Discovery of BIIB068: A Selective, Potent, Reversible Bruton's Tyrosine Kinase Inhibitor as an Orally Efficacious Agent for Autoimmune Diseases.

Autoreactive B cell-derived antibodies form immune complexes that likely play a pathogenic role in autoimmune diseases. In systemic lupus erythematosus (SLE), these antibodies bind Fc receptors on myeloid cells and induce proinflammatory cytokine production by monocytes and NETosis by neutrophils. Bruton's tyrosine kinase (BTK) is a non-receptor tyrosine kinase that signals downstream of Fc receptors and plays a transduction role in antibody expression following B cell activation. Given the roles of BTK in both the production and sensing of autoreactive antibodies, inhibitors of BTK kinase activity may provide therapeutic value to patients suffering from autoantibody-driven immune disorders. Starting from an in-house proprietary screening hit followed by structure-based rational design, we have identified a potent, reversible BTK inhibitor, BIIB068 (1), which demonstrated good kinome selectivity with good overall drug-like properties for oral dosing, was well tolerated across preclinical species at pharmacologically relevant doses with good ADME properties, and achieved >90% inhibition of BTK phosphorylation (pBTK) in humans.

Incidence of spontaneous arrhythmias in freely moving healthy untreated Sprague-Dawley rats.

Spontaneous arrhythmia characterization in healthy rats can support interpretation when studying novel therapies. Male (n = 55) and female (n = 40) Sprague-Dawley rats with telemetry transmitters for a derivation II ECG. Arrhythmias were assessed from continuous ECG monitoring over a period of 24-48 h, and data analyzed using an automated detection algorithm with 100% manual over-read. While a total of 1825 spontaneous ventricular premature beats (VPB) were identified, only 7 rats (or 7.4%) did not present with any over the recording period. Spontaneous episode(s) of ventricular tachycardia (VT) were noted in males (27%) and females (3%). The incidence of VPB was significantly higher (p < 0.01) during the night time (7 pm-7 am) compared to daytime, while males presented with significantly (p < 0.001) more VPB than females. Most VPB were observed as single ectopic beats, followed by salvos (2 or 3 consecutive VPBs), and VT (i.e. 4 consecutive VPBs). Most VPBs were single premature ventricular contractions (PVCs) (57%), while the remaining were escape complexes (43%). Spontaneous premature junctional complexes (PJC) were also observed and were significantly more frequent during the night, and in males. Lastly, 596 episodes of spontaneous 2nd-degree atrioventricular (AV) block were identified and were significantly more frequent during the day time in males. Most 2nd-degree AV block episodes were Mobitz type I (57%), with a significantly (p < 0.05) higher incidence in males. This work emphasizes the importance of obtaining sufficient baseline data when undertaking arrhythmia analysis in safety study and provides a better understanding of both sex- and time- dependent effects of spontaneous arrhythmias in rats.

An Investigative Study of Pancreatic Exocrine Biomarkers, Histology, and Histomorphometry in Male Zucker Diabetic Fatty (ZDF) Rats Given Dulaglutide by Subcutaneous Injection Twice Weekly for 13 Weeks.

Glucagon-like peptide-1 (GLP-1) receptor agonist therapy has been implicated as a possible risk factor for acute pancreatitis in patients with type 2 diabetes. Dulaglutide is a long-acting GLP-1 receptor agonist in development for treatment of type 2 diabetes. The effects of dulaglutide were evaluated in male Zucker diabetic fatty (ZDF) rats to examine whether dulaglutide may induce or modulate pancreatitis. Rats were randomized to dose groups receiving twice-weekly subcutaneously administered dulaglutide 0.5, 1.5, and 5.0 mg/kg/dose (corresponding human plasma exposures following twice-weekly dosing are 3-, 8-, and 30-fold, respectively) for 13 weeks or to vehicle control. Following termination, serially trimmed sections of pancreases were stained with hematoxylin and eosin or co-stained with an epithelial marker and a marker of either proliferation or apoptosis. Efficacious reductions in glucose and hemoglobin A1c occurred at all dulaglutide doses. Lipase activity was unaffected, and there were modest increases in total and pancreatic amylase activities at all doses without individual microscopic inflammatory correlates. Microscopic dulaglutide-related pancreatic changes included increased interlobular ductal epithelium without ductal cell proliferation (≥0.5 mg/kg), increased acinar atrophy with/without inflammation (≥1.5 mg/kg), and increased incidence/severity of neutrophilic acinar pancreatic inflammation (5.0 mg/kg). In summary, dulaglutide treatment was associated with mild alterations in ductal epithelium and modest exacerbation of spontaneous lesions of the exocrine pancreas typically found in the ZDF rat model.

