Bronchoalveolar Lavage and Oleic Acid Two-hit Model for Inducing Acute Respiratory Distress Syndrome in Swine Models.

INTRODUCTION: Acute respiratory distress syndrome (ARDS) is a widespread and often fatal clinical syndrome marked by the acute onset of pulmonary edema and inflammatory-mediated disruptions in alveolar-capillary permeability resulting in impaired gas exchange and tissue oxygenation with subsequent acute respiratory failure that accounts for 10.4% of all intensive care unit admissions worldwide and boasts a mortality rate of 38.5%. The current treatment for ARDS remains largely supportive. This is largely because of the many challenges of achieving a stable and sustainable animal model that recreates the pathophysiology of ARDS experimentally in a controlled setting to allow research to elucidate potential treatments of ARDS moving forward. MATERIALS AND METHODS: The bronchoalveolar lavage and oleic acid models are currently the 2 most frequently used experimental models in inducing ARDS in animal models. This study demonstrated that combining them into a "two-hit model" can produce sustained ARDS in swine models per the Horowitz index (PaO2/FiO2 ratio of ≤300 mmHg). Additionally, expected changes in pH, pCO2, lung compliance, cytokines, and tissue histopathology were observed and add to our confidence and reliability that the "two-hit model" produces symptomatic ARDS in a manner very similar to that observed in humans. RESULTS AND CONCLUSIONS: In conclusion, we demonstrated a viable animal model of human ARDS that is maintained for a prolonged period, suitable for continuous monitoring of the progression, and evaluation of potential future treatments and procedures to reduce patient morbidity and mortality. To carry out this two-hit model, lung injury was induced through a combination of bronchoalveolar lavage and oleic acid administration and the disease process of ARDS is subsequently tracked through clinically relevant parameters such as respiratory mechanics, cytokine response, aretrial blood gas (ABG) changes, and observation of postmortem histopathologic changes. This promising new model has the capacity to successfully replicate human ARDS which is a well-known and notoriously multifactorial pathogenic process to reproduce experimentally for an extended period of time. The "two-hit model" is a viable and appropriate model for the research of novel treatments for ARDS.

Next-Generation REBOA (Resuscitative Endovascular Balloon Occlusion of the Aorta) Device Precisely Achieves Targeted Regional Optimization in a Porcine Model of Hemorrhagic Shock.

INTRODUCTION: Limitations such as time-dependent distal ischemia have slowed the adoption of resuscitative endovascular balloon occlusion of the aorta (REBOA) for noncompressible hemorrhage. Next-generation REBOA technologies may allow for controlled partial flow, known as targeted regional optimization, to reduce distal ischemia. We aimed to characterize the efficacy of one such catheter in a porcine model of lethal hemorrhagic shock. METHODS: Noncompressible hemorrhage from an iliac injury was induced in anesthetized swine (Sus scrofa) (70-90 kg), targeting 30% total blood volume. Animals were then randomized to partial aortic occlusion (PO) with targeted distal mean arterial pressure (MAP) of 35-40 mm of mercury (mm Hg) and complete aortic occlusion (CO) (n = 8 per group) for 90 min. All groups were then resuscitated during a two-h critical care (CC) phase, with flow rate and MAP recorded continuously at the distal infrarenal aorta and proximal carotid artery, and analyzed with two-way repeated measures analysis of variance with S-N-K post-hoc test. RESULTS: During aortic occlusion, MAP distal to the balloon was consistently maintained at 35.8 ± 0.3 mm Hg in the PO group compared to 27.1 ± 0.3 mm Hg in the CO group (P < 0.05), which also corresponded to higher flow rates (202.9 ± 4.8 mL/min PO versus 25.9 ± 0.8 mL/min CO; P < 0.05). MAP proximal to the balloon was significantly higher with CO versus PO (109.2 ± 2.3 mm Hg versus 85.2 ± 2.3 mm Hg; P < 0.05). During the CC phase, distal aortic flow and MAP were not significantly different between groups. However, creatinine returned to baseline levels by the end of the study in the PO group, but not the CO group. One animal died in the CO group, whereas none died in the PO group. CONCLUSIONS: This is the first examination of the next-generation pREBOA-PRO in a porcine model of lethal hemorrhagic shock. We show technical feasibility of this technique to precisely achieve targeted regional optimization without device failure or complication. The ability to titrate balloon inflation and thus distal flow/pressure may extend the therapeutic window of REBOA by mitigating distal ischemia.

Locking and Unlocking Thrombin Function Using Immunoquiescent Nucleic Acid Nanoparticles with Regulated Retention In Vivo.

