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Four antibodies that inhibit interleukin (IL)-23 are approved for the treatment of moderate-to-severe plaque psoriasis. Here, we present non-clinical data comparing ustekinumab, guselkumab, tildrakizumab and risankizumab with regard to thermostability, IL-23 binding affinity, inhibitory-binding mode, in vitro potency and in vivo efficacy. Risankizumab and guselkumab exhibited 5-fold higher affinity for IL-23 and showed more potent inhibition of IL-23 signaling than ustekinumab and tildrakizumab. Risankizumab and guselkumab completely blocked the binding of IL-23 to IL-23Rα as expected, whereas tildrakizumab did not. In vitro, risankizumab and guselkumab blocked the terminal differentiation of TH17 cells in a similar manner, while tildrakizumab had minimal impact on TH17 differentiation. In a human IL-23-induced ear-swelling mouse model, risankizumab and guselkumab were more effective than ustekinumab and tildrakizumab at reducing IL-17, IL-22, and keratinocyte gene expression. Our results indicate that the four clinically approved antibodies targeting IL-23 differ in affinity and binding epitope. These attributes contribute to differences in in vitro potency, receptor interaction inhibition mode and in vivo efficacy in preclinical studies as described in this report, and similarly may affect the clinical performance of these drugs.

Background:Losartan, a blocker of the angiotensin II type I receptor, is an important part of the standard of care for diabetic nephropathy (DN). The obese ZSF-1 rats display many aspects of the clinical features of human Type II DN. The current study was designed to examine the treatment effects of losartan on obese ZSF-1 rats and to evaluate the impact of the onset of dosing on efficacy.Methods:The rats (7–10 weeks) underwent a right uninephrectomy (Unx) or sham surgery. Losartan (3, 10, 30 mg/kg) was dosed 3 or 9 weeks post-Unx and continued for 12 weeks.Results:Treatment with losartan reduced urinary protein excretion and blood lipids (triglyceride and cholesterol) dose-dependently in both studies. The glomerular filtration rate (GFR) was significantly lower in obese ZSF-1 rats compared with those in lean rats, and losartan was efficacious against this endpoint, in particular with the earlier onset of treatment. Losartan also decreased tubulointerstitial fibrosis, and similar to GFR, earlier treatment conferred beneficial actions even at the lowest dose of 3 mg/kg. Several urinary biomarkers were elevated in the obese ZSF-1 rats, but the levels of sTNFR1, TIMP-1, L-FABP and KIM-1 were the only markers decreased by losartan.Conclusions:Losartan was renoprotective in the ZSF-1 rats with DN, improving both the pathological and functional parameters of the disease. Importantly, the data also highlight the importance of treatment at earlier stages of the disease for protecting against decline in the GFR and the development of fibrosis.

Introduction The adenine model of kidney disease typically involves dietary delivery of adenine over several weeks. This model can be variable in its disease progression and can result in significant mortality. In the current study, the amount of adenine delivered to rats was controlled by utilizing oral gavage administration over a short period in an attempt to induce robust renal pathology while addressing variability and viability of the animals. Methods Adenine (150 or 200 mg/kg) was administered via oral gavage for 10 consecutive days, and assessed over a total of 20 days. Results Both adenine dose groups manifested pathophysiological features of kidney disease such as proteinuria, elevated serum creatinine and BUN, and tubulointerstitial fibrosis. The animals also displayed a decline in glomerular filtration rate. Renal mRNA expression of genes associated with injury, inflammation, and fibrosis (i.e., Col1a1, Acta2, Serpine1, Timp1, Fn-Eda, Tgfb1, Ccl2, Nlrp3, Aqp1 and Ccnd1) were elevated as were urinary biomarkers that have translational utility (i.e., clusterin, KIM-1, MCP-1, OPN, NGAL, B2M, calbindin, and cystatin C). All disease endpoints were more pronounced in the 200 mg/kg group, however, while measures of tissue fibrosis were sustained, there was partial recovery by day 20 in functional readouts. No mortality was observed in either dose group. Discussion Short-term delivery of adenine via precise gavage delivery induced a robust model with hallmarks of fibrotic kidney disease, had limited variance between animals, and no animal morbidity within the 20 days studied. This model represents a methodical alternative to long-term dietary dosing of adenine.

