid antagonist naltrindole (10 nM) surmountably antagonised the responses to DPDPE, but not to PF-3911459. Responses to DAMGO were completely abolished in the presence of the µ-opioid antagonist CTAP (3 µM), which only weakly antagonised responses to SC-50484. We conclude that under these conditions, δ and µ-opioid receptors, but not κ-opioid receptors, are functional in the neuronally stimulated longitudinal human vas deferens. Additionally, the human vas deferens preparation can be used as part of a drug discovery screening project to assess opioid potency, efficacy and selectivity at native human tissues, thus providing more confidence in translation.

Estimation of binding rate constants using a simultaneous mixed-effects method: application to monoamine transporter reuptake inhibitor reboxetine.

BACKGROUND AND PURPOSE: Reboxetine is a clinically used antidepressant and is a racemic mixture of two enantiomers, SS- and RR-reboxetine. The aim of the work described in this manuscript was to determine the kinetics of binding of the RR- and SS-reboxetine to the human noradrenaline transporter (hNET). EXPERIMENTAL APPROACH: We have applied a simultaneous mixed-effects method to the analysis of the transient kinetics of binding of SS-, RR- and racemic reboxetine to hNET. This method allowed simultaneous modelling of multiple datasets, taking into account inter-experiment variability, thereby facilitating robust parameter estimation and minimizing the assumptions made. KEY RESULTS: The mixed-effects method proved simple and robust. SS-reboxetine bound to hNET according to a one-step binding model with the SS-enantiomer having 130-fold higher steady state affinity than the RR-enantiomer (K(d)= 0.076 +/- 0.009 nM vs. 9.7 +/- 0.8 nM respectively). The k(on) for SS-reboxetine was c. 1.4 x 10(5) M(-1).s(-1) and k(off) 1.05 x 10(-5) s(-1) (t(1/2) approximately 18 h). The k(on) for RR-reboxetine was c. 4.3 x 10(5) M(-1).s(-1) and k(off) 4.2 x 10(-3) s(-1) (t(1/2) approximately 3 min). The racemate behaved as expected for an equimolar mixture of RR- and SS-reboxetine, assuming mutually exclusive binding. CONCLUSIONS AND IMPLICATIONS: These data will be useful for the interpretation of the behaviour of reboxetine and its enantiomers in man and the method used could be applied to other candidate drugs.

Localisation of melanin-concentrating hormone receptor 1 in rat brain and evidence that sleep parameters are not altered despite high central receptor occupancy.

The present study describes the optimisation of an autoradiography assay that provides a means to measure the in vitro potency of melanin-concentrating hormone receptor 1 (MCH(1)) antagonists in native tissues and their ex vivo receptor occupancy. Initial localisation studies demonstrated that the MCH(1) receptor radioligand [(125)I]-S36057 bound to rat caudate putamen with specific binding of consistently >60%. In vitro, the MCH(1) receptor antagonists GW3430, SNAP-94847 and 4'-{[1-(cyclopropylmethyl)piperidin-4-ylidene] [5-fluoro-6-(trifluoromethyl)-1H-benzimidazol-2-yl]methyl}biphenyl-3-carbonitrile (referred to as Compound A) exhibited concentration dependent inhibition of the specific binding of [(125)I]-S36057, with a rank order of affinity of SNAP-94847>Compound A>GW3430. In an ex vivo occupancy assay, Compound A dosed orally to rats caused a concentration dependent inhibition of the specific binding of [(125)I]-S36057 to rat caudate putamen. The occupancy reached 87+/-11% at 30 mg/kg and the estimated ED(50) was 9.3 mg/kg, which was equivalent to a free plasma concentration of 40 nM. As MCH has been reported to play a role in the regulation of the sleep cycle, the effect of Compound A on sleep parameters was investigated. However Compound A, at exposures that achieved near maximal receptor occupancy, failed to demonstrate any effects on the sleep/wake pattern in telemetered rats. We conclude that our ex vivo receptor occupancy assay is suitable for selecting centrally penetrant MCH(1) receptor antagonists and that, despite high levels of receptor occupancy, the selective MCH(1) receptor antagonist Compound A failed to elicit any changes in sleep electroencephalogram (EEG) parameters.

CCR5 pharmacology methodologies and associated applications.

The G protein-coupled chemokine (C-C motif) receptor, CCR5, was originally characterized as a protein responding functionally to a number of CC chemokines. As with chemokine receptors in general, studies indicate that CCR5 plays a role in inflammatory responses to infection, although its exact role in normal immune function is not completely defined. The vast majority of research into CCR5 has been focused on its role as an essential and predominant coreceptor for HIV-1 entry into host immune cells. Discovery of this role was prompted by the elucidation that individuals homozygous for a 32 bp deletion in the CCR5 gene do not express the receptor at the cell surface, and as a consequence, are remarkably resistant to HIV-1 infection, and apparently possess no other clear phenotype. Multiple studies followed with the ultimate aim of identifying drugs that functionally and physically blocked CCR5 to prevent HIV-1 entry, and thus provide a completely new approach to treating infection and AIDS, the world's biggest infectious disease killer. To this end, functional antagonists with potent anti-HIV-1 activity have been discovered, as best exemplified by maraviroc, the first new oral drug for the treatment of HIV-1 infection in 10 years. In this chapter, the specific methods used to characterize CCR5 primary pharmacology and apply the data generated to enable drug discovery, notably maraviroc, for the treatment of HIV infection and potentially inflammatory-based indications, are described.

