RLS-0071 Moderated Elevated Myeloperoxidase Level and Activity in an Asymptomatic Subject in Clinical Trial RLS-0071-101.

BACKGROUND RLS-0071 is a dual-targeting peptide developed for the regulation of humoral and cellular inflammation via inhibition of neutrophil effectors, including myeloperoxidase and neutrophil extracellular trap formation (NETosis). The safety, pharmacokinetics, and pharmacodynamics of single and multiple doses of RLS-0071 were evaluated in a first-in-human clinical trial in healthy volunteers. Myeloperoxidase is the major peroxidase enzyme present in neutrophilic granules and contributes to cellular inflammation. Extracellular myeloperoxidase has been associated with chronic inflammation in a variety of diseases, including atherosclerosis. RLS-0071 has previously been shown to inhibit extracellular myeloperoxidase function both in vitro and in vivo in animal disease models. CASE REPORT Healthy subjects participating in the RLS-0071-101 study were screened for baseline myeloperoxidase level, leading to the identification of a 21-year-old woman with elevated baseline levels. After randomization, the subject received 9 intravenous infusions of 10 mg/kg RLS-0071. The subject tolerated the peptide infusions well with no adverse changes in vital signs, significantly abnormal clinical laboratory results, or severe adverse events. Analysis of this subject's myeloperoxidase plasma concentrations demonstrated that her myeloperoxidase levels decreased by 43% and myeloperoxidase activity levels decreased 49% after infusions of RLS-0071. The reduction in the patient's plasma myeloperoxidase levels demonstrated a partial return to baseline levels 24 hours after cessation of dosing. There were no other clinically meaningful safety observations for this subject. CONCLUSIONS This observation suggests RLS-0071 has the therapeutic potential to moderate plasma myeloperoxidase levels and activity and modulate diseases in which myeloperoxidase contributes to pathogenesis.

Vaccine-Preventable Diseases Requiring Hospitalization.

BACKGROUND: Plain children often have lower immunization rates than non-Plain children. Penn State Health Children's Hospital is a tertiary medical center with large nearby Plain (Amish and Mennonite) communities. We sought to describe the characteristics of children hospitalized with vaccine-preventable diseases (VPDs). We hypothesized that Amish children would have a higher risk of VPDs than non-Amish children. METHODS: International Classification of Diseases, Ninth Revision codes were used to identify patients <18 years diagnosed with a VPD from January 1, 2005, to December 31, 2015, at Penn State Children's Hospital. Demographic information, immunization status, and outcomes were obtained from medical records. By using the number of children in our primary service area, we calculated the risk of VPD requiring hospitalization for Amish and non-Amish children. We assessed the relationship between Plain affiliation and vaccination status by using the Pearson correlation coefficient. RESULTS: There were 215 children with 221 VPDs. Most occurred in non-Plain children: 179 of 221 (81%). Except for pneumococcal infections, VPD occurred mostly in unvaccinated or immunocompromised children, regardless of Plain affiliation. There were 15 Haemophilus influenzae type b and 5 tetanus infections that occurred in children with an unvaccinated or unknown vaccination status. The risk of a VPD requiring hospitalization was greater for Amish than for non-Plain children (risk ratio: 2.67 [95% confidence interval: 1.87-3.82]). There was a strong correlation between Plain affiliation and lack of vaccination (r = -0.63, P < .01). CONCLUSIONS: Amish children had an increased risk of a VPD requiring hospitalization than non-Plain children. With the exception of those with pneumococcal disease, most vaccinated children hospitalized with a VPD were immunocompromised.

Therapeutic hypothermia modulates complement factor C3a and C5a levels in a rat model of hypoxic ischemic encephalopathy.