Background: Obese ZSF-1 rats display many features of human type II diabetes including nephropathy (DN). The study aimed to further understand the relevance of this model to DN, for which glomerular filtration rate (GFR), renal fibrosis and several urinary/tissue biomarkers was followed over 24 weeks in ZSF-1 rats. Methods: Intact/sham or uninephrectomized male and female ZSF-1 rats were studied. GFR was measured by transdermal clearance of fluorescein isothiocyanate-sinistrin. Urine was collected every 2-4 weeks for biomarker analysis. Renal tissue was examined histologically for fibrosis and for levels of inflammatory and fibrotic genes. Results: Male obese ZSF-1 rats demonstrated metabolic syndrome and proteinuria. Female counterparts were hyperlipidemic with delayed proteinuria, but were not hyperglycemic. Kidney hyperfiltration was observed in male obese rats in weeks 2-4 after surgery, and subsequently declined to levels significantly lower than controls. Tubulointerstitial/glomerular fibrosis in male obese rats was significantly elevated by week 12 post surgery and continued to expand in the ensuing weeks, particularly in uninephrectomized rats. Female rats had less severe fibrosis. Except for epidermal growth factor which decreased, the levels of several key inflammatory, injury and fibrotic factors were elevated in both tissue (mRNA) and urine (protein) of male obese rats. Conclusion: Male obese ZSF-1 rats represent an important DN model, manifesting key pathophysiological features including metabolic syndrome, proteinuria, progressive tubular and glomerular fibrosis, and transient hyperfiltration followed by progressive decline in renal function. Uninephrectomy significantly accelerated disease progression. Females were less severe in disease manifestation. Several urinary and tissue biomarkers were identified in the male obese rats that tracked with disease progression.

ATP-sensitive P2X7 receptors are localized on cells of immunological origin including peripheral macrophages and glial cells in the CNS. Activation of P2X7 receptors leads to rapid changes in intracellular calcium concentrations, release of the pro-inflammatory cytokine IL-1beta, and following prolonged agonist exposure, the formation of cytolytic pores in plasma membranes. Data from gene knockout studies and recently described selective antagonists indicate a role for P2X7 receptor activation in inflammation and pain. While several species selective P2X7 antagonists exist, A-804598 represents a structurally novel, competitive, and selective antagonist that has equivalent high affinity at rat (IC50 = 10 nM), mouse (IC50 = 9 nM) and human (IC50 = 11 nM) P2X7 receptors. A-804598 also potently blocked agonist stimulated release of IL-1beta and Yo-Pro uptake from differentiated THP-1 cells that natively express human P2X7 receptors. A-804598 was tritiated ([3H]A-804598; 8.1Ci/mmol) and utilized to study recombinant rat P2X7 receptors expressed in 1321N1 cells. [3H]A-804598 labeled a single class of high affinity binding sites (Kd=2.4 nM and apparent Bmax=0.56 pmol/mg). No specific binding was observed in untransfected 1321N1 cells. The pharmacological profile for P2X antagonists to inhibit [3H]A-804598 binding correlated with their ability to block functional activation of P2X7 receptors (r=0.95, P

N'-aryl acyl hydrazides were identified as P2X7 receptor antagonists. Structure-activity relationship (SAR) studies evaluated functional activity by monitoring calcium flux inhibition in cell lines expressing recombinant human and rat P2X7 receptors. Selected analogs were assayed in vitro for their capacity to inhibit release of cytokine IL-1beta. Compounds with potent antagonist function were evaluated in vivo using the zymosan-induced peritonitis model. A representative compound effectively attenuated mechanical allodynia in a rat model of neuropathic pain.

