Jeffrey B. Madwed

Anti-Inflammatory Effects of a p38 Mitogen-Activated Protein Kinase Inhibitor During Human Endotoxemia

The p38 mitogen-activated protein kinase (MAPK) participates in intracellular signaling cascades resulting in inflammatory responses. Therefore, inhibition of the p38 MAPK pathway may form the basis of a new strategy for treatment of inflammatory diseases. However, p38 MAPK activation during systemic inflammation in humans has not yet been shown, and its functional significance in vivo remains unclear. Hence, we exposed 24 healthy male subjects to an i.v. dose of LPS (4 ng/kg), preceded 3 h earlier by orally administered 600 or 50 mg BIRB 796 BS (an in vitro p38 MAPK inhibitor) or placebo. Both doses of BIRB 796 BS significantly inhibited LPS-induced p38 MAPK activation in the leukocyte fraction of the volunteers. Cytokine production (TNF-α, IL-6, IL-10, and IL-1R antagonist) was strongly inhibited by both low and high dose p38 MAPK inhibitor. In addition, p38 MAPK inhibition diminished leukocyte responses, including neutrophilia, release of elastase-α1-antitrypsin complexes, and up-regulation of CD11b with down-regulation of L-selectin. Finally, blocking p38 MAPK decreased C-reactive protein release. These data identify p38 MAPK as a principal mediator of the inflammatory response to LPS in humans. Furthermore, the anti-inflammatory potential of an oral p38 MAPK inhibitor in humans in vivo suggests that p38 MAPK inhibitors may provide a new therapeutic option in the treatment of inflammatory diseases.

NOVEL CROSS-TALK WITHIN THE IKK FAMILY CONTROLS INNATE IMMUNITY

Members of the IKK {IκB [inhibitor of NF-κB (nuclear factor κB)] kinase} family play a central role in innate immunity by inducing NF-κB- and IRF [IFN (interferon) regulatory factor]-dependent gene transcription programmes required for the production of pro-inflammatory cytokines and IFNs. However, the molecular mechanisms that activate these protein kinases and their complement of physiological substrates remain poorly defined. Using MRT67307, a novel inhibitor of IKKϵ/TBK1 (TANK {TRAF [TNF (tumour-necrosis-factor)-receptor-associated factor]-associated NF-κB activator}-binding kinase 1) and BI605906, a novel inhibitor of IKKβ, we demonstrate that two different signalling pathways participate in the activation of the IKK-related protein kinases by ligands that activate the IL-1 (interleukin-1), TLR (Toll-like receptor) 3 and TLR4 receptors. One signalling pathway is mediated by the canonical IKKs, which directly phosphorylate and activate IKKϵ and TBK1, whereas the second pathway appears to culminate in the autocatalytic activation of the IKK-related kinases. In contrast, the TNFα-induced activation of the IKK-related kinases is mediated solely by the canonical IKKs. In turn, the IKK-related kinases phosphorylate the catalytic subunits of the canonical IKKs and their regulatory subunit NEMO (NF-κB essential modulator), which is associated with reduced IKKα/β activity and NF-κB-dependent gene transcription. We also show that the canonical IKKs and the IKK-related kinases not only have unique physiological substrates, such as IκBα, p105, RelA (IKKα and IKKβ) and IRF3 (IKKϵ and TBK1), but also have several substrates in common, including the catalytic and regulatory (NEMO and TANK) subunits of the IKKs themselves. Taken together, our studies reveal that the canonical IKKs and the IKK-related kinases regulate each other by an intricate network involving phosphorylation of their catalytic and regulatory (NEMO and TANK) subunits to balance their activities during innate immunity.