BACKGROUND: Therapeutic hypothermia (HT) is the only intervention that improves outcomes in neonatal hypoxic-ischemic encephalopathy (HIE). However, the multifactorial mechanisms by which HT impacts HIE are incompletely understood. The complement system plays a major role in the pathogenesis of ischemia-reperfusion injuries such as HIE. We have previously demonstrated that HT modulates complement activity in vitro. METHODS: Term equivalent rat pups were subjected to unilateral carotid ligation followed by hypoxia (8% O2) for 45 min to simulate HIE. A subset of animals was subjected to HT (31-32°C for 6 h). Plasma and brain levels of C3a and C5a were measured. Receptors for C3a (C3aR) and C5a (C5aR) along with C1q, C3, and C9 were characterized in neurons, astrocytes, and microglia. RESULTS: We found that HT increased systemic expression of C3a and decreased expression of C5a after HIE. In the brain, C3aR and C5aR are predominantly expressed on microglia after HIE. HT increased local expression of C3aR and decreased expression on C5aR after HIE. Furthermore, HT decreased local expression of C1q, C3-products, and C9 in the brain. CONCLUSION: HT is associated with significant alteration of complement effectors and their cognate receptors. Complement modulation may improve outcomes in neonatal HIE.

Peptide inhibitor of complement C1 modulates acute intravascular hemolysis of mismatched red blood cells in rats.

BACKGROUND: Acute hemolytic transfusion reactions have a broad clinical presentation from mild and transitory signs and symptoms to shock, disseminated intravascular coagulation, renal failure, and death. We have recently developed a rat model of acute intravascular hemolysis showing that the classical complement pathway mediates antibody-dependent hemolysis. The objective of this study was to evaluate the role of the classical pathway inhibitor peptide inhibitor of complement C1 (PIC1) in this animal model. STUDY DESIGN AND METHODS: Male Wistar rats received a 15% transfusion of human red blood cells (RBCs) and blood was isolated from the animals up to 120 minutes. Animals received PIC1 either 2 minutes before or 0.5 minutes after transfusion. Sham-, vehicle-, and cobra venom factor (CVF)-treated animals were used as control groups with a subset of rats also receiving an equivalent dose of intravenous immunoglobulin (IVIG) before transfusion. Blood was analyzed for transfused RBC survival by flow cytometry and free hemoglobin (Hb) in isolated plasma by spectrophotometry. RESULTS: Vehicle-treated rats showed decreased human RBC survival and increased free Hb as expected. Rats receiving PIC1 before transfusion showed increased human RBC survival and decreased Hb similar to CVF-treated rats. Notably, rats receiving PIC1 after initiation of transfusion showed similar decreases in hemolysis as animals receiving PIC1 before transfusion. Compared to IVIG and saline controls, PIC1-treated animals demonstrated decreased hemolysis and protection from acute kidney injury. CONCLUSIONS: These results demonstrate that PIC1 has efficacy in an animal model of acute intravascular hemolysis in both prevention and rescue scenarios.

Glucose-based dialysis fluids inhibit innate defense against Staphylococcus aureus.

BACKGROUND: Staphylococcus aureus peritonitis is a serious complication of Chronic Peritoneal Dialysis (CPD) and associated with a higher risk for severe and recurrent infections compared with other bacteria. We have previously shown that complement-mediated effectors essential for optimal opsonophagocytosis of S. aureus are inhibited by high glucose concentrations. Since most commonly used peritoneal dialysis (PD) fluids are glucose-based, we hypothesized that glucose-based PD fluids likely inhibit complement host defenses against S. aureus. METHODS: Commercially available PD fluids were tested: glucose-based (Dianeal), Dianeal supplemented with amino acids, icodextrin-based (Extraneal) and amino acid-based (Nutrineal). Control PD fluid was generated to simulate Dianeal excluding the glucose. Three commercially available glucose concentrations were tested: Dianeal 1.5% (15 gm/1000 ml), Dianeal 2.5% (25 gm/1000 ml) and Dianeal 4.25% (42.5 gm/1000 ml). Complement effectors against S. aureus were analyzed including opsonization with C3-fragments, anaphylatoxin generation, and phagocytosis efficiency. We also evaluated clinical strains, including MRSA strains, and specific complement activation pathways. RESULTS: Glucose-based PD fluids inhibited complement opsonization of S. aureus (≥7-fold reduction) and inhibited S. aureus-induced generation of anaphylatoxins C3a and C5a (>10-fold reduction) compared to non-glucose based PD fluids. Dianeal 1.5%, 2.5% and 4.25%, all similarly inhibited C3-mediated opsonization. Glucose-based PD fluids showed a ≥4-fold reduction in opsonization of clinical strains of S.aureus, including MRSA strains. Decreased opsonization of S.aureus in the glucose-based PD fluid compared with non-glucose based fluids correlated with decreased phagocytosis by neutrophils. CONCLUSION: Complement-mediated opsonophagocytosis of S. aureus and anaphylatoxin generation were severely inhibited in glucose-based PD fluids compared with non-glucose-based PD fluids. By inhibiting complement host defenses, glucose-based PD fluids may increase the risk of and severity of S. aureus peritonitis for CPD patients using these fluids.