The goal of this study was to identify a structurally distinct D(4)-selective agonist with superior oral bioavailability to our first-generation clinical candidate 1a (ABT-724) for the potential treatment of erectile dysfunction. Arylpiperazines such as (heteroarylmethyl)piperazine 1a, benzamide 2, and acetamides such as 3a,b exhibit poor oral bioavailability. Structure-activity relationship (SAR) studies with the arylpiperidine template provided potent partial agonists such as 4d and 5k that demonstrated no improvement in oral bioavailability. Further optimization with the (N-oxy-2-pyridinyl)piperidine template led to the discovery of compound 6b (ABT-670), which exhibited excellent oral bioavailability in rat, dog, and monkey (68%, 85%, and 91%, respectively) with comparable efficacy, safety, and tolerability to 1a. The N-oxy-2-pyridinyl moiety not only provided the structural motif required for agonist function but also reduced metabolism rates. The SAR study leading to the discovery of 6b is described herein.

The first selective dopamine D4 agonist radioligand is described. The synthesis of these piperazine radioligands relied on the transformation of brominated precursors 4a and 4b with tritium gas in the presence of a sensitive cyano functional group. The specific activity of these two radioligands was measured and [3H]6b found to be suitable for use in D4 saturation and competition binding studies. The synthesis, biological, and radioactivity of this new agonist radioligand as well as preliminary SAR will be discussed.

The goal of this study was to develop a new approach to study the pharmacology of the dopamine D 4 receptor that could be used in comparative studies with dopamine D 2 and D 3 receptors. Stable HEK-293 cell lines co-expressing recombinant human D 2L , D 3 or D 4 receptors along with Gα qo5 cDNA were prepared. Dopamine induced a robust, transient calcium signal in these cell lines with EC 50 s for D 2L , D 3 and D 4 of 18.0, 11.9 and 2.2 nM, respectively. Reported D 4 -selective agonists CP226269 and PD168077 were potent, partial D 4 agonists exhibiting 31–1700-fold selectivity for D 4 over D 3 or D 2 . Non-selective D 2 -like agonists apomorphine and quinpirole showed full efficacy but did not discriminate across the three receptors. D 3 -selective agonists 7-hydroxy-DPAT and PD128907 were potent but non-selective D 2 -like agonists. The reported D 3 partial agonist BP-897 exhibited minimal agonist activity at D 3 but was a potent D 3 antagonist and a partial D 4 agonist. Other D 2 -like antagonists, haloperidol, clozapine, and domperidone showed concentration-dependent inhibition of dopamine responses at all three receptors with K i ranging from 0.05 to 48.3 nM. The D 3 selective antagonist S33084 and D 4 -selective antagonist L-745870 were highly selective for D 3 and D 4 receptors with K b of 0.7 and 0.1 nM, respectively. Stable co-expression of D 2 -like receptors with chimeric Gα qo5 proteins in HEK-293 cells is an efficient method to study receptor activation in a common cellular background and an efficient method for direct comparison of ligand affinity and efficacy across human D 2L , D 3 and D 4 receptors.

Nonsteriodal anti-inflammatory drugs (NSAIDs) are efficacious for the treatment of pain associated with inflammatory disease. Clinical experience with marketed selective cyclooxygenase-2 (COX-2) inhibitors (celecoxib, rofecoxib, and valdecoxib) has confirmed the utility of these agents in the treatment of inflammatory pain with an improved gastrointestinal safety profile relative to NSAID comparators. These COX-2 inhibitors belong to the same structural class. Each contains a core heterocyclic ring with two appropriately substituted phenyl rings appended to adjacent atoms. Here, we report the identification of vicinally disubstituted pyridazinones as potent and selective COX-2 inhibitors. The lead compound in the series, ABT-963 [2-(3,4-difluoro-phenyl)-4-(3-hydroxy-3-methyl-butoxy)-5-(4-methanesulfonyl-phenyl)-2H-pyridazin-3-one], has excellent selectivity (ratio of 276, COX-2/COX-1) in human whole blood, improved aqueous solubility compared with celecoxib and rofecoxib, high oral anti-inflammatory potency in vivo, and gastric safety in the animal studies. After oral administration, ABT-963 reduced prostaglandin E2 production in the rat carrageenan air pouch model (ED50 of 0.4 mg/kg) and reduced the edema in the carrageenan induced paw edema model with an ED30 of 1.9 mg/kg. ABT-963 dose dependently reduced nociception in the carrageenan hyperalgesia model (ED50 of 3.1 mg/kg). After 14 days of dosing in the adjuvant arthritis model, ABT-963 had an ED(50) of 1.0 mg/kg in reducing the swelling of the hind paws. Magnetic resonance imaging examination of the diseased paws in the adjuvant model showed that ABT-963 significantly reduced bone loss and soft tissue destruction. ABT-963 is a highly selective COX-2 inhibitor that may have utility in the treatment of the pain and inflammation associated with arthritis.

