Preclinical Efficacy and Anti-Inflammatory Mechanisms of Action of the Bruton Tyrosine Kinase Inhibitor Rilzabrutinib for Immune-Mediated Disease.

Bruton tyrosine kinase (BTK) is expressed in B cells and innate immune cells, acting as an essential signaling element in multiple immune cell pathways. Selective BTK inhibition has the potential to target multiple immune-mediated disease pathways. Rilzabrutinib is an oral, reversible, covalent BTK inhibitor designed for immune-mediated diseases. We examined the pharmacodynamic profile of rilzabrutinib and its preclinical mechanisms of action. In addition to potent and selective BTK enzyme and cellular activity, rilzabrutinib inhibited activation and inflammatory activities of B cells and innate cells such as macrophages, basophils, mast cells, and neutrophils, without cell death (in human and rodent assay systems). Rilzabrutinib demonstrated dose-dependent improvement of clinical scores and joint pathology in a rat model of collagen-induced arthritis and demonstrated reductions in autoantibody-mediated FcγR signaling in vitro and in vivo, with blockade of rat Arthus reaction, kidney protection in mouse Ab-induced nephritis, and reduction in platelet loss in mouse immune thrombocytopenia. Additionally, rilzabrutinib inhibited IgE-mediated, FcεR-dependent immune mechanisms in human basophils and mast cell-dependent mouse models. In canines with naturally occurring pemphigus, rilzabrutinib treatment resulted in rapid clinical improvement demonstrated by anti-inflammatory effects visible within 2 wk and all animals proceeding to complete or substantial disease control. Rilzabrutinib is characterized by reversible covalent BTK binding, long BTK residence time with low systemic exposure, and multiple mechanistic and biological effects on immune cells. Rilzabrutinib's unique characteristics and promising efficacy and safety profile support clinical development of rilzabrutinib for a broad array of immune-mediated diseases.

Prolonged and tunable residence time using reversible covalent kinase inhibitors.

Drugs with prolonged on-target residence times often show superior efficacy, yet general strategies for optimizing drug-target residence time are lacking. Here we made progress toward this elusive goal by targeting a noncatalytic cysteine in Bruton's tyrosine kinase (BTK) with reversible covalent inhibitors. Using an inverted orientation of the cysteine-reactive cyanoacrylamide electrophile, we identified potent and selective BTK inhibitors that demonstrated biochemical residence times spanning from minutes to 7 d. An inverted cyanoacrylamide with prolonged residence time in vivo remained bound to BTK for more than 18 h after clearance from the circulation. The inverted cyanoacrylamide strategy was further used to discover fibroblast growth factor receptor (FGFR) kinase inhibitors with residence times of several days, demonstrating the generalizability of the approach. Targeting of noncatalytic cysteines with inverted cyanoacrylamides may serve as a broadly applicable platform that facilitates 'residence time by design', the ability to modulate and improve the duration of target engagement in vivo.

A phase I trial of PRN1008, a novel reversible covalent inhibitor of Bruton's tyrosine kinase, in healthy volunteers.

AIM: To evaluate the safety, tolerability, and pharmacokinetics/pharmacodynamics of PRN1008, a novel Bruton's tyrosine kinase (BTK) inhibitor, in healthy volunteers, and thus determine the dose range for future clinical studies. METHODS: This was a two-part randomized, placebo controlled study in healthy volunteers using a liquid formulation. Part I was a single ascending dose design with dose levels of 50-1200 mg (n = 6 active, two placebos per cohort); Part II was a multiple ascending dose design, with dose regimens ranging from 300 to 900 mg daily, either four times or twice daily for 10 days. Plasma pharmacokinetics, adverse events, vital signs, electrocardiograms and laboratory parameters were assessed. BTK occupancy in peripheral blood mononuclear cells was evaluated as a marker of target engagement. RESULTS: PRN1008 was rapidly absorbed following oral administration, and was safe and well tolerated in all dose regimens evaluated in both single and multiple doses. PRN1008 demonstrated a large volume of distribution, and a half-life of approximately 3-4 h. BTK occupancy of >90% was observed within 4 h after dosing in both single and multiple dose regimens, and was closely linked to maximum plasma concentration. BTK occupancy decay was slow (-1.6% h-1 ), and occupancy was sustained despite drug concentrations being undetectable. No severe or serious adverse events occurred, and the most common adverse events were gastrointestinal in nature. CONCLUSIONS: PRN1008 was safe and well-tolerated following oral administration, and achieved high, sustained levels of BTK occupancy in peripheral blood mononuclear cells.