Overcoming hERG affinity in the discovery of maraviroc; a CCR5 antagonist for the treatment of HIV.

Avoiding cardiac liability associated with blockade of hERG (human ether a go-go) is key for successful drug discovery and development. This paper describes the work undertaken in the discovery of a potent CCR5 antagonist, maraviroc 34, for the treatment of HIV. In particular the use of a pharmacophore model of the hERG channel and a high throughput binding assay for the hERG channel are described that were critical to elucidate SAR to overcome hERG liabilities. The key SAR involves the introduction of polar substituents into regions of the molecule where it is postulated to undergo hydrophobic interactions with the ion channel. Within the CCR5 project there appeared to be no strong correlation between hERG affinity and physiochemical parameters such as pKa or lipophilicity. It is believed that chemists could apply these same strategies early in drug discovery to remove hERG interactions associated with lead compounds while retaining potency at the primary target.

Overcoming HERG affinity in the discovery of the CCR5 antagonist maraviroc.

The discovery of maraviroc 17 is described with particular reference to the generation of high selectivity over affinity for the HERG potassium channel. This was achieved through the use of a high throughput binding assay for the HERG channel that is known to show an excellent correlation with functional effects.

Maraviroc (UK-427,857), a potent, orally bioavailable, and selective small-molecule inhibitor of chemokine receptor CCR5 with broad-spectrum anti-human immunodeficiency virus type 1 activity.

Maraviroc (UK-427,857) is a selective CCR5 antagonist with potent anti-human immunodeficiency virus type 1 (HIV-1) activity and favorable pharmacological properties. Maraviroc is the product of a medicinal chemistry effort initiated following identification of an imidazopyridine CCR5 ligand from a high-throughput screen of the Pfizer compound file. Maraviroc demonstrated potent antiviral activity against all CCR5-tropic HIV-1 viruses tested, including 43 primary isolates from various clades and diverse geographic origin (geometric mean 90% inhibitory concentration of 2.0 nM). Maraviroc was active against 200 clinically derived HIV-1 envelope-recombinant pseudoviruses, 100 of which were derived from viruses resistant to existing drug classes. There was little difference in the sensitivity of the 200 viruses to maraviroc, as illustrated by the biological cutoff in this assay (= geometric mean plus two standard deviations [SD] of 1.7-fold). The mechanism of action of maraviroc was established using cell-based assays, where it blocked binding of viral envelope, gp120, to CCR5 to prevent the membrane fusion events necessary for viral entry. Maraviroc did not affect CCR5 cell surface levels or associated intracellular signaling, confirming it as a functional antagonist of CCR5. Maraviroc has no detectable in vitro cytotoxicity and is highly selective for CCR5, as confirmed against a wide range of receptors and enzymes, including the hERG ion channel (50% inhibitory concentration, >10 microM), indicating potential for an excellent clinical safety profile. Studies in preclinical in vitro and in vivo models predicted maraviroc to have human pharmacokinetics consistent with once- or twice-daily dosing following oral administration. Clinical trials are ongoing to further investigate the potential of using maraviroc for the treatment of HIV-1 infection and AIDS.

Functional selectivity of muscarinic receptor antagonists for inhibition of M3-mediated phosphoinositide responses in guinea pig urinary bladder and submandibular salivary gland.

Binding and functional affinities of the muscarinic acetylcholine (mACh) receptor antagonists darifenacin, tolterodine, oxybutynin, and atropine were assessed in Chinese hamster ovary (CHO) cells expressing the human recombinant M2 (CHO-m2) or M3 (CHO-m3) receptors, and in guinea pig bladder and submandibular gland. In [N-methyl-3H]scopolamine methyl chloride binding studies in CHO cells, darifenacin displayed selectivity (14.8-fold) for the M3 versus M2 mACh receptor subtype. Oxybutynin was nonselective, whereas atropine and tolterodine were weakly M2-selective (5.1- and 6.6-fold, respectively). Antagonist functional affinity estimates were determined by the inhibition of agonist-induced [3H]inositol phosphate accumulation in CHO-m3 cells and antagonism of the agonist-induced inhibition of forskolin-stimulated cyclic AMP accumulation in CHO-m2 cells. Darifenacin was the most M3-selective antagonist (32.4-fold), whereas oxybutynin, atropine, and tolterodine exhibited lesser selectivity. Functional affinity estimates in guinea pig urinary bladder and submandibular salivary gland using indices of phosphoinositide turnover revealed that oxybutynin, darifenacin, and tolterodine each displayed selectivity for the response in the bladder, relative to that seen in the submandibular gland (9.3-, 7.9-, and 7.4-fold, respectively). In contrast, atropine displayed a similar affinity in both tissues. These data demonstrate that in bladder, compared with submandibular gland from a single species, the mACh receptor antagonists darifenacin, tolterodine, and oxybutynin display selectivity to inhibit agonist-mediated phosphoinositide responses. It is proposed that both responses are mediated via M3 mACh receptor activation and that differential functional affinities displayed by some, but not all, antagonists are indicative of the influence of cell background upon the pharmacology of the M3 mACh receptor.