Assessing the Impact of Losmapimod on Proteinuria in Idiopathic Focal Segmental Glomerulosclerosis.

INTRODUCTION: Idiopathic focal segmental glomerulosclerosis (FSGS) is a leading cause of nephrotic syndrome and end-stage renal disease. In preclinical models and biopsies of human FSGS kidneys, p38 mitogen-activated protein kinase (MAPK) has demonstrated enhanced activity; and p38 MAPK inhibition has improved disease markers. This proof-of-concept trial aimed to assess efficacy, safety, tolerability, and pharmacokinetics of losmapimod, an oral p38 MAPK inhibitor, in humans with FSGS. METHODS: A single-arm, multicenter, open-label, Phase II trial (NCT02000440) was conducted in adults with FSGS; proteinuria ≥2.0 g/d; estimated glomerular filtration rate (eGFR) ≥45 ml/min per 1.73 m2; blood pressure <140/90 mm Hg. Collapsing and genetic forms of FSGS were excluded. The primary endpoint was number of patients with ≥50% proteinuria reduction and eGFR ≥70% of baseline after receiving losmapimod twice-daily for 16 to 24 weeks. RESULTS: Seventeen patients received ≥1 losmapimod dose. No patients achieved the primary endpoint; therefore, the study was terminated following a prespecified interim analysis. At week 24, proteinuria reductions between 20% and <50% were observed in 4 patients and proteinuria increases >20% in 3 patients. One patient achieved a proteinuria response (≥50% reduction) at week 2 but subsequently relapsed. Losmapimod pharmacokinetics were consistent with prior studies. No serious adverse events (AEs) were reported. CONCLUSION: p38 MAPK inhibition with losmapimod did not result in ≥50% reduction of proteinuria in patients with FSGS. However, study population heterogeneity may have contributed to our negative findings and therefore this does not eliminate the potential to demonstrate benefit in a population more sensitive to p38 MAPK inhibition if identifiable in the future by precision-medicine methods.

A randomized dose-finding study demonstrating the efficacy and tolerability of albiglutide in Japanese patients with type 2 diabetes mellitus.

OBJECTIVE: To investigate the optimal dosage/regimen and to evaluate the efficacy and safety of albiglutide in Japanese patients with type 2 diabetes mellitus. RESEARCH DESIGN AND METHODS: This was a randomized, double-blind, placebo-controlled, multicenter, parallel-group, dose-ranging, superiority study in Japanese patients with type 2 diabetes mellitus. Patients (n = 215) who were treatment naive or washed out of one oral antidiabetic drug were randomized to placebo or albiglutide 15 mg weekly, 30 mg weekly, or 30 mg every other week (biweekly). CLINICAL TRIAL REGISTRATION: NCT01098461. MAIN OUTCOME MEASURES: The primary end point was the change from baseline in HbA1c at week 16, measured using the Japan Diabetes Society standardization scheme and presented here using the National Glycohemoglobin Standardization Program equivalents. Other measures of efficacy as well as safety and population pharmacokinetics and pharmacokinetics/pharmacodynamics of albiglutide were assessed. RESULTS: Baseline HbA1c was 8.53%. There was a statistically significant difference between each albiglutide treatment group and placebo for change from baseline in HbA1c at week 16, with treatment effects of -0.89% for 15 mg weekly, -1.55% for 30 mg weekly, and -1.10% for 30 mg biweekly (P < 0.0001 for all groups vs placebo). By week 16, 63.0% and 33.3% of patients in the 30 mg weekly albiglutide group compared with 6.0% and 0% of patients in the placebo group achieved HbA1c <7.4% and <6.9%, respectively. No serious adverse events were related to study therapy; no deaths occurred. Nasopharyngitis was the most frequently reported adverse event in all treatment groups (n = 43 [20.3%]). CONCLUSIONS: Albiglutide exhibited therapeutic hypoglycemic effects with good tolerability among Japanese patients with type 2 diabetes mellitus; the 30 mg weekly dose was the most efficacious in this study. The 16 week duration of the study prevents generalizing these conclusions to longer treatment periods.

Pharmacodynamics, pharmacokinetics, safety, and tolerability of albiglutide, a long-acting glucagon-like peptide-1 mimetic, in patients with type 2 diabetes.