Phase 1 clinical trial evaluating safety, exposure and pharmacodynamics of BTK inhibitor tolebrutinib (PRN2246, SAR442168).

Bruton's tyrosine kinase (BTK), expressed in B cells and cells of innate immunity, including microglia, is an essential signaling element downstream of the B-cell receptor and Fc-receptors. Tolebrutinib (PRN2246, SAR442168) is a potent BTK inhibitor that covalently binds the kinase, resulting in durable inhibition with the potential to target inflammation in the periphery and central nervous system (CNS). Tolebrutinib crosses the blood-brain barrier and potently inhibits BTK in microglial cells isolated from the CNS. A first-in-human randomized, double-blind, placebo-controlled study of tolebrutinib was conducted. The trial design consisted of five single ascending dose arms with oral administration of a single dose of 5, 15, 30, 60, and 120 mg (n = 6 per arm, n = 2 placebo), five multiple ascending dose arms with oral administration of 7.5, 15, 30, 60, and 90 mg (n = 8 per arm, n = 2 placebo) over 10 days, and one arm (n = 4) in which cerebral spinal fluid (CSF) exposure was measured 2 h after a single 120 mg dose. Tolebrutinib was well-tolerated in the study and all treatment-related treatment emergent adverse events were mild. Tolebrutinib was rapidly absorbed following oral administration with a rapid half-life of ~ 2 h. Peripheral BTK occupancy was assessed at various timepoints by an enzyme-linked immunosorbent assay-based readout using an irreversible probe. Assessments demonstrated extensive and prolonged peripheral BTK occupancy at steady-state with once daily doses as low as 7.5 mg. Further, CSF exposure was demonstrated 2 h after administration at 120 mg.

Endogenous purinergic control of bladder activity via presynaptic P2X3 and P2X2/3 receptors in the spinal cord.

P2X(3) and P2X(2/3) receptors are localized on sensory afferents both peripherally and centrally and have been implicated in various sensory functions. However, the physiological role of these receptors expressed presynaptically in the spinal cord in regulating sensory transmission remains to be elucidated. Here, a novel selective P2X(3) and P2X(2/3) antagonist, AF-792 [5-(5-ethynyl-2-isopropyl-4-methoxy-phenoxy)-pyrimidine-2,4-diamine, previously known as RO-5], in addition to less selective purinoceptor ligands, was applied intrathecally in vivo. Cystometry recordings were made to assess changes in the micturition reflex contractions after drug treatments. We found that AF-792 inhibited micturition reflex activity significantly (300 nmol; from baseline contraction intervals of 1.18 +/- 0.07 to 9.33 +/- 2.50 min). Furthermore, inhibition of P2X(3) and P2X(2/3) receptors in the spinal cord significantly attenuated spinal activation of extracellular-signal regulated kinases induced by acute peripheral stimulation of the bladder with 1% acetic acid by 46.4 +/- 12.0% on average. Hence, the data suggest that afferent signals originating from the bladder are regulated by spinal P2X(3) and P2X(2/3) receptors and establish directly an endogenous central presynaptic purinergic mechanism to regulate visceral sensory transmission. Identification of this spinal purinergic control in visceral activities may help the development of P2X(3) and P2X(2/3) antagonist to treat urological dysfunction, such as overactive bladder, and possibly other debilitating sensory disorders, including chronic pain states.

Systemic blockade of P2X3 and P2X2/3 receptors attenuates bone cancer pain behaviour in rats.