Pharmacological activation of neuronal nicotinic acetylcholine receptors can produce non-opioid antinociception in rodents. However, multiple nAChR subtypes exist, the most abundant of which contain alpha4 and beta2 subunits. The purpose of the present study was to investigate the role of alpha4-containing nAChRs in mediating nicotinic antinociception using an in vivo antisense strategy. Both i.c.v. infusion and repeated bolus injections into the cerebral aqueduct of an antisense oligonucleotide against the alpha4 subunit significantly attenuated the antinociceptive effects of the nAChR agonist A-85380 in the paw withdrawal test of acute thermal pain. Rats treated with a scrambled oligonucleotide displayed a full antinociceptive response to A-85380, while discontinuing antisense treatment restored the antinociceptive effects of the nicotinic agonist. Double immunohistochemical labeling revealed near-complete overlap of expression of the serotonin marker tryptophan hydroxylase and the alpha4 nAChR subunit in the dorsal raphe nucleus. The expression of alpha4-containing nAChRs by serotonergic neurons in the dorsal raphe offered a means to address nonspecific alpha4 knock-down, i.e., oligonucleotide-induced neurotoxicity. Immunohistochemical detection of alpha4 expression was reduced by nearly 50% in the dorsal raphe of antisense-treated rats as compared to either saline or missense-treated controls. In contrast, the expression of tryptophan hydroxylase, as well as, the alpha7 nAChR subunit in antisense-infused rats was similar to that observed in saline- and missense-treated controls. The results of these studies suggest that alpha4-containing nAChRs, possibly expressed by serotonergic neurons, are involved in nicotinic-mediated analgesia. However, these data do not eliminate the possibility that other nicotinic subunit combinations may also play a role in antinociception produced by nAChR activation.

Growing evidence supports a role for the immune system in the induction and maintenance of chronic pain. ATP is a key neurotransmitter in this process. Recent studies demonstrate that the glial ATP receptor, P2X7, contributes to the modulation of pathological pain. To further delineate the endogenous mechanisms that are involved in P2X7-related antinociception, we utilized a selective P2X7 receptor antagonist, A-438079, in a series of in vivo and in vitro experiments. Injection of A-438079 (10-300 micromol/kg, i.p.) was anti-allodynic in three different rat models of neuropathic pain and it attenuated formalin-induced nocifensive behaviors. Using in vivo electrophysiology, A-438079 (80 micromol/kg, i.v.) reduced noxious and innocuous evoked activity of different classes of spinal neurons (low threshold, nociceptive specific, wide dynamic range) in neuropathic rats. The effects of A-438079 on evoked firing were diminished or absent in sham rats. Spontaneous activity of all classes of spinal neurons was also significantly reduced by A-438079 in neuropathic but not sham rats. In vitro, A-438079 (1 microM) blocked agonist-induced (2,3-O-(4-benzoylbenzoyl)-ATP, 30 microM) current in non-neuronal cells taken from the vicinity of the dorsal root ganglia. Furthermore, A-438079 dose-dependently (0.3-3 microM) decreased the quantity of the cytokine, interleukin-1beta, released from peripheral macrophages. Thus, ATP, acting through the P2X7 receptor, exerts a wide-ranging influence on spinal neuronal activity following a chronic injury. Antagonism of the P2X7 receptor can in turn modulate central sensitization and produce antinociception in animal models of pathological pain. These effects are likely mediated through immuno-neural interactions that affect the release of endogenous cytokines.