CONTEXT: Native glucagon-like peptide-1 increases insulin secretion, decreases glucagon secretion, and reduces appetite but is rapidly inactivated by dipeptidyl peptidase-4. Albiglutide is a novel dipeptidyl peptidase-4-resistant glucagon-like peptide-1 dimer fused to human albumin designed to have sustained efficacy in vivo. OBJECTIVES: The objectives were to investigate pharmacodynamics, pharmacokinetics, safety, and tolerability of albiglutide in type 2 diabetes subjects. METHODS: In a single-blind dose-escalation study, 54 subjects were randomized to receive placebo or 9-, 16-, or 32-mg albiglutide on d 1 and 8. In a complementary study, 46 subjects were randomized to a single dose (16 or 64 mg) of albiglutide to the arm, leg, or abdomen. RESULTS: Significant dose-dependent reductions in 24-h mean weighted glucose [area under the curve((0-24 h))] were observed, with placebo-adjusted least squares means difference values in the 32-mg cohort of -34.8 and -56.4 mg/dl [95% confidence interval (-54.1, -15.5) and (-82.2, -30.5)] for d 2 and 9, respectively. Placebo-adjusted fasting plasma glucose decreased by -26.7 and -50.7 mg/dl [95% confidence interval (-46.3, -7.06) and (-75.4, -26.0)] on d 2 and 9, respectively. Postprandial glucose was also reduced. No hypoglycemic episodes were detected in the albiglutide cohorts. The frequency and severity of the most common adverse events, headache and nausea, were comparable with placebo controls. Albiglutide half-life ranged between 6 and 7 d. The pharmacokinetics or pharmacodynamic of albiglutide was unaffected by injection site. CONCLUSIONS: Albiglutide improved fasting plasma glucose and postprandial glucose with a favorable safety profile in subjects with type 2 diabetes. Albiglutide's long half-life may allow for once-weekly or less frequent dosing.

Compstatin inhibits complement activation by binding to the beta-chain of complement factor 3.

Compstatin is a peptidic complement inhibitor that prevents the cleavage of complement factor 3 (C3) by C3 convertase. Compstatin differs from other C3-regulatory proteins, such as complement receptor (CR) 1 and decay-accelerating factor (DAF), in that it binds native as well as activated C3 fragments and acts through mechanisms that do not involve the destabilization of the C3 convertase or the accelerated degradation of C3b. Compstatin's activity most likely relies on its affinity for native C3 and the conformational change that results upon binding with C3. Although the intermolecular interactions between compstatin and C3 have been studied, the identity of the targeted region on C3 is still elusive. To address this issue, we synthesized a photo-crosslinking compstatin analog and used it to probe C3 for sites of interaction. We identified a 40-kDa region at the C-terminus of the beta-chain of C3 that included the binding site of the compstatin analog. The specificity of the binding was confirmed by inhibition studies, which showed reduced crosslinking signal after pre-incubation of C3 with compstatin but not with various inactive analogs. Binding studies performed with a recombinant homolog of the 40-kDa region confirmed these findings. Five smaller recombinant proteins corresponding to various overlapping regions of the 40-kDa fragment did not bind compstatin, suggesting that a proper protein conformation, only found in larger fragments, is required for compstatin binding. The identified region on the beta-chain has, thus far, not been implicated in C3 cleavage or interactions with other proteins. Therefore, further research on this part of the C3 molecule may have implications for studies on the regulation of C3 cleavage, as well as for complement-based drug design.

Synthetic small-molecule complement inhibitors.

During the past few years, several large molecular-weight compounds with complement-inhibitory activities have entered clinical trials for a wide variety of acute and chronic inflammatory conditions. Various small synthetic compounds that offer several advantages over the larger complement inhibitors are also being discovered at a rapid pace. In this review, the focus will be on three of these small molecules, a C3-binding peptide, compstatin; a synthetic peptidic antagonist of the C5a anaphylatoxin receptor, 3D53; and a non-peptidergic antagonist of the C3a anaphylatoxin receptor, SB-290157. In recent years, compstatin has undergone a series of optimizations that have led to more active and stable analogs, while 3D53 and SB-290157 have been more extensively tested in animal models of various human inflammatory diseases. These compounds have been shown to be effective and display little or no toxicity, and as such may be promising new candidates for further therapeutic development.

