Michael Patrick Dillon

Pharmacology of P2X channels

Significant progress in understanding the pharmacological characteristics and physiological importance of homomeric and heteromeric P2X channels has been achieved in recent years. P2X channels, gated by ATP and most likely trimerically assembled from seven known P2X subunits, are present in a broad distribution of tissues and are thought to play an important role in a variety of physiological functions, including peripheral and central neuronal transmission, smooth muscle contraction, and inflammation. The known homomeric and heteromeric P2X channels can be distinguished from each other on the basis of pharmacological differences when expressed recombinantly in cell lines, but whether this pharmacological classification holds true in native cells and in vivo is less well-established. Nevertheless, several potent and selective P2X antagonists have been discovered in recent years and shown to be efficacious in various animal models including those for visceral organ function, chronic inflammatory and neuropathic pain, and inflammation. The recent advancement of drug candidates targeting P2X channels into human trials, confirms the medicinal exploitability of this novel target family and provides hope that safe and effective medicines for the treatment of disorders involving P2X channels may be identified in the near future.

Purinoceptors as therapeutic targets for lower urinary tract dysfunction

Lower urinary tract symptoms (LUTS) are present in many common urological syndromes. However, their current suboptimal management by muscarinic and α1‐adrenoceptor antagonists leaves a significant opportunity for the discovery and development of superior medicines. As potential targets for such therapeutics, purinoceptors have emerged over the last two decades from investigations that have established a prominent role for ATP in the regulation of urinary bladder function under normal and pathophysiological conditions. In particular, evidence suggests that ATP signaling via P2X1 receptors participates in the efferent control of detrusor smooth muscle excitability, and that this function may be heightened in disease and aging. ATP also appears to be involved in bladder sensation, via activation of P2X3 and P2X2/3 receptors on sensory afferent neurons, both within the bladder itself and possibly at central synapses. Such findings are based on results from classical pharmacological and localization studies in non‐human and human tissues, knockout mice, and studies using recently identified pharmacological antagonists – some of which possess attributes that offer the potential for optimization into candidate drug molecules. Based on recent advances in this field, it is clearly possible that the development of selective antagonists for these receptors will occur that could lead to therapies offering better relief of sensory and motor symptoms for patients, while minimizing the systemic side effects that limit current medicines.

Muscarinic receptor ligands and their therapeutic potential

Over the past year, the introduction of novel ligands has accelerated the classification of muscarinic receptor subtypes and has led to a better understanding of their physiological role. Important in this respect is the recent recognition of the exquisite selectivity of a series of snake toxins, enabling better definition of the muscarinic subtype 4 receptor. Moreover, several compounds, both agonists and antagonists, are progressing in advanced clinical trials for the treatment of several conditions, including Alzheimers disease, pain, urinary incontinence and chronic obstructive pulmonary disease.

AF-353, a novel, potent and orally bioavailable P2X3/P2X2/3 receptor antagonist

Background and purpose:  Purinoceptors containing the P2X3 subunit (P2X3 homotrimeric and P2X2/3 heterotrimeric) are members of the P2X family of ion channels gated by ATP and may participate in primary afferent sensitization in a variety of pain‐related diseases. The current work describes the in vitro pharmacological characteristics of AF‐353, a novel, orally bioavailable, highly potent and selective P2X3/P2X2/3 receptor antagonist.

Cardiovascular effects of rilmenidine, moxonidine and clonidine in conscious wild‐type and D79N α2A‐adrenoceptor transgenic mice

We investigated the cardiovascular effects of rilmenidine, moxonidine and clonidine in conscious wild‐type and D79N α2A‐adrenoceptor mice. The in vitro pharmacology of these agonists was determined at recombinant (human) α2‐adrenoceptors and at endogenous (dog) α2A‐adrenoceptors. In wild‐type mice, rilmenidine, moxonidine (100, 300 and 1000 μg kg−1, i.v.) and clonidine (30, 100 and 300 μg kg−1, i.v.) dose‐dependently decreased blood pressure and heart rate. In D79N α2A‐adrenoceptor mice, responses to rilmenidine and moxonidine did not differ from vehicle control. Clonidine‐induced hypotension was absent, but dose‐dependent hypertension and bradycardia were observed. In wild‐type mice, responses to moxonidine (1 mg kg−1, i.v.) were antagonized by the non‐selective, non‐imidazoline α2‐adrenoceptor antagonist, RS‐79948‐197 (1 mg kg−1, i.v.). Affinity estimates (pKi) at human α2A‐, α2B‐ and α2C‐adrenoceptors, respectively, were: rilmenidine (5.80, 5.76 and 5.33), moxonidine (5.37, <5 and <5) and clonidine (7.21, 7.16 and 6.87). In a [35S]‐GTPγS incorporation assay, moxonidine and clonidine were α2A‐adrenoceptor agonists (pEC50/intrinsic activity relative to noradrenaline): moxonidine (5.74/0.85) and clonidine (7.57/0.32). In dog saphenous vein, concentration‐dependent contractions were observed (pEC50/intrinsic activity relative to noradrenaline): rilmenidine (5.83/0.70), moxonidine (6.48/0.98) and clonidine (7.22/0.83). Agonist‐independent affinities were obtained with RS‐79948‐197. Thus, expression of α2A‐adrenoceptors is a prerequisite for the cardiovascular effects of moxonidine and rilmenidine in conscious mice. There was no evidence of I1‐imidazoline receptor‐mediated effects. The ability of these compounds to act as α2A‐adrenoceptor agonists in vitro supports this conclusion.

