Safety Evaluation of Lipid Nanoparticle-Formulated Modified mRNA in the Sprague-Dawley Rat and Cynomolgus Monkey.

The pharmacology, pharmacokinetics, and safety of modified mRNA formulated in lipid nanoparticles (LNPs) were evaluated after repeat intravenous infusion to rats and monkeys. In both species, modified mRNA encoding the protein for human erythropoietin (hEPO) had predictable and consistent pharmacologic and toxicologic effects. Pharmacokinetic analysis conducted following the first dose showed that measured hEPO levels were maximal at 6 hours after the end of intravenous infusion and in excess of 100-fold the anticipated efficacious exposure (17.6 ng/ml) at the highest dose tested.24 hEPO was pharmacologically active in both the rat and the monkey, as indicated by a significant increase in red blood cell mass parameters. The primary safety-related findings were caused by the exaggerated pharmacology of hEPO and included increased hematopoiesis in the liver, spleen, and bone marrow (rats) and minimal hemorrhage in the heart (monkeys). Additional primary safety-related findings in the rat included mildly increased white blood cell counts, changes in the coagulation parameters at all doses, as well as liver injury and release of interferon γ-inducible protein 10 in high-dose groups only. In the monkey, as seen with the parenteral administration of cationic LNPs, splenic necrosis and lymphocyte depletion were observed, accompanied with mild and reversible complement activation. These findings defined a well-tolerated dose level above the anticipated efficacious dose. Overall, these combined studies indicate that LNP-formulated modified mRNA can be administered by intravenous infusion in 2 toxicologically relevant test species and generate supratherapeutic levels of protein (hEPO) in vivo.

Nitinol Memory Rods Versus Titanium Rods: A Biomechanical Comparison of Posterior Spinal Instrumentation in a Synthetic Corpectomy Model.

STUDY DESIGN: Biomechanical investigation. OBJECTIVE: To compare the biomechanical performance of nitinol memory metal rods and titanium rods when used as posterior spinal instrumentation in a synthetic model. METHODS: Biomechanical testing was performed using ultra-high-molecular-weight polyethylene blocks. Nineteen spinal constructs were created to allow comparison of 5.5-mm nitinol rods with 5.5-mm titanium rods. Static compression and rotational testing were performed on an Instron 8874 and Instron 4202 at 37°C to simulate body temperature. RESULTS: The average titanium construct stiffness under static compression or bending was 47.2 ± 9.1 N/mm while nitinol's stiffness averaged 48.9 ± 12.4 N/mm (P = .83). During axial rotation testing, the nitinol rod system showed no torsional stiffness difference from the titanium system: 0.95 ± 0.03 Nm/deg versus 0.96 ± 0.17 Nm/deg, respectively (P = 0.91). There was a statistically significant difference between the average torsional yield point for the titanium constructs (14.4 ± 1.6 Nm/deg) and nitinol constructs (21.3 ± 0.8 Nm/deg) (P = .004). The torsional toughness of the nitinol constructs was also statistically greater than the titanium rods: 473 GN/m3 versus 784 GN/m3 (P = .0006). There was no statistically significant difference between the nitinol group sustaining a higher number of fatigue cycles until failure and the titanium group (181 660 cycles vs 64 104 cycles, respectively, P = .22). CONCLUSIONS: This study provides biomechanical evidence that nitinol rods used in a posterior construct are comparable to titanium rods with regard to compression and have increased torsional failure load and torsional toughness. While nitinol trended toward superior fatigue resistance, there was no significant difference in nitinol versus titanium construct fatigue resistance.

Addressing the challenge of community reentry among released inmates with serious mental illness.

The purpose of the paper is to discuss the formidable challenges to community reentry and reintegration faced by U.S. prison inmates with serious mental illness and to describe various strategies for improving transitional services for these individuals. We review epidemiologic data supporting the high prevalence of severe mental illness in U.S. prisons as well as the historical factors underlying the criminalization of the mentally ill. The importance and challenges of providing adequate psychiatric care for mentally ill prisoners during their incarceration are discussed. We also review the numerous psychosocial and economic challenges confronting these individuals upon their release from prison, such as unemployment and vulnerability to homelessness, as well as specific barriers they may encounter in attempting to access community-based mental health services. We follow with a discussion of some of the more promising strategies for improving the transition of the mentally ill from prison to the community. In the final sections, we review the evidence for a relationship between serious mental illness and recidivism and briefly discuss emerging alternatives to incarceration of the mentally ill.