Complement C5a receptor is essential for the optimal generation of antiviral CD8+ T cell responses.

The complement system has been long regarded as an important effector of the innate immune response. Furthermore, complement contributes to various aspects of B and T cell immunity. Nevertheless, the role of complement in CD8(+) T cell antiviral responses has yet to be fully delineated. We examined the CD8(+) T cell response in influenza type A virus-infected mice treated with a peptide antagonist to C5aR to test the potential role of complement components in CD8(+) T cell responses. We show that both the frequency and absolute numbers of flu-specific CD8(+) T cells are greatly reduced in C5aR antagonist-treated mice compared with untreated mice. This reduction in flu-specific CD8(+) T cells is accompanied by attenuated antiviral cytolytic activity in the lungs. These results demonstrate that the binding of the C5a component of complement to the C5a receptor plays an important role in CD8(+) T cell responses.

Dopamine inhibits luteinizing hormone synthesis and release in the juvenile European eel: a neuroendocrine lock for the onset of puberty.

In various adult teleost fishes, LH ovulatory peak is under a dual neurohormonal control that is stimulatory by GnRH and inhibitory by dopamine (DA). We investigated whether DA could also be involved in the inhibitory control of LH at earlier steps of gametogenesis by studying the model of the European eel, Anguilla anguilla, which remains at a prepubertal stage until the oceanic reproductive migration. According to a protocol previously developed in the striped bass, eels received sustained treatments with GnRH agonist (GnRHa), DA-receptor antagonist (pimozide), and testosterone (T) either alone or in combination. Only the triple treatment with T, GnRHa, and pimozide could trigger dramatic increases in LH synthesis and release as well as in plasma vitellogenin levels and a stimulation of ovarian vitellogenesis. Thus, in the prepubertal eel, removal of DA inhibition is required for triggering GnRH-stimulated LH synthesis and release as well as ovarian development. To locate the anatomical support for DA inhibition, the distribution of tyrosine hydroxylase (TH) in the brain and pituitary was studied by immunocytochemistry. Numerous TH-immunoreactive cell bodies were observed in the preoptic anteroventral nucleus, with a dense tract of immunoreactive fibers reaching the pituitary proximal pars distalis, where the gonadotrophs are located. This pathway corresponds to that mediating the inhibition of LH and ovulation in adult teleosts. To our knowledge, this is the first demonstration of a pivotal role for DA in the control of LH and puberty in a juvenile teleost. These data support the view that DA inhibition on LH secretion is an ancient evolutionary component in the neuroendocrine regulation of reproduction that may have been partially maintained throughout vertebrate evolution.

A functional C5a anaphylatoxin receptor in a teleost species.

The anaphylatoxins are potent, complement-derived low m.w. proteins that bind to specific seven-transmembrane receptors to elicit and amplify a variety of inflammatory reactions. C5a is the most potent of these phlogistic peptides and is a strong chemoattractant for neutrophils and macrophages/monocytes. Although lower vertebrates possess complement systems that are believed to function similarly to those of mammals, anaphylatoxin receptors have not previously been characterized in any nonmammalian vertebrate. To study the functions of C5a in teleost fish, we generated recombinant C5a of the rainbow trout, Oncorhynchus mykiss (tC5a), and used fluoresceinated tC5a (tC5aF) and flow cytometry to identify the C5a receptor (C5aR) on trout leukocytes. Granulocytes/Macrophages present in cell suspensions of the head kidney (HKL), the main hemopoietic organ in teleosts, showed a univariate type of receptor expression, whereas those from the peripheral blood demonstrated either a low or high level of expression. The binding of tC5aF was inhibited by excess amounts of unlabeled tC5a or tC5a(desArg), demonstrating that sites other than the C-terminal of tC5a interact with the C5aR. Both tC5a and tC5a(desArg) were able to induce chemotactic responses in granulocytes in a concentration-dependent manner, but the desArg derivative was at least 10-fold less active. Homologous desensitization occurred after HKL were exposed to continuous or high concentrations of tC5a, with a loss of tC5aF binding and an 80% reduction in chemotactic responses toward tC5a. Pertussis toxin reduced the migration of HKL toward tC5a by 40%, suggesting only a partial involvement of pertussis toxin-sensitive G(i) proteins in tC5a-mediated chemotaxis.

