Joel R Gever

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.

Pharmacological pleiotropism of the human recombinant α1A-adrenoceptor : implications for α1-adrenoceptor classification

Three fully‐defined α1‐adrenoceptors (α1A, α1B and α1D) have been established in pharmacological and molecular studies. A fourth α1‐adrenoceptor, the putative α1L‐adrenoceptor, has been defined in functional but not molecular studies, and has been proposed to mediate contraction of human lower urinary tract tissues; its relationship to the three fully characterized α1‐adrenoceptors is not known. In the present study, binding affinities were estimated by displacement of [3H]‐prazosin in membrane homogenates of Chinese hamster ovary (CHO‐K1) cells stably expressing the human α1A‐, α1B‐ and α1D‐adrenoceptors and were compared with affinity estimates obtained functionally in identical cells by measuring inhibition of noradrenaline (NA)‐stimulated accumulation of [3H]‐inositol phosphates. For the α1A‐adrenoceptor, binding studies revealed a pharmacological profile typical for the classically defined α1A‐adrenoceptor, such that prazosin, RS‐17053, WB4101, 5‐methylurapidil, Rec15/2739 and S‐niguldipine all displayed subnanomolar affinity. A different profile of affinity estimates was obtained in inositol phosphates accumulation studies: prazosin, WB4101, 5‐methylurapidil, RS‐17053 and S‐niguldipine showed 10 to 40 fold lower affinity than in membrane binding. However, affinity estimates were not ‘frameshifted’, as tamsulosin, indoramin and Rec15/2739 yielded similar, high affinity estimates in binding and functional assays. In contrast, results from human α1B‐ and α1D‐adrenoceptors expressed in CHO‐K1 cells gave antagonist affinity profiles in binding and functional assays that were essentially identical. A concordance of affinity estimates from the functional (inositol phosphates accumulation) studies of the α1A‐adrenoceptor in CHO‐K1 cells was found with estimates published recently from contractile studies in human lower urinary tract tissues (putative α1L‐adrenoceptor). These data show that upon functional pharmacological analysis, the cloned α1A‐adrenoceptor displays pharmacological recognition properties consistent with those of the putative α1L‐adrenoceptor. Why this profile differs from that obtained in membrane binding, and whether it explains the α1L‐adrenoceptor pharmacology observed in many native tissues, requires further investigation.

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.

RO1138452 and RO3244794: characterization of structurally distinct, potent and selective IP (prostacyclin) receptor antagonists

Prostacyclin (PGI2) possesses various physiological functions, including modulation of nociception, inflammation and cardiovascular activity. Elucidation of these functions has been hampered by the absence of selective IP receptor antagonists. Two structurally distinct series of IP receptor antagonists have been developed: 4,5‐dihydro‐1H‐imidazol‐2‐yl)‐[4‐(4‐isopropoxy‐benzyl)‐phenyl]‐amine (RO1138452) and R‐3‐(4‐fluoro‐phenyl)‐2‐[5‐(4‐fluoro‐phenyl)
‐benzofuran‐2‐ylmethoxycarbonylamino]‐propionic acid (RO3244794). RO1138452 and RO3244794 display high affinity for IP receptors. In human platelets, the receptor affinities (pKi) were 9.3±0.1 and 7.7±0.03, respectively; in a recombinant IP receptor system, pKi values were 8.7±0.06 and 6.9±0.1, respectively. Functional antagonism of RO1138452 and RO3244794 was studied by measuring inhibition of carbaprostacyclin‐induced cAMP accumulation in CHO‐K1 cells stably expressing the human IP receptor. The antagonist affinities (pKi) of RO1138452 and RO3244794 were 9.0±0.06 and 8.5±0.11, respectively. Selectivity profiles for RO1138452 and RO3244794 were determined via a panel of receptor binding and enzyme assays. RO1138452 displayed affinity at I2 (8.3) and PAF (7.9) receptors, while RO3244794 was highly selective for the IP receptor: pKi values for EP1 (<5), EP3 (5.38), EP4 (5.74) and TP (5.09). RO1138452 (1–10 mg kg−1, i.v.) and RO3244794 (1–30 mg kg−1, i.v.) significantly reduced acetic acid‐induced abdominal constrictions. RO1138452 (3–100 mg kg−1, p.o.) and RO3244794 (0.3–30 mg kg−1, p.o.) significantly reduced carrageenan‐induced mechanical hyperalgesia and edema formation. RO3244794 (1 and 10 mg kg−1, p.o.) also significantly reduced chronic joint discomfort induced by monoiodoacetate. These data suggest that RO1138452 and RO3244794 are potent and selective antagonists for both human and rat IP receptors and that they possess analgesic and anti‐inflammatory potential.

In vitroα1‐adrenoceptor pharmacology of Ro 70–0004 and RS‐100329, novel α1A‐adrenoceptor selective antagonists

It has been hypothesized that in patients with benign prostatic hyperplasia, selective antagonism of the α1A‐adrenoceptor‐mediated contraction of lower urinary tract tissues may, via a selective relief of outlet obstruction, lead to an improvement in symptoms. The present study describes the α1‐adrenoceptor (α1‐AR) subtype selectivities of two novel α1‐AR antagonists, Ro 70‐0004 (aka RS‐100975) and a structurally‐related compound RS‐100329, and compares them with those of prazosin and tamsulosin. Radioligand binding and second‐messenger studies in intact CHO‐K1 cells expressing human cloned α1A‐, α1B‐ and α1D‐AR showed nanomolar affinity and significant α1A‐AR subtype selectivity for both Ro 70‐0004 (pKi 8.9: 60 and 50 fold selectivity) and RS‐100329 (pKi 9.6: 126 and 50 fold selectivity) over the α1B‐ and α1D‐AR subtypes respectively. In contrast, prazosin and tamsulosin showed little subtype selectivity. Noradrenaline‐induced contractions of human lower urinary tract (LUT) tissues or rabbit bladder neck were competitively antagonized by Ro 70‐0004 (pA2 8.8 and 8.9), RS‐100329 (pA2 9.2 and 9.2), tamsulosin (pA2 10.4 and 9.8) and prazosin (pA2 8.7 and 8.3 respectively). Affinity estimates for tamsulosin and prazosin in antagonizing α1‐AR‐mediated contractions of human renal artery (HRA) and rat aorta (RA) were similar to those observed in LUT tissues, whereas Ro 70‐0004 and RS‐100329 were approximately 100 fold less potent (pA2 values of 6.8/6.8 and 7.3/7.9 in HRA/RA respectively). The α1A‐AR subtype selectivity of Ro 70‐0004 and RS‐100329, demonstrated in both cloned and native systems, should allow for an evaluation of the clinical utility of a ‘uroselective’ agent for the treatment of symptoms associated with benign prostatic hyperplasia.

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.

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.

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.

Pharmacological characteristics of Ro 115-1240, a selective alpha1A/1L-adrenoceptor partial agonist: a potential therapy for stress urinary incontinence.

To describe the preclinical pharmacology of Ro 115–1240, a peripherally acting selective α1A/1L‐adrenoceptor (AR) partial agonist, compared with the α1A/1L‐AR full agonist amidephrine, as AR agonists have some utility in the treatment of stress urinary incontinence (SUI) but are limited by undesirable cardiovascular and central nervous system side‐effects.

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.