Impact of mRNA chemistry and manufacturing process on innate immune activation.

Messenger RNA (mRNA) represents an attractive therapeutic modality for potentially a wide range of clinical indications but requires uridine chemistry modification and/or tuning of the production process to prevent activation of cellular innate immune sensors and a concomitant reduction in protein expression. To decipher the relative contributions of these factors on immune activation, here, we compared, in multiple cell and in vivo models, mRNA that encodes human erythropoietin incorporating either canonical uridine or N1-methyl-pseudouridine (1mΨ), synthesized by either a standard process shown to have double-stranded RNA (dsRNA) impurities or a modified process that yields a highly purified mRNA preparation. Our data demonstrate that the lowest stimulation of immune endpoints was with 1mΨ made by the modified process, while mRNA containing canonical uridine was immunostimulatory regardless of process. These findings confirm that uridine modification and the reduction of dsRNA impurities are both necessary and sufficient at controlling the immune-activating profile of therapeutic mRNA.

Accelerated Blood Clearance of Lipid Nanoparticles Entails a Biphasic Humoral Response of B-1 Followed by B-2 Lymphocytes to Distinct Antigenic Moieties.

Accelerated blood clearance (ABC) is a phenomenon in which certain pharmaceutical agents are rapidly cleared from the blood upon second and subsequent administrations. ABC has been observed for many lipid-delivery vehicles, including liposomes and lipid nanoparticles (LNP). Previous studies have demonstrated a role for humoral responses against the polyethylene glycol motifs in clearance, but significant gaps remain in our understanding of the mechanism of ABC, and strategies for limiting the impact of ABC in a clinical setting have been elusive. mRNA therapeutics have great promise, but require chronic administration in encapsulating delivery systems, of which LNP are the most clinically advanced. In this study, we investigate the mechanisms of ABC for mRNA-formulated LNP in vivo and in vitro. We present evidence that ABC of mRNA-formulated LNP is dramatic and proceeds rapidly, based on a previously unrecognized ability of LNP to directly activate B-1 lymphocytes, resulting in the production of antiphosphorylcholine IgM Abs in response to initial injection. Upon repeated injections, B-2 lymphocytes also become activated and generate a classic anti-polyethylene glycol adaptive humoral response. The ABC response to phosphorylcholine/LNP-encapsulated mRNA is therefore a combination of early B-1 lymphocyte and later B-2 lymphocyte responses.

Immediate versus delayed repair of destructive bowel injuries in patients with an open abdomen.

Trauma surgeons frequently encounter destructive bowel injuries. The timing of the repair of the bowel injury should be performed in patients with planned open abdomen management and second-look laparotomy has not been specifically addressed. Our primary objective was to determine if there was a significant difference in the incidence of major complications between immediate and delayed repair among patients with traumatic bowel injuries and planned open abdomens. This was a retrospective cohort study of adult patients with traumatic bowel injuries treated between 2001 and 2011 and who underwent laparotomy and were left with an open abdomen with a planned second operation. Pediatric patients (age less than 15 years) and patients who died in the first 24 hours of admission were excluded. The primary exposure of interest was dichotomously defined based on either definitive repair of the bowel injury during the initial trauma operation (immediate) or definitive repair during a subsequent surgery (delayed). Major complications were defined as enterocutaneous fistula, dehiscence, and abscess. Ninety-two patients met study eligibility. Of these, 50 (54%) underwent immediate bowel repair. Univariate analysis suggested no significant differences in the proportion of major complications between the two groups. After adjusting for Injury Severity Score, penetrating injury, initial base deficit, and presence of colon injury, there was no statistical difference in incidence of major complications between the two groups. Patients undergoing immediate Versus delayed repair of traumatic bowel injuries and who are left with an open abdomen have comparable outcomes in terms of major complications.

Prior exposure to phencyclidine decreases voluntary sucrose consumption and operant performance for food reward.