Pain remains an area of considerable unmet clinical need, and this is particularly true of pain associated with bone metastases, in part because existing analgesic drugs show only limited efficacy in many patients and in part because of the adverse side effects associated with these agents. An important issue is that the nature and roles of the algogens produced in bone that drive pain-signalling systems remain unknown. Here, we tested the hypothesis that adenosine triphosphate is one such key mediator through actions on P2X3 and P2X2/3 receptors, which are expressed selectively on primary afferent nocioceptors, including those innervating the bone. Using a well-established rat model of bone cancer pain, AF-353, a recently described potent and selective P2X3 and P2X2/3 receptor antagonist, was administered orally to rats and found to produce highly significant prevention and reversal of bone cancer pain behaviour. This attenuation occurred without apparent modification of the disease, since bone destruction induced by rat MRMT-1 carcinoma cells was not significantly altered by AF-353. Using in vivo electrophysiology, evidence for a central site of action was provided by dose-dependent reductions in electrical, mechanical and thermal stimuli-evoked dorsal horn neuronal hyperexcitability following direct AF-353 administration onto the spinal cord of bone cancer animals. A peripheral site of action was also suggested by studies on the extracellular release of adenosine triphosphate from MRMT-1 carcinoma cells. Moreover, elevated phosphorylated-extracellular signal-regulated kinase expression in dorsal root ganglion neurons, induced by co-cultured MRMT-1 carcinoma cells, was significantly reduced in the presence of AF-353. These data suggest that blockade of P2X3 and P2X2/3 receptors on both the peripheral and central terminals of nocioceptors contributes to analgesic efficacy in a model of bone cancer pain. Thus, systemic P2X3 and P2X2/3 receptor antagonists with central nervous system penetration may offer a promising therapeutic tool in treating bone cancer pain.

Overexpression of NGF in mouse urothelium leads to neuronal hyperinnervation, pelvic sensitivity, and changes in urinary bladder function.

NGF has been suggested to play a role in urinary bladder dysfunction by mediating inflammation, as well as morphological and functional changes, in sensory and sympathetic neurons innervating the urinary bladder. To further explore the role of NGF in bladder sensory function, we generated a transgenic mouse model of chronic NGF overexpression in the bladder using the urothelium-specific uroplakin II (UPII) promoter. NGF mRNA and protein were expressed at higher levels in the bladders of NGF-overexpressing (NGF-OE) transgenic mice compared with wild-type littermate controls from postnatal day 7 through 12-16 wk of age. Overexpression of NGF led to urinary bladder enlargement characterized by marked nerve fiber hyperplasia in the submucosa and detrusor smooth muscle and elevated numbers of tissue mast cells. There was a marked increase in the density of CGRP- and substance P-positive C-fiber sensory afferents, neurofilament 200-positive myelinated sensory afferents, and tyrosine hydroxylase-positive sympathetic nerve fibers in the suburothelial nerve plexus. CGRP-positive ganglia were also present in the urinary bladders of transgenic mice. Transgenic mice had reduced urinary bladder capacity and an increase in the number and amplitude of nonvoiding bladder contractions under baseline conditions in conscious open-voiding cystometry. These changes in urinary bladder function were further associated with an increased referred somatic pelvic hypersensitivity. Thus, chronic urothelial NGF overexpression in transgenic mice leads to neuronal proliferation, focal increases in urinary bladder mast cells, increased urinary bladder reflex activity, and pelvic hypersensitivity. NGF-overexpressing mice may, therefore, provide a useful transgenic model for exploring the role of NGF in urinary bladder dysfunction.

Selective pharmacological blockade of the TRPV1 receptor suppresses sensory reflexes of the rodent bladder.

PURPOSE: We investigated the pharmacological effect of TRPV1 antagonists in anesthetized rodent models of bladder function. MATERIALS AND METHODS: The TRPV1 antagonists JNJ17203212 and JYL1421 were evaluated in the anesthetized rat volume induced micturition reflex model. JNJ17203212 was further evaluated in this model in capsaicin (Sigma) desensitized rats, and in rat capsaicin and mouse citric acid models of irritant induced detrusor overactivity. RESULTS: Systemic JNJ17203212 and JYL1421 administration in the anesthetized rat volume induced micturition reflex model resulted in an increased micturition threshold volume. JNJ17203212 also decreased bladder contraction amplitude but JYL1421 had no effect. Capsaicin desensitization significantly increased baseline micturition threshold volume and decreased bladder contraction amplitude in the volume induced micturition reflex model compared to those in sham treated controls and JNJ17203212 produced no further effect after capsaicin desensitization. JNJ17203212 was also effective in 2 models of irritant induced detrusor overactivity, preventing the decrease in micturition threshold volume and the increase in bladder contraction amplitude observed with intravesical instillation of 10 microM capsaicin, and the decreased voiding interval induced by intravesical citric acid. CONCLUSIONS: The TRPV1 antagonists JNJ17203212 and JYL1421 increased the threshold for activation of the micturition reflex in the anesthetized rat volume induced micturition reflex model. This effect appeared to be mediated by capsaicin sensitive afferents. JNJ17203212 also inhibited detrusor overactivity induced by intravesical capsaicin and intravesical citric acid. These data extend our understanding of the role of TRPV1 in sensory modulation of the micturition reflex under nonirritant and inflammatory conditions.