Collagen-Induced Arthritis is Exacerbated in C-Reactive Protein Deficient Mice

OBJECTIVE Blood C-reactive protein (CRP) is routinely measured to gauge inflammation. In rheumatoid arthritis (RA), a heightened CRP level is predictive of a poor outcome, while a lowered CRP level is indicative of a positive response to therapy. CRP interacts with the innate and adaptive immune systems in ways that suggest it may be causal in RA and, although this is not proven, it is widely assumed that CRP makes a detrimental contribution to the disease process. Paradoxically, results from animal studies have indicated that CRP might be beneficial in RA. This study was undertaken to study the role of CRP in a mouse model of RA, the collagen-induced arthritis (CIA) model. METHODS We compared the impact of CRP deficiency with that of transgenic overexpression of CRP on inflammatory and immune responses in mice, using CRP-deficient (Crp-/-) and human CRP-transgenic (CRP-Tg) mice, respectively. Susceptibility to CIA, a disease that resembles RA in humans, was compared between wild-type, Crp-/-, and CRP-Tg mice. RESULTS CRP deficiency significantly altered the inflammatory cytokine response evoked by challenge with endotoxin or anti-CD3 antibody, and heightened some immune responses. Compared to that in wild-type mice, CIA in Crp-/- mice progressed more rapidly and was more severe, whereas CIA in CRP-Tg mice was dramatically attenuated. Despite these disparate clinical outcomes, anticollagen autoantibody responses during CIA did not differ among the genotypes. CONCLUSION CRP exerts an early and beneficial effect in mice with CIA. The mechanism of this effect remains unknown but does not involve improvement of the autoantibody profile. In humans, the presumed detrimental role of a heightened blood CRP level during active RA might be balanced by a beneficial effect of the baseline CRP (i.e., levels manifest during the preclinical stages of disease).

Anti-LFA-1α reduces the dose of cyclosporin A needed to produce immunosuppression in heterotopic cardiac transplanted rats

BACKGROUND Monoclonal antibodies (MAb) against cell adhesion molecules prolong the time to acute rejection of transplanted organs in animals. Postulated mechanisms of action include blockade of trafficking of host leukocytes into or recognizing of effector/target cells within the allograft. We examined whether an anti-ICAM-1 (1A29), anti-LFA-1 alpha (WT.1), or anti-CD-18 (WT.3) could reduce the immunosuppressive dose of cyclosporin A (CsA) when used in combination. METHODS A rat heterotopic cardiac transplant model with ACI donors and Lewis recipients was used. MAb dose was 3 mg/kg, i.p. with treatment on Days -3 and -1 prior to transplant, followed by daily dosing for 10 days post-transplantation (Tx). Cyclosporin A doses were either 1.5 or 2.75 mg/kg, PO beginning the day of and for 10 days post-Tx. RESULTS Untreated allografted rats demonstrated a mean rejection time (MRT) +/- SEM of 8.8 +/- 0.6 days. Cyclosporin A at 1.5 and 2.75 mg/kg showed mean rejection times of 8.5 +/- 0.3 (NS) and 20.5 +/- 1.9 (p < 0.05) days, respectively. Monotherapy with 1A29 or WT.3 did not prolong MRT, whereas WT.1 increased MRT to 21.7 +/- 4.3 days (p < 0.05). MAb combination therapy did not extend MRT greater than that demonstrated by WT.1 alone. However, MAb and CsA combination therapy significantly increased MRT with WT.1 and CsA resulting in the greatest extension. WT.1 combination with CsA at 1.5 mg/kg and 2.75 mg/kg increased MRT to > 46.8 +/- 6.3 and > 44.2 +/- 9.4 days, respectively. CONCLUSIONS Anti-LFA-1 alpha and CsA combination therapy significantly extends the time to rejection of transplanted rat hearts. We conclude that combining an anti-LFA-1 alpha and CsA may be beneficial in prolonging allograft rejection times and in reducing the amount of CsA necessary for immune suppression, thereby minimizing its toxic effects.

BI 5700, a Selective Chemical Inhibitor of IκB Kinase 2, Specifically Suppresses Epithelial-Mesenchymal Transition and Metastasis in Mouse Models of Tumor Progression