Prior exposure to the psychotomimetic drug phencyclidine (PCP) produces a number of schizophrenia-like behaviors in animals. The goal of the present study was to determine whether prior exposure to PCP produces decreased reward function, thereby modeling one aspect of negative schizophrenic symptomatology. To this aim, the consequences of prior exposure to PCP were assessed on two types of appetitive consumptive behavior. In the first set of experiments, the effects of PCP (15 mg/kg, 20 h before testing) on sucrose consumption were tested for three consecutive days under conditions of deprivation and nondeprivation. In the deprivation condition, animals were water deprived for 4 h prior to injection of PCP or saline (SAL). Twenty hours following the injection (24 h after the onset of water deprivation), animals were allowed access to either 5% sucrose or water for 30 min. In the nondeprivation condition, 5% sucrose consumption was measured for 30 min, 20 h after PCP or SAL injection and water consumption was measured during the 23.5 h preceding sucrose consumption. PCP decreased both sucrose and water consumption under deprivation conditions on the second and third day of testing but selectively decreased sucrose consumption under nondeprivation conditions on all three testing days. LiCl (50 mg/kg, 20 h before testing) did not significantly reduce sucrose consumption in the nondeprivation paradigm, indicating that the effect of PCP was not due to conditioned taste aversion. In the second experiment, PCP (15 mg/kg, 20 h before testing) decreased operant performance when animals were switched from a continuous reinforcement schedule of food delivery to a fixed ratio (FR4) schedule. Apomorphine (APO, 30 microg/kg, 30 min before testing), a positive control, induced a similar performance deficit. However, the PCP-induced deficit was not apparent until the third day of FR4 testing while the APO deficit was apparent on the first day. The effects of PCP on sucrose consumption demonstrate PCP-induced decreases in reward function. However, the delayed appearance of the PCP-induced decrease in operant performance suggests that these results may be better explained by a PCP-induced attentional deficit, also characteristic of schizophrenic psychopathology.

Activation of nociceptive neurons in T9 and T10 in cerulein pancreatitis.

Mechanisms of pain transduction in acute pancreatitis are poorly understood. Increased Fos expression in the spinal cord is a marker of activation of nociceptive neurons. We hypothesized that cerulein pancreatitis leads to increased Fos expression at T9 and T10, which receive sensory input from the pancreas. Rats were injected with cerulein (100 microg/kg, s.c.) or saline carrier (NS). Endpoints at 4, 6, and 10 h were serum amylase, myeloperoxidase activity (MPO), and spinal cord Fos expression (number of immunoreactive nuclei/section dorsal gray matter). Fos-like immunoreactivity (FLI) at T9-T10 was compared to internal controls (T6, T12). An average of 20 spinal cord histologic sections were evaluated per rat. Some animals were injected with the mu-opioid receptor agonist, buprenorphine (90 microg/kg, s.c.), 3 h after cerulein, and their endpoints were measured at 6 h. Analysis of variance and t tests were used for statistical analysis. Results are means +/- SEM. As expected, cerulein induced edematous pancreatitis, with a 4-fold increase in serum amylase at 6 h [cer (n = 8): 14,000 +/- 1,300 U/ml versus NS (n = 10): 3,700 +/- 300, P < 0.005)] and a 2-fold increase in MPO activity (0.25 +/- 0.05) activity units/dry wt versus 0.13 +/- 0.02, P < 0.05). Cerulein induced nearly a 2-fold increase in FLI at T9 and T10 [n = 10 (cer) and n = 13 (NS): T9, 14 +/- 1.5 versus 7.8 +/- 0.88; T10, 15 +/- 1.7 versus 8.3 +/- 0.70; P < 0.05]. Peak effects of cerulein on FLI occurred at 6 h and were greatest at T9/T10 with relative sparing of T6/T12. T6/T12 expression was similar in experimental and control groups. Buprenorphine significantly reduced both serum amylase and FLI and T9/T10. Cerulein-induced acute pancreatitis in rat increases visceral nociceptive signaling at spinal cord levels T9 and T10, with a peak at 6 h. Blockade of this effect by the mu-opioid receptor agonist buprenorphine could occur either by direct activation of central opioid receptors and/or an anti-inflammatory mechanism. FLI is a useful tool for studying the pathophysiology of pain in experimental acute pancreatitis.