The unbalanced coagulation of blood is a life-threatening event that requires accurate and timely treatment. We introduce a user-friendly biomolecular platform based on modular RNA-DNA anticoagulant fibers programmed for reversible extracellular communication with thrombin and subsequent control of anticoagulation via a "kill-switch" mechanism that restores hemostasis. To demonstrate the potential of this reconfigurable technology, we designed and tested a set of anticoagulant fibers that carry different thrombin-binding aptamers. All fibers are immunoquiescent, as confirmed in freshly collected human peripheral blood mononuclear cells. To assess interindividual variability, the anticoagulation is confirmed in the blood of human donors from the U.S. and Brazil. The anticoagulant fibers reveal superior anticoagulant activity and prolonged renal clearance in vivo in comparison to free aptamers. Finally, we confirm the efficacy of the "kill-switch" mechanism in vivo in murine and porcine models.

Advanced partial occlusion controller allows for increased precision during targeted regional optimization in a porcine model of hemorrhagic shock.

BACKGROUND: Targeted regional optimization (TRO), a partial resuscitative endovascular balloon occlusion of the aorta strategy, may mitigate distal ischemia and extend the window of effectiveness for this adjunct. An automated device may allow greater control and precise regulation of flow past the balloon, while being less resource-intensive. The objective of this study was to assess the technical feasibility of the novel advanced partial occlusion controller (APOC) in achieving TRO at multiple distal pressures. METHODS: Female swine (n = 48, 68.1 ± 0.7 kg) were randomized to a target distal mean arterial pressure (MAP) of 25 mm Hg, 35 mm Hg, or 45 mm Hg by either manual (MAN) or APOC regulation (n = 8 per group). Uncontrolled hemorrhage was generated by liver laceration. Targeted regional optimization was performed for 85 minutes, followed by surgical control and a 6-hour critical care phase. Proximal and distal MAP and flow rates were measured continuously. RESULTS: At a target distal MAP of 25 mm Hg, there was no difference in the MAP attained (APOC: 26.2 ± 1.05 vs. MAN: 26.1 ± 1.78 mm Hg) but the APOC had significantly less deviance (10.9%) than manual titration (14.9%, p < 0.0001). Similarly, at a target distal MAP of 45 mm Hg, there was no difference in mean pressure (44.0 ± 0.900 mm Hg vs. 45.2 ± 1.31 mm Hg) but APOC had less deviance (9.34% vs. 11.9%, p < 0.0001). There was no difference between APOC and MAN in mean (34.6 mm Hg vs. 33.7 mm Hg) or deviance (9.95% vs. 10.4%) at a target distal MAP of 35 mm Hg, respectively. The APOC made on average 77 balloon volume adjustments per experiment compared with 29 by manual titrations. CONCLUSION: The novel APOC consistently achieved and sustained precisely regulated TRO across all groups and demonstrated reduced deviance at the 25 mm Hg and 45 mm Hg groups compared with manual titration.

Implementation of the Uniformed Services University Pain Registry Biobank: A Military and Veteran Population-Focused Biobank and Registry.

OBJECTIVE: The objective of this overview is to discuss the development, implementation, data content, and structure of the Uniformed Services University Pain Registry Biobank. Additionally, procedures and policies for accessing samples for pain-related research purposes are detailed. DESIGN: Cross-sectional overview. SETTING: Multiple military treatment facilities. SUBJECTS: Adult beneficiaries seeking care within the Military Health System. METHODS: Participants complete a baseline battery of biopsychosocial survey measures, including Patient-Reported Outcomes Measurement Information System (PROMIS®) measures, and provide biological samples (e.g., blood and saliva). Relevant health history, including medications and surgical history, is extracted from medical records. During the course of the subsequent year, enrolled participants complete surveys and provide biological samples at 3 months, 6 months, and 12 months. Thereafter, participants are contacted once annually to complete self-reported assessments and provide biological samples. RESULTS: In the first year alone, 86 subjects have participated in the Uniformed Services University Pain Registry Biobank and provided 390 observations (e.g., biological samples and biopsychosocial patient-reported outcomes). The Uniformed Services University Pain Registry Biobank's integration of biological samples, patient-reported outcomes, and health record data over a longitudinal period across a diverse sample recruited from multiple military facilities addresses many of the limitations faced by other pain-related registries or biorepositories. CONCLUSIONS: The Uniformed Services University Pain Registry Biobank will serve as a platform for conducting research closely aligned with the Federal Pain Research Strategy. The inclusion of active duty service members, beneficiaries, and civilians living with and without acute or chronic pain provides a unique data repository for all investigators interested in advancing pain science.