Peptide Inhibitor of Complement C1 (PIC1) Rapidly Inhibits Complement Activation after Intravascular Injection in Rats.

The complement system has been increasingly recognized to play a pivotal role in a variety of inflammatory and autoimmune diseases. Consequently, therapeutic modulators of the classical, lectin and alternative pathways of the complement system are currently in pre-clinical and clinical development. Our laboratory has identified a peptide that specifically inhibits the classical and lectin pathways of complement and is referred to as Peptide Inhibitor of Complement C1 (PIC1). In this study, we determined that the lead PIC1 variant demonstrates a salt-dependent binding to C1q, the initiator molecule of the classical pathway. Additionally, this peptide bound to the lectin pathway initiator molecule MBL as well as the ficolins H, M and L, suggesting a common mechanism of PIC1 inhibitory activity occurs via binding to the collagen-like tails of these collectin molecules. We further analyzed the effect of arginine and glutamic acid residue substitution on the complement inhibitory activity of our lead derivative in a hemolytic assay and found that the original sequence demonstrated superior inhibitory activity. To improve upon the solubility of the lead derivative, a pegylated, water soluble variant was developed, structurally characterized and demonstrated to inhibit complement activation in mouse plasma, as well as rat, non-human primate and human serum in vitro. After intravenous injection in rats, the pegylated derivative inhibited complement activation in the blood by 90% after 30 seconds, demonstrating extremely rapid function. Additionally, no adverse toxicological effects were observed in limited testing. Together these results show that PIC1 rapidly inhibits classical complement activation in vitro and in vivo and is functional for a variety of animal species, suggesting its utility in animal models of classical complement-mediated diseases.

Complement inhibition significantly decreases red blood cell lysis in a rat model of acute intravascular hemolysis.

BACKGROUND: Prevention of acute hemolytic transfusion reactions is a worldwide concern. The objective of this study was to develop a simple rat model of complement-mediated acute intravascular hemolysis. STUDY DESIGN AND METHODS: Human AB red blood cells (RBCs) were incubated with complement-sufficient or complement-deficient Wistar rat serum (WRS) in the presence and absence of human RBC antibody in vitro to elucidate the mechanism of hemolysis. To study the role of complement in acute intravascular hemolysis in vivo, Wistar rats were treated either with or without cobra venom factor (CVF) to deplete complement activity. Human AB RBCs were then injected into both groups of rats, followed by serial blood draws up to 2 hours. Venous blood clearance and lysis of transfused RBCs at each time point were measured by flow cytometry and spectrophotometry. RBC sequestration was determined in the liver, spleen, and kidney by immunohistochemistry. RESULTS: In vitro incubation of human RBCs with WRS demonstrated that RBC lysis was mediated via the classical complement pathway and that hemolysis was antibody dependent. Transfusion of human RBCs into rats showed significantly less hemolysis in the CVF group versus untreated group. RBC sequestration in the spleen and liver 2 hours posttransfusion were not quantitatively different between the two groups. CONCLUSIONS: Given the much higher degree of similarity for rat and human complement compared to mice, this simple rat model is ideal for testing novel inhibitors of classical pathway activation for the prevention and treatment of acute intravascular hemolysis.

Acute MRSA sinusitis with intracranial extension and marginal vancomycin susceptibility.

Methicillin resistant Staphylococcus aureus (MRSA) is increasingly being described as a cause of acute sinusitis. We present a patient with acute MRSA sinusitis complicated by rapid intracranial extension, marginal vancomycin susceptibility (MIC = 2 mg/L), delayed drainage of intracranial abscess, and subsequent development of rifampin resistance. Given the relatively high risk of intracranial extension of severe acute bacterial sinusitis and high mortality associated with invasive MRSA infections, we suggest early surgical drainage of intracranial abscesses in these circumstances. We believe this is important given the limited intracranial penetration of currently available treatment options for MRSA, especially those with a vancomycin minimal inhibitory concentration (MIC) of ≥2 mg/L.