Transduction of photoreceptors with equine infectious anemia virus lentiviral vectors: safety and biodistribution of StarGen for Stargardt disease.

PURPOSE: StarGen is an equine infectious anemia virus (EIAV)-based lentiviral vector that expresses the photoreceptor-specific adenosine triphosphate (ATP)-binding cassette transporter (ABCA4) protein that is mutated in Stargardt disease (STGD1), a juvenile macular dystrophy. EIAV vectors are able to efficiently transduce rod and cone photoreceptors in addition to retinal pigment epithelium in the adult macaque and rabbit retina following subretinal delivery. The safety and biodistribution of StarGen following subretinal delivery in macaques and rabbits was assessed. METHODS: Regular ophthalmic examinations, IOP measurements, ERG responses, and histopathology were carried out in both species to compare control and vector-treated eyes. Tissue and fluid samples were obtained to evaluate the persistence, biodistribution, and shedding of the vector following subretinal delivery. RESULTS: Ophthalmic examinations revealed a slightly higher level of inflammation in StarGen compared with control treated eyes in both species. However, inflammation was transient and no overt toxicity was observed in StarGen treated eyes and there were no abnormal clinical findings. There was no StarGen-associated rise in IOP or abnormal ERG response in either rabbits or macaques. Histopathologic examination of the eyes did not reveal any detrimental changes resulting from subretinal administration of StarGen. Although antibodies to StarGen vector components were detected in rabbit but not macaque serum, this immunologic response did not result in any long-term toxicity. Biodistribution analysis demonstrated that the StarGen vector was restricted to the ocular compartment. CONCLUSIONS: In summary, these studies demonstrate StarGen to be well tolerated and localized following subretinal administration.

Molecular imaging of monocrotaline-induced pulmonary vascular disease with radiolabeled linear adrenomedullin.

UNLABELLED: No test currently exists for molecular imaging of pulmonary arterial hypertension (PAH). Adrenomedullin is a vasodilator peptide predominantly cleared by pulmonary endothelial receptors. We developed a linear adrenomedullin derivative radiolabeled with (99m)Tc ((99m)Tc-AM-L) for imaging of pulmonary circulation and tested its capacity to detect anomalies of pulmonary circulation caused by PAH. METHODS: PAH was induced by monocrotaline in rats and compared with controls. After 5 wk, (99m)Tc-AM-L was injected intravenously. Plasma kinetics were measured, lung activity was determined in vivo after 30 min using a nuclear camera, and lung activity was determined ex vivo in explanted lungs. Expression of adrenomedullin receptors was measured in lung homogenates. RESULTS: The plasma levels of (99m)Tc-AM-L significantly increased in PAH by approximately 2-fold. Uptake by the lungs was homogeneous but greatly reduced in PAH by about 70%. In vivo retention was 14% +/- 1% (mean +/- SD) of the injected dose in controls and 4% +/- 1% in PAH (P < 0.0001). A similar reduction was measured ex vivo (6.0 +/- 1.6 percentage injected dose per gram [%ID/g] vs. 0.95 +/- 0.21 %ID/g, P < 0.0001). The expression of the heterodimeric component of the adrenomedullin receptor, receptor activity modifying protein 2, was also greatly reduced in PAH lungs (P < 0.001). Interestingly, right ventricular uptake of (99m)Tc-AM-L was increased by PAH (P = 0.02) and correlated with the degree of right ventricular hypertrophy (r = 0.83, P = 0.001). CONCLUSION: Pulmonary uptake of (99m)Tc-AM-L is greatly reduced in monocrotaline-induced PAH. This novel molecular imaging agent may be useful in the diagnosis and follow-up of pulmonary vascular disorders.

Downregulation of the endothelin system of lung myofibroblasts in congestive heart failure.

Congestive heart failure (CHF) causes lung remodelling with thickening of the alveolar septa and proliferation of myofibroblasts (MYF). Endothelin-1 (ET-1) is increased in CHF and may contribute to this process. CHF was induced in rats by myocardial infarction. After three weeks there was lung remodelling with thickening of alveolar septa and increases in 5'-bromodeoxyuridine uptake and vimentin expression (P < 0.05). The mitogenic and protein synthesis response of MYF to ET-1 (10 nM) were assessed by H-thymidine and H-leucine incorporation respectively. The mitogenic response in CHF (19.0 +/- 3.0%, mean +/- SEM) was less than for sham rats (35 +/- 5.4%, P < 0.05). This was associated with a lower production of ET-1 by CHF MYF (15.15 +/- 5.67 fmol/ml) compared to sham (33.66 +/- 13.22 fmol/ml; P < 0.05). Additionally, protein expression of ETA (0.36 +/- 0.038 AU) and ETB receptors (0.24 +/- 0.075 AU) were reduced in CHF compared to shams (0.65 +/- 0.086 AU and 0.81 +/- 0.21 AU respectively; P < 0.05). There is a downregulation of the ET system of lung MYF in CHF with reduced proliferation in response to ET-1. This may represent a protective adaptation to counteract lung remodelling in response to chronic exposure to high levels of ET-1.