ATP-sensitive P2X(7) receptors are localized on cells of immunological origin including glial cells in the central nervous system. Activation of P2X(7) receptors leads to rapid changes in intracellular calcium concentrations, release of the proinflammatory cytokine interleukin-1beta (IL-1beta), and following prolonged agonist exposure, cytolytic plasma membrane pore formation. P2X(7) knockout mice show reduced inflammation as well as decreased nociceptive sensitivity following peripheral nerve injury. A-740003 (N-(1-{[(cyanoimino)(5-quinolinylamino) methyl] amino}-2,2-dimethylpropyl)-2-(3,4-dimethoxyphenyl)acetamide) is a novel competitive antagonist of P2X(7) receptors (IC(50) values = 40 nM for human and 18 nM for rat) as measured by agonist-stimulated changes in intracellular calcium concentrations. A-740003 showed weak or no activity (IC(50) > 10 muM) at other P2 receptors and an array of other neurotransmitter and peptide receptors, ion channels, reuptake sites, and enzymes. A-740003 potently blocked agonist-evoked IL-1beta release (IC(50) = 156 nM) and pore formation (IC(50) = 92 nM) in differentiated human THP-1 cells. Systemic administration of A-740003 produced dose-dependent antinociception in a spinal nerve ligation model (ED(50) = 19 mg/kg i.p.) in the rat. A-740003 also attenuated tactile allodynia in two other models of neuropathic pain, chronic constriction injury of the sciatic nerve and vincristine-induced neuropathy. In addition, A-740003 effectively reduced thermal hyperalgesia observed following intraplantar administration of carrageenan or complete Freund's adjuvant (ED(50) = 38-54 mg/kg i.p.). A-740003 was ineffective in attenuating acute thermal nociception in normal rats and did not alter motor performance at analgesic doses. These data demonstrate that selective blockade of P2X(7) receptors in vivo produces significant antinociception in animal models of neuropathic and inflammatory pain.

A new series of dopamine D4 receptor agonists, 1-aryl-3-(4-pyridinepiperazin-1-yl)propanone oximes, was designed through the modification of known dopamine D4 receptor agonist PD 168077. Replacement of the amide group with a methylene-oxime moiety produced compounds with improved stability and efficacy. Structure-activity relationsips (SAR) of the aromatic ring linked to the N-4-piperazine ring confirmed the superiority of 2-pyridine as a core for D4 agonist activity. A two-methylene linker between the oxime group and the N-1-piperazine ring displayed the best profile. New dopamine D4 receptor agonists, exemplified by (E)-1-(4-chlorophenyl)-3-(4-pyridin-2-ylpiperazin-1-yl)propan-1-one O-methyloxime (59a) and (E)-1-(3-chloro-4-fluorophenyl)-3-(4-pyridin-2-ylpiperazin-1-yl)propan-1-one O-methyloxime (64a), exhibited favorable pharmacokinetic profiles and showed oral bioavailability in rat and dog. Subsequent evaluation of 59a in the rat penile erection model revealed in vivo activity, comparable in efficacy to apomorphine. Our results suggest that the oximes provide a novel structural linker for 4-arylpiperazine-based D4 agonists, possessing leadlike quality and with potential to develop a new class of potent and selective dopamine D4 receptor agonists.