The role of metabotropic glutamate receptor mGlu5 in control of micturition and bladder nociception.

In micturition control, the roles of ionotropic glutamate (iGlu) receptors NMDA and AMPA are well established, whereas little is known about the function of metabotropic glutamate (mGlu) receptors. Since antagonists for mGlu5 receptors are efficacious in animal models of inflammatory and neuropathic pain, we examined whether mGlu5 receptors play a role in the voiding reflex and bladder nociception and, if so, via centrally or peripherally localized receptors. The mGlu5 receptor antagonist MPEP dose-dependently increased the micturition threshold (MT) volume in the volume-induced micturition reflex (VIMR) model in anesthetized rats. Following doses of 5.2, 15.5 and 51.7micromol/kg of MPEP (intraduodenal), the MT was increased by 24.7+/-5.0%, 97.2+/-12.5% (P<0.01) and 128.0+/-28.3% (P<0.01) from the baseline, respectively (n=4-5; compared with 0.8+/-9.1% in the vehicle group). Infusing MPEP (0.3, 1mM) directly into the bladder also raised MT. However, the efficacious plasma concentrations of MPEP following intravesical dosing were similar to that after intraduodenal dosing (EC(50) of 0.11 and 0.27microM, respectively, P>0.05). MPEP also dose-dependently attenuated the visceromotor responses (VMR, total number of abdominal EMG spikes during phasic bladder distension) in anesthetized rats. The VMR was decreased to 1332.4+/-353.9 from control of 2886.5+/-692.2 spikes/distension (n=6, P<0.01) following MPEP (10micromol/kg, iv). Utilizing the isolated mouse bladder/pelvic nerve preparation, we found that neither MPEP (up to 3microM) nor MTEP (up to 10microM) affected afferent discharge in response to bladder distension (n=4-6). In contrast, MPEP attenuated the responses of the mesenteric nerves to distension of the mouse jejunum in vitro. These data suggest that mGlu5 receptors play facilitatory roles in the processing of afferent input from the urinary bladder, and that central rather than peripheral mGlu5 receptors appear to be responsible.

Effects of the selective prostacyclin receptor antagonist RO3244019 on the micturition reflex in rats.

PURPOSE: We investigated the role of prostacyclin on afferent modulation of the micturition reflex using the novel selective prostacyclin receptor antagonist RO3244019 in rat models of bladder function. MATERIALS AND METHODS: The effects of RO3244019 on urodynamic parameters were evaluated in 3 rat models. In the anesthetized isovolumetric bladder contraction and the volume induced micturition reflex (Refill) models the effects of RO3244019 and chronic capsaicin desensitization were compared. In the citric acid induced detrusor overactivity model the effects of RO3244019 and the cyclooxygenase inhibitor indomethacin were evaluated. RESULTS: In the isovolumetric bladder contraction model RO3244019 dose dependently decreased bladder contraction frequency with a mean +/- SEM maximum decrease of 72.2% +/- 4.3% at 3.16 mg/kg. RO3244019 also dose dependently increased the micturition threshold in the Refill model with a maximum increase of 86.9% +/- 19.1% at 3.0 mg/kg. In animals that were chronically treated with capsaicin bladder contraction frequency was decreased by 88.9% in the isovolumetric bladder contraction model and micturition threshold was increased by 68.1% in the Refill model relative to sham treated rats. RO3244019 (3.0 mg/kg) further increased the micturition threshold in capsaicin treated animals by 53.7% +/- 18.1% from baseline. In the citric acid induced detrusor overactivity model citric acid decreased the voiding interval to 28.5% of baseline. This effect was reversed by RO3244019 (73.0% +/- 6.4%) and indomethacin (97.7% +/- 5.5%) at 3.0 mg/kg compared to vehicle (55.0% +/- 4.1%). CONCLUSIONS: The prostacyclin receptor antagonist RO3244019 decreased bladder contraction frequency and increased micturition threshold in the anesthetized isovolumetric bladder contraction and Refill models, respectively, and increased the micturition voiding interval in the conscious citric acid induced detrusor overactivity model. Additionally, RO3244019 remained effective for increasing the micturition threshold in the Refill model even following chronic capsaicin desensitization. Taken together these data suggest that prostacyclin may have a facilitory role in the micturition reflex by modulating the threshold for activation of capsaicin sensitive and insensitive bladder sensory afferents.