Role of endothelin receptors on basal and endothelin-1-stimulated lung myofibroblast proliferation.

Proliferation of myofibroblasts (MYF) contributes to numerous lung disorders. Endothelin-1 (ET-1) production is increased in various lung diseases and could contribute to lung remodelling. The respective roles of ETA and ETB receptors (ETA-R, ETB-R) and the role of endogenous ET-1 production by lung MYF on proliferation of MYF remain uncertain. Rat lung MYF were isolated and 3H-thymidine and 3H-leucine incorporation assays were completed in the presence of a selective ETA-R antagonist, a selective ETB-R antagonist, or a combination of both. Receptor expression was evaluated by confocal imaging, and ET-1 levels were measured by ELISA. Confocal microscopy revealed abundant ETA-R and ETB-R expression on lung MYF. ET-1 (10 nmol/L) stimulated MYF proliferation and protein synthesis through PI3-kinase and p38 pathways. Although neither selective ETA-R blockade (BQ-123, 1 micromol/L) nor selective ETB-R blockade (BQ-788, 1 micromol/L) alone inhibited proliferation or protein synthesis, their combination almost completely abolished ET-1 mitogenic effect. Surprisingly, basal MYF proliferation was increased by selective blockade of either ETA-R or ETB-R alone, but not by dual blockade. ET-1 levels were not affected by the antagonists. Our findings indicate that both the ETA-R and the ETB-R regulate basal and stimulated lung MYF proliferation and suggest possible interactions between the receptors.

Bone marrow-derived progenitor cells contribute to lung remodelling after myocardial infarction.

BACKGROUND: Congestive heart failure (CHF) causes structural modifications of the lungs that contribute to the functional limitations of affected subjects. We hypothesized that bone marrow-derived progenitor cells could contribute to lung structural remodelling after myocardial infarction (MI). METHODS: Wistar rats were irradiated and received a bone marrow transplant (BMT) from green fluorescent protein (GFP) transgenic rats, followed 5 weeks later by coronary artery ligation or sham operation. Five weeks after MI, lung immunofluorescence studies were performed and GFP expression evaluated by Western immunoblotting. RESULTS: After MI, rats developed lung structural remodelling characterized by myofibroblast (MF) proliferation in the alveolar septa. After BMT, some GFP+ cells were found in the lungs of sham animals. The amount of GFP+ cells in the lungs of MI rats was greatly increased with evidence of differentiation into MFs, as evaluated by co-localization correlation analysis with smooth muscle alpha-actin (P<.01). These cells were particularly abundant in the perivenular regions where they incorporated into the wall of blood vessels. There was a threefold increase in lung GFP protein expression after MI (P=.01). CONCLUSIONS: After MI, bone marrow-derived progenitor differentiates into lung MFs. This novel pathophysiologic process may contribute to the pulmonary manifestations of CHF and could have significant therapeutic implications.

Modification of the pulmonary renin-angiotensin system and lung structural remodelling in congestive heart failure.

Lung structural remodelling, characterized by myofibroblast proliferation and collagen deposition, contributes to impaired functional capacity in CHF (congestive heart failure). As the lung is the primary site for the formation of Ang II (angiotensin II), local modifications of this system could contribute to lung remodelling. Rats with CHF, induced following myocardial infarction (MI) via coronary artery ligation, were compared with sham-operated controls. The MI group developed lung remodelling as confirmed by morphometric measurements and immunohistochemistry. Pulmonary Ang II concentrations increased more than 6-fold (P<0.01), and AT1 (Ang II type 1) receptor expression was elevated by 3-fold (P<0.01) with evidence of distribution in myofibroblasts. AT2 (Ang II type 2) receptor expression was unchanged. In isolated lung myofibroblasts, AT1 and AT2 receptors were expressed, and Ang II stimulated proliferation as measured by [3H]thymidine incorporation. In normal rats, chronic intravenous infusion of Ang II (0.5 mg.kg(-1) of body weight.day(-1)) for 28 days significantly increased mean arterial pressure (P<0.05), without pulmonary hypertension, lung remodelling or a change in AT1 receptor expression. We conclude that there is a modification of the pulmonary renin-angiotensin system in CHF, with increased Ang II levels and AT1 receptor expression on myofibroblasts. Although this may contribute to lung remodelling, the lack of effect of increased plasma Ang II levels alone suggests the importance of local pulmonary Ang II levels combined with the effect of other factors activated in CHF.