2-[4-(3,4-Dimethylphenlyl)piperazin-1-ylmethyl]-1H benzoimidazole (A-381393) was identified as a potent dopamine D4 receptor antagonist with excellent receptor selectivity. [3H]-spiperone competition binding assays showed that A-381393 potently bound to membrane from cells expressing recombinant human dopamine D4.4 receptor (Ki=1.5 nM), which was 20-fold higher than that of clozapine (Ki=30.4 nM). A-381393 exhibited highly selective binding for the dopamine D4.4 receptor (>2700-fold) when compared to D1, D2, D3 and D5 dopamine receptors. Furthermore, in comparison to clozapine and L-745870, A-381393 exhibits better receptor selectivity, showing no affinity up to 10 microM for a panel of more than 70 receptors and channels, with the exception of moderate affinity for 5-HT2A (Ki=370 nM). A-381393 potently inhibited the functional activity of agonist-induced GTP-gamma-S binding assay and 1 microM dopamine induced-Ca2+ flux in human dopamine D4.4 receptor expressing cells, but not in human dopamine D2L or D3 receptor cells. In contrast to L-745870, A-381393 did not exhibit any significant intrinsic activity in a D4.4 receptor. In vivo, A-381393 has good brain penetration after subcutaneous administration. A-381393 inhibited penile erection induced by the selective D4 agonist PD168077 in conscious rats. Thus, A-381393 is a novel selective D4 antagonist that will enhance the ability to study dopamine D4 receptors both in vitro and in vivo.

A new class of agents with potential utility for the treatment of erectile dysfunction has been discovered, guided by the hypothesis that selective D4 agonists are erectogenic but devoid of the side effects typically associated with dopaminergic agents. The lead agent 2-(4-pyridin-2-ylpiperazin-1-ylmethyl)-1H-benzimidazole (1, ABT-724) was discovered by optimization of a series of benzimidazole arylpiperazines. This highly selective D4 agonist was found to be very potent and efficacious in vivo, eliciting penile erections in rats at a dose of 0.03 micromol/kg, with a positive response rate of 77% erectile incidence. Even at high doses, it was devoid of side effects in animal models of central nervous system behaviors, emesis, or nausea. The structure-activity relationship of the parent benzimidazole series leading to 1 is described, with the detailed in vitro and in vivo profiles described. Distinctive structural features were discovered that are associated with D4 selective agonism in this series of analogues.

A series of subtype selective dopamine D(4) receptor ligands from the hetroarylmethylphenylpiperazine class have been discovered that exhibit a remarkable structure-activity relationship (SAR), revealing a substituent effect in which regiosubstitution on the terminal arylpiperazine ring can modulate functional or intrinsic activity. Other structure-dependent efficacy studies in the dopamine D(4) field have suggested a critical interaction of the heteroarylmethyl moiety with specific protein microdomains in controlling intrinsic activity. Our studies indicate that for some binding orientations, the phenylpiperazine moiety also plays a key role in determining efficacy. These data also implicate a kinetic or efficiency term, contained within measured functional affinities for agonists, which support a sequential binding and conformational stabilization model for receptor activation. The structural similarity between partial agonist and antagonist, within this subset of ligands, and lack of bioisosterism for this substituent effect are key phenomena for these hypotheses.

Diaryl piperazine acetamides were identified as potent and selective dopamine D(4) receptor agonists. Our strategy is based on an amide bond reversal of an acid sensitive, dopamine D(4) receptor partial agonist, PD 168077. This reversal provided compounds with excellent potency and improved stability. Systematic evaluation of the substitution on the aryl piperazine portion revealed a significant effect on functional activity. The synthesis and biological activity of these new dopamine D(4) agonists is discussed.

A model of lung inflammation was developed in Brown Norway rats. Intense lung eosinophilia was induced by a single intravenous injection of Sephadex G-200 particles. The eosinophilia observed was preceded by an increase in cysteinyl leukotrienes found in lung lavage fluids. Theophylline and albuterol were tested in the model and found to be inactive, while dexamethasone was effective. Zileuton, a specific leukotriene inhibitor, was found to effectively inhibit leukotriene formation and the influx of eosinophils into the lungs of these Sephadex-treated animals. Studies with specific leukotriene D4 antagonists of the cysLT1 type receptor indicate that this leukotriene receptor is probably not involved directly in the eosinophilic inflammation. This model appears to be useful in characterizing potential anti-inflammatory effects of inhibitors by evaluating their ability to prevent eosinophil influx into the lung.