The Critical Choice of Animal Models in Nanomedicine Safety Assessment: A Lesson Learned From Hemoglobin-Based Oxygen Carriers.

Intravenous injection of nanopharmaceuticals can induce severe hypersensitivity reactions (HSRs) resulting in anaphylactoid shock in a small percentage of patients, a phenomenon explicitly reproducible in pigs. However, there is a debate in the literature on whether the pig model of HSRs can be used as a safety test for the prediction of severe adverse reactions in humans. Given the importance of using appropriate animal models for toxicity/safety testing, the choice of the right species and model is a critical decision. In order to facilitate the decision process and to expand the relevant information regarding the pig or no pig dilemma, this review examines an ill-fated clinical development program conducted by Baxter Corporation in the United States 24 years ago, when HemeAssist, an αα (diaspirin) crosslinked hemoglobin-based O2 carrier (HBOC) was tested in trauma patients. The study showed increased mortality in the treatment group relative to controls and had to be stopped. This disappointing result had far-reaching consequences and contributed to the setback in blood substitute research ever since. Importantly, the increased mortality of trauma patients was predicted in pig experiments conducted by US Army scientists, yet they were considered irrelevant to humans. Here we draw attention to that the underlying cause of hemoglobin-induced aggravation of hemorrhagic shock and severe HSRs have a common pathomechanism: cardiovascular distress due to vasoconstrictive effects of hemoglobin (Hb) and reactogenic nanomedicines, manifested, among others, in pulmonary hypertension. The main difference is that in the case of Hb this effect is due to NO-binding, while nanomedicines can trigger the release of proinflammatory mediators. Because of the higher sensitivity of cloven-hoof animals to this kind of cardiopulmonary distress compared to rodents, these reactions can be better reproduced in pigs than in murine or rat models. When deciding on the battery of tests and the appropriate models to identify the potential hazard for nanomedicine-induced severe HSR, the pros and cons of the various species must be considered carefully.

Invasive mechanical ventilation using a bilevel PAP ST device in a healthy swine model.

PURPOSE: The Coronavirus Disease 2019 (COVID-19) pandemic may cause an acute shortage of ventilators. Standard noninvasive bilevel positive airway pressure devices with spontaneous and timed respirations (bilevel PAP ST) could provide invasive ventilation but evidence on their effectiveness in this capacity is limited. We sought to evaluate the ability of bilevel PAP ST to effect gas exchange via invasive ventilation in a healthy swine model. METHODS: Two single limb respiratory circuits with passive filtered exhalation were constructed and evaluated. Next, two bilevel PAP ST devices, designed for sleep laboratory and home use, were tested on an intubated healthy swine model using these circuits. These devices were compared to an anesthesia ventilator. RESULTS: We evaluated respiratory mechanics, minute ventilation, oxygenation, and presence of rebreathing for all of these devices. Both bilevel PAP ST devices were able to control the measured parameters. There were noted differences in performance between the two devices. Despite these differences, both devices provided effective invasive ventilation by controlling minute ventilation and providing adequate oxygenation in the animal model. CONCLUSIONS: Commercially available bilevel PAP ST can provide invasive ventilation with a single limb respiratory circuit and in-line filters to control oxygenation and ventilation without significant rebreathing in a swine model. Further study is needed to evaluate safety and efficacy in clinical disease models. In the setting of a ventilator shortage during the COVID-19 pandemic, and in other resource-constrained situations, these devices may be considered as an effective alternative means for invasive ventilation.

Feasibility of App-Based Postsurgical Assessment of Pain, Pain Impact, and Regional Anesthesia Effects: A Pilot Randomized Controlled Trial.

OBJECTIVE: Postsurgical follow-up calls enable nurses to assess a patient's condition, provide tailored education, and improve the patient's experience. Despite the benefits, barriers to phone-based assessments may include patient nonresponse and lack of time due to demanding clinical schedules. The purpose of this trial was to examine the feasibility and utility of a smartphone app, mCare, for assessing pain, pain impact, and peripheral nerve block effects in patients. DESIGN: Pilot randomized control trial. SETTING AND PATIENTS: Eligible patients at a military treatment facility undergoing same-day surgery were randomized to the mCare group (N = 24) or the standard-of-care telephone (N = 26) group. RESULTS: Outcomes included initial response (assessment completion) rates and participant and nurse satisfaction. There were no differences in the response rates upon initial contact attempt, and patients in both groups reported similar levels of satisfaction and convenience. Nurses reported greater satisfaction with the app compared with standard-of-care telephone calls. CONCLUSIONS: Before wider implementation, further considerations of app-based assessment need to be fully explored.