Identification and SAR of novel diaminopyrimidines. Part 1: The discovery of RO-4, a dual P2X3/P2X2/3 antagonist for the treatment of pain

P2X purinoceptors are ligand-gated ion channels whose endogenous ligand is ATP. Both the P2X(3) and P2X(2/3) receptor subtypes have been shown to play an important role in the regulation of sensory function and dual P2X(3)/P2X(2/3) antagonists offer significant potential for the treatment of pain. A high-throughput screen of the Roche compound collection resulted in the identification of a novel series of diaminopyrimidines; subsequent optimization resulted in the discovery of RO-4, a potent, selective and drug-like dual P2X(3)/P2X(2/3) antagonist.

Development of a human IgG4 bispecific antibody for dual targeting of interleukin-4 (IL-4) and interleukin-13 (IL-13) cytokines

Background: Dual neutralization of IL-4 and IL-13 is a promising therapeutic approach for asthma and allergy. Results: Knobs-into-holes IgG1 and IgG4 bispecific antibodies targeting both cytokines were developed. Conclusion: Bispecific antibodies of both isotypes have comparable in vitro potencies, in vivo pharmacokinetics, and lung partitioning. Significance: Further extension of knobs-into-holes technology to human IgG4 isotype as reported here provides greater options for therapeutics. Human bispecific antibodies have great potential for the treatment of human diseases. Although human IgG1 bispecific antibodies have been generated, few attempts have been reported in the scientific literature that extend bispecific antibodies to other human antibody isotypes. In this paper, we report our work expanding the knobs-into-holes bispecific antibody technology to the human IgG4 isotype. We apply this approach to generate a bispecific antibody that targets IL-4 and IL-13, two cytokines that play roles in type 2 inflammation. We show that IgG4 bispecific antibodies can be generated in large quantities with equivalent efficiency and quality and have comparable pharmacokinetic properties and lung partitioning, compared with the IgG1 isotype. This work broadens the range of published therapeutic bispecific antibodies with natural surface architecture and provides additional options for the generation of bispecific antibodies with differing effector functions through the use of different antibody isotypes.

Identification and SAR of novel diaminopyrimidines. Part 2: The discovery of RO-51, a potent and selective, dual P2X3/P2X2/3 antagonist for the treatment of pain

The purinoceptor subtypes P2X(3) and P2X(2/3) have been shown to play a pivotal role in models of various pain conditions. Identification of a potent and selective dual P2X(3)/P2X(2/3) diaminopyrimidine antagonist RO-4 prompted subsequent optimization of the template. This paper describes the SAR and optimization of the diaminopyrimidine ring and particularly the substitution of the 2-amino group. The discovery of the highly potent and drug-like dual P2X(3)/P2X(2/3) antagonist RO-51 is presented.

Discovery and optimization of RO-85, a novel drug-like, potent, and selective P2X3 receptor antagonist

Despite the extensive literature describing the role of the ATP-gated P2X(3) receptors in a variety of physiological processes the potential of antagonists as therapeutic agents has been limited by the lack of drug-like selective molecules. In this paper we report the discovery and optimization of RO-85, a novel drug-like, potent and selective P2X(3) antagonist. High-throughput screening of the Roche compound collection identified a small hit series of heterocyclic amides from a large parallel synthesis library. Rapid optimization, facilitated by high-throughput synthesis, focusing on increasing potency and improving drug-likeness resulted in the discovery of RO-85.

Discovery of First-in-Class, Potent, and Orally Bioavailable Embryonic Ectoderm Development (EED) Inhibitor with Robust Anticancer Efficacy

Overexpression and somatic heterozygous mutations of EZH2, the catalytic subunit of polycomb repressive complex 2 (PRC2), are associated with several tumor types. EZH2 inhibitor, EPZ-6438 (tazemetostat), demonstrated clinical efficacy in patients with acceptable safety profile as monotherapy. EED, another subunit of PRC2 complex, is essential for its histone methyltransferase activity through direct binding to trimethylated lysine 27 on histone 3 (H3K27Me3). Herein we disclose the discovery of a first-in-class potent, selective, and orally bioavailable EED inhibitor compound 43 (EED226). Guided by X-ray crystallography, compound 43 was discovered by fragmentation and regrowth of compound 7, a PRC2 HTS hit that directly binds EED. The ensuing scaffold hopping followed by multiparameter optimization led to the discovery of 43. Compound 43 induces robust and sustained tumor regression in EZH2MUT preclinical DLBCL model. For the first time we demonstrate that specific and direct inhibition of EED can be effective as an anticancer strategy.