Increasing evidence suggests that processes termed epithelial-mesenchymal transitions (EMTs) play a key role in therapeutic resistance, tumor recurrence, and metastatic progression. NF-κB signaling has been previously identified as an important pathway in the regulation of EMT in a mouse model of tumor progression. However, it remains unclear whether there is a broad requirement for this pathway to govern EMT and what the relative contribution of IKK family members acting as upstream NF-κB activators is toward promoting EMT and metastasis. To address this question, we have used a novel, small-molecule inhibitor of IκB kinase 2 (IKK2/IKKβ), termed BI 5700. We investigated the role of IKK2 in a number of mouse models of EMT, including TGFβ-induced EMT in the mammary epithelial cell line EpRas, CT26 colon carcinoma cells, and 4T1 mammary carcinoma cells. The latter model was also used to evaluate in vivo activities of BI 5700.We found that BI 5700 inhibits IKK2 with an IC(50) of 9 nM and was highly selective as compared to other IKK family members (IKK1, IKKε, and TBK1) and other kinases. BI 5700 effectively blocks NF-κB activity in EpRas cells and prevents TGFβ-induced EMT. In addition, BI 5700 reverts EMT in mesenchymal CT26 cells and prevents EMT in the 4T1 model. Oral application of BI 5700 significantly interferes with metastasis after mammary fat-pad injection of 4T1 cells, yielding fewer, smaller, and more differentiated metastases as compared to vehicle-treated control animals. We conclude that IKK2 is a key regulator of both the induction and maintenance of EMT in a panel of mouse tumor progression models and that the IKK2 inhibitor BI 5700 constitutes a promising candidate for the treatment of metastatic cancers.

Identification of a Potent Sodium Hydrogen Exchanger Isoform 1 (NHE1) Inhibitor with a Suitable Profile for Chronic Dosing and Demonstrated Cardioprotective Effects in a Preclinical Model of Myocardial Infarction in the Rat

Sodium-hydrogen exchanger isoform 1 (NHE1) is a ubiquitously expressed transmembrane ion channel responsible for intracellular pH regulation. During myocardial ischemia, low pH activates NHE1 and causes increased intracellular calcium levels and aberrant cellular processes, leading to myocardial stunning, arrhythmias, and ultimately cell damage and death. The role of NHE1 in cardiac injury has prompted interest in the development of NHE1 inhibitors for the treatment of heart failure. This report outlines our efforts to identify a compound suitable for once daily, oral administration with low drug-drug interaction potential starting from NHE1 inhibitor sabiporide. Substitution of a piperidine for the piperazine of sabiporide followed by replacement of the pyrrole moiety and subsequent optimization to improve potency and eliminate off-target activities resulted in the identification of N-[4-(1-acetyl-piperidin-4-yl)-3-trifluoromethyl-benzoyl]-guanidine (60). Pharmacological evaluation of 60 revealed a remarkable ability to prevent ischemic damage in an ex vivo model of ischemia reperfusion injury in isolated rat hearts.

Mitigation of Off-Target Adrenergic Binding and Effects on Cardiovascular Function in the Discovery of Novel Ribosomal S6 Kinase 2 Inhibitors

We previously reported the discovery of a novel ribosomal S6 kinase 2 (RSK2) inhibitor, (R)-5-Methyl-1-oxo-2,3,4,5-tetrahydro-1H-[1,4]diazepino[1,2-a] indole-8-carboxylic acid [1-(3-dimethylamino-propyl)-1H-benzoimidazol-2-yl]-amide (BIX 02565), with high potency (IC50 = 1.1 nM) targeted for the treatment of heart failure. In the present study, we report that despite nanomolar potency at the target, BIX 02565 elicits off-target binding at multiple adrenergic receptor subtypes that are important in the control of vascular tone and cardiac function. To elucidate in vivo the functional consequence of receptor binding, we characterized the cardiovascular (CV) profile of the compound in an anesthetized rat CV screen and telemetry-instrumented conscious rats. Infusion of BIX 02565 (1, 3, and 10 mg/kg) in the rat CV screen resulted in a precipitous decrease in both mean arterial pressure (MAP; to -65 ± 6 mm Hg below baseline) and heart rate (−93 ± 13 beats/min). In telemetry-instrumented rats, BIX 02565 (30, 100, and 300 mg/kg p.o. QD for 4 days) elicited concentration-dependent decreases in MAP after each dose (to −39 ± 4 mm Hg on day 4 at Tmax); analysis by Demming regression demonstrated strong correlation independent of route of administration and influence of anesthesia. Because of pronounced off-target effects of BIX 02565 on cardiovascular function, a high-throughput selectivity screen at adrenergic α1A and α2A was performed for 30 additional RSK2 inhibitors in a novel chemical series; a wide range of adrenergic binding was achieved (0–92% inhibition), allowing for differentiation within the series. Eleven lead compounds with differential binding were advanced to the rat CV screen for in vivo profiling. This led to the identification of potent RSK2 inhibitors (cellular IC50 <0.14 nM) without relevant α1A and α2A inhibition and no adverse cardiovascular effects in vivo.