Complement: structure, functions, evolution, and viral molecular mimicry.

The complement (C') system has long been recognized as an important mediator of innate immune defense and inflammation. In recent years there is increasing evidence suggesting that complement components may also participate in non-inflammatory and developmental processes. Here we review our current work on the structural-functional aspects of C3-ligand interactions and the rational design of small-sized complement inhibitors. We present a novel, proteomics-based, approach to studying protein-protein interactions within the C' system and discuss our progress in the study of viral immune evasion strategies. Furthermore we discuss the involvement of complement proteins in organ regeneration and hematopoietic development.

Molecular cloning of the beta subunit of complement component eight of rainbow trout.

Complement-mediated killing of pathogens through the lytic pathway is an important effector mechanism of the innate immune response. C8 is one of the components of the lytic pathway and is composed of an alpha, beta, and gamma subunit. In the present study we report the cloning and characterization of the primary structure of the C8beta subunit in the rainbow trout (Oncorhynchus mykiss). The deduced amino acid sequence of trout C8beta shows 72 and 47% identity with that of Japanese flounder and human, respectively. It also contains many of the same structural motifs as those found in mammalian lytic components. The C8beta gene appears to exists as a single copy in the trout genome and is expressed primarily in the liver. The protein encoded by the gene was identified by Western blotting using an anti-peptide antibody and was approximately 65kDa.

The effects of long-term testosterone, gonadotropin-releasing hormone agonist and pimozide treatments on testicular development and luteinizing hormone levels in juvenile and early maturing striped bass, Morone saxatilis.

The present study was conducted to test the responsiveness of the juvenile male reproductive axis to hormonal stimulation and to compare it to that of early maturing males. Long-term treatments with various combinations of T, GnRHa and pimozide did not result in an increased incidence of early maturing males, but did stimulate spermatogenesis slightly in juvenile fish. In early maturing males, the treatments appeared to be inhibitory since they resulted in a reduction of the GSI and a lower incidence of spermiating males. In early maturing males, pituitary LH content was elevated by GnRHa treatments alone while in juvenile males a combination of T and GnRHa was needed to increase the levels of LH in the pituitary. Thus, T may play an important role during puberty by potentiating the effects of GnRH on LH synthesis. In both juvenile and early maturing males, plasma LH levels could be increased only by high doses of GnRHa (in combination with T). Therefore, LH synthesis and release probably require different levels of GnRH stimulation. A GnRH challenge (single injection of 50 microg GnRHa/kg) at the end of the experiment resulted in a dramatic elevation of plasma LH levels in almost all animals. This finding demonstrates that pituitaries from juvenile and early maturing males were responsive to GnRHa stimulation, even after long-term hormonal treatments. The addition of pimozide did not affect the T- and GnRHa-induced increase in pituitary LH content but inhibited the release of LH in response to a GnRHa challenge. In conclusion, high doses of GnRHa in combination with T can increase plasma LH levels in juvenile males but do not induce complete testicular maturation. Factors other than T, GnRHa or LH are probably involved in the induction and completion of spermatogenesis.

The complement system in teleosts.

Complement, an important component of the innate immune system, is comprised of about 35 individual proteins. In mammals, activation of complement results in the generation of activated protein fragments that play a role in microbial killing, phagocytosis, inflammatory reactions, immune complex clearance, and antibody production. Fish appear to possess activation pathways similar to those in mammals, and the fish complement proteins identified thus far show many homologies to their mammalian counterparts. Because information about complement proteins, regulatory proteins, and complement receptors in fish is far from complete, it is unclear whether all the complement functions that have been identified in mammals also occur in fish. However, it has been clearly demonstrated that fish complement can lyse foreign cells and opsonise foreign organisms for destruction by phagocytes. There are also indications that complement fragments participate in inflammatory reactions. Fish possess multiple isoforms of several complement proteins, such as C3 and factor B. It has been hypothesised that the function of this diversity in complement proteins serves to expand their innate immune recognition capacity and response. Understanding the functions of complement in fish and the roles the individual proteins, including the various isoforms, play in host defence, is important not only for understanding the evolution of this system but also for the development of new strategies in fish health management.