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 alpha(1)-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 P2X(1) 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 P2X(3) and P2X(2/3) 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.

An investigation of the effects of zaprinast, a PDE inhibitor, on the nitrergic control of the urethra in anaesthetized female rats.

1. The effects of L-NAME and zaprinast were investigated (i.v.) on reflex-evoked changes in bladder and urethral pressures in urethane-anaesthetized female rats. 2. L-NAME attenuated reflex-evoked urethral relaxations (65+/-10%), while zaprinast potentiated these responses (68+/-24%). L-NAME and zaprinast also increased baseline urethral pressure and urethral striated muscle (EUS-EMG) activity. These drugs had little effect on the bladder. 3. Following pre-treatment with alpha-bungarotoxin (i.v.) to block urethral striated muscle, L-NAME and zaprinast failed to increase baseline urethral pressure. Further zaprinast failed to alter the size of reflex-evoked urethral relaxations. 4. Intra-urethral zaprinast caused a significant increase while sodium nitroprusside (SNP) and isoprenaline caused decreases in urethral pressure (+14+/-3%, -25+/-5%, -29+/-7%, respectively). These changes were associated with increases in EUS-EMG activity. After chlorisondamine (i.v.), zaprinast caused a significant fall in urethral pressure, while the decrease in urethral pressure caused by SNP and isoprenaline was potentiated. No changes in EUS-EMG activity occurred. 5. These results indicate that a nitrergic pathway mediates reflex-evoked urethral smooth muscle relaxations. The data also indicates that there is a background release of NO, which reduces sphincter skeletal muscle activity. Further, the ability of zaprinast to potentiate nitrergic evoked urethral relaxations involves an increase in striated muscle tone. This appears to be an indirect result of smooth muscle relaxation and is mediated, at least in part, by a chlorisondamine-sensitive mechanism.

Characterization of a novel CRAC inhibitor that potently blocks human T cell activation and effector functions.

Store operated calcium entry (SOCE) downstream of T cell receptor (TCR) activation in T lymphocytes has been shown to be mediated mainly through the Calcium Release Activated Calcium (CRAC) channel. Here, we compared the effects of a novel, potent and selective CRAC current inhibitor, 2,6-Difluoro-N-{5-[4-methyl-1-(5-methyl-thiazol-2-yl)-1,2,5,6-tetrahydro-pyridin-3-yl]-pyrazin-2-yl}-benzamide (RO2959), on T cell effector functions with that of a previously reported CRAC channel inhibitor, YM-58483, and a calcineurin inhibitor Cyclosporin A (CsA). Using both electrophysiological and calcium-based fluorescence measurements, we showed that RO2959 is a potent SOCE inhibitor that blocked an IP3-dependent current in CRAC-expressing RBL-2H3 cells and CHO cells stably expressing human Orai1 and Stim1, as well as SOCE in human primary CD4(+) T cells triggered by either TCR stimulation or thapsigargin treatment. Furthermore, we demonstrated that RO2959 completely inhibited cytokine production as well as T cell proliferation mediated by TCR stimulation or MLR (mixed lymphocyte reaction). Lastly, we showed by gene expression array analysis that RO2959 potently blocked TCR triggered gene expression and T cell functional pathways similar to CsA and another calcineurin inhibitor FK506. Thus, both from a functional and transcriptional level, our data provide evidence that RO2959 is a novel and selective CRAC current inhibitor that potently inhibits human T cell functions.