Indole RSK inhibitors. Part 1: Discovery and initial SAR

A series of inhibitors for the 90 kDa ribosomal S6 kinase (RSK) based on an 1-oxo-2,3,4,5-tetrahydro-1H-[1,4]diazepino[1,2-a]indole-8-carboxamide scaffold were identified through high throughput screening. An RSK crystal structure and exploratory SAR were used to define the series pharmacophore. Compounds with good cell potency, such as compounds 43, 44, and 55 were identified, and form the basis for subsequent kinase selectivity optimization.

Early and Transient Sodium-Hydrogen Exchanger Isoform 1 Inhibition Attenuates Subsequent Cardiac Hypertrophy and Heart Failure Following Coronary Artery Ligation

Na+/H+ exchanger 1 (NHE-1) inhibition attenuates the hypertrophic response and heart failure in various experimental models. As the hypertrophic program is rapidly initiated following insult, we investigated whether early and transient administration of a NHE-1 inhibitor will exert salutary effects on cardiomyocyte hypertrophy or heart failure using both in vitro and in vivo approaches. Neonatal cardiomyocytes were treated with the novel, potent, and highly specific NHE-1 inhibitor BIX (N-[4-(1-acetyl-piperidin-4-yl)-3-trifluoromethyl-benzoyl]-guanidine; 100 nM) for 1 hour in the presence of 10 µM phenylephrine, after which the cells were maintained for a further 23 hours in the absence of NHE-1 inhibition. One-hour treatment with the NHE-1 inhibitor prevented phenylephrine-induced hypertrophy, which was associated with prevention of activation of calcineurin, a key component of the hypertrophic process. Experiments were then performed in rats subjected to coronary artery ligation, in which the NHE-1 inhibitor was administered immediately after infarction for a 1-week period followed by a further 5 weeks of sustained coronary artery occlusion in the absence of drug treatment. This approach significantly attenuated left ventricular hypertrophy and improved both left ventricular systolic and diastolic dysfunction, which was also associated with inhibition of calcineurin activation. Our findings indicate that early and transient administration of an NHE-1 inhibitor bestows subsequent inhibition of cardiomyocyte hypertrophy in culture as well as cardiac hypertrophy and heart failure in vivo, suggesting a critical early NHE-1–dependent initiation of the hypertrophic program. The study also suggests a preconditioning-like phenomenon in preventing hypertrophy and heart failure by early and transient NHE-1 inhibition.

Protective Effects of Monoclonal Antibodies to Cell Adhesion Glycoproteins in Models of Cardiac Reperfusion Injury and Leukocyte/Vascular Responses

Prompt reperfusion of an ischemic tissue is necessary for restoration of function but can be associated, paradoxically, with the progressive deterioration of reversibly damaged cells resulting in dysfunction and necrosis (Braunwald 1985; Heras et al. 1988). This reperfusion injury has been studied extensively in models of myocardial ischemia (Lehr et al. 1993; Lefer et al. 1994) but has also been implicated in delayed graft function and rejection following cardiac allograft transplantation (Land 1994). The cause of reperfusion injury is no doubt multifactorial and is believed to involve an inappropriate infiltration of leukocytes and the subsequent release of their cytotoxic mediators into the previously ischemic tissue (Pinckard et al. 1983; Mullane et al. 1984). The leukocytic infiltrate has been implicated in the alteration of tissue function which can lead to cell death and organ failure. Leukocyte adhesion to the vascular endothelium, and subsequent migration into the ischemic myocardium or into a transplanted heart is orchestrated by a series of ligand-receptor events between leukocyte and endothelial cell surface glycoproteins (Lipsky et al. 1993). The importance of the various cell adhesion glycoproteins in mediating tissue damage in preclinical models of these disease states has been addressed with the administration of monoclonal antibodies (MAbs) which recognize and neutralize the function of the particular glycoproteins.