Predosing Chemical Stability of Admixtures of Propofol, Ketamine, Fentanyl, and Remifentanil.

Admixtures of propofol-ketamine, propofol-ketamine-fentanyl, and propofol-ketamine-remifentanil were subjected to various clinically relevant conditions to study their chemical stability. A novel high-performance liquid chromatography-mass spectrometry method revealed no degradation of any compound by incubation at 37°C, constant mixing, or table-top storage for 6- and 24-hour time periods, except variable recovery of both propofol and fentanyl in the admixtures of propofol-ketamine-fentanyl suggesting possible degradation.

Hypersensitivity reactions to intravenous lipid emulsion in swine: relevance for lipid resuscitation studies.

BACKGROUND: Reports in the recent experimental literature have provided contradicting results in different animal species regarding the efficacy of IV lipid emulsion (ILE) in the reversal of cardiovascular and central nervous system symptoms of local anesthetic and other lipophilic drug overdoses. In particular, ILE seemed to be effective in rats, rabbits, dogs, and humans, but not in swine, for which it not only failed to reverse the adverse effects of anesthetics, but the animals also developed a generalized cutaneous mottling or a dusky appearance immediately after ILE, suggestive of another type of toxicity. The latter symptoms arise in complement (C) activation-related pseudoallergy, a hypersensitivity reaction to particulate drugs and agents. METHODS: Ten Yorkshire swine (15-20 kg) were sedated with ketamine and anesthetized with isoflurane. ILE 1.5 and 5 mL/kg 20% was administered via the ear vein while pulmonary arterial pressure, systemic arterial blood pressure, electrocardiogram, and end-tidal CO2 were recorded continuously. Thromboxane was measured in blood collected at baseline and 2 and 10 minutes after injections. Complement activation by lipid emulsion was also assessed in vitro with soluble terminal complement complex (SC5b-9) and sheep red blood cell assays. RESULTS: Significant increases were observed in the pulmonary pressure (median [interquartile range]) within minutes after the administration of ILE, both at doses 1.5 and 5 mL/kg (15 [12-16.5] to 18.5 [16-20] mm Hg, P = 0.0058 and 15.5 [13-17.25] to 39.5 [30.5-48.5], respectively). The systemic arterial blood pressure increased, and the heart rate decreased after both injections. Thromboxane B2 concentration (median [interquartile range]) in the blood plasma increased from a baseline of 617.3 [412.4-920] to 1132 [597.9-1417] pg/mL (P = 0.0055) and from 1276 [1200-2581] to 4046 [2946-8442] pg/mL (P = 0.0017) after the administration of 1.5 and 5 mL/kg ILE, respectively. Intralipid did not cause in vitro complement activation in human serum. CONCLUSIONS: ILE causes clinically significant hemodynamic changes in pigs, in concert with significant increases in the plasma thromboxane concentration. However, the in vitro tests did not confirm involvement of the complement system in human sera, leaving the underlying mechanism of these findings in doubt. Nonetheless, the observed hemodynamic and biochemical effects of ILE serve as a caveat that the pig is not an ideal model for the study of interventions involving ILE.

Improving awareness of nonanesthesia-related malignant hyperthermia presentations: a tale of two brothers.

A 30-year-old man developed unexplained rhabdomyolysis, persistently increased creatine kinase and severe debilitating muscle cramps. After a nondiagnostic neurologic evaluation, he was referred for a muscle biopsy, to include histology/histochemistry, a myoglobinuria panel, and a caffeine halothane contracture test. Only the caffeine halothane contracture test was positive, and a subsequent ryanodine receptor type 1 gene evaluation revealed a mutation functionally causative for malignant hyperthermia. His identical twin brother, who was suffering from similar complaints, was found to share the same mutation. They each require oral dantrolene therapy to control symptoms, despite difficulty in identifying health care providers familiar with treating this disorder.

Multiacquisition T1-mapping MRI during tidal respiration for quantification of myocardial T1 in swine with heart failure.

OBJECTIVE: The purpose of this article is to evaluate a free-breathing pulse sequence to quantify myocardial T1 changes in a swine model of tachycardia-induced heart failure. MATERIALS AND METHODS: Yorkshire swine were implanted with pacemakers and were ventricularly paced at 200 beats/min to induce heart failure. Animals were scanned twice with a 1.5-T MRI scanner, once at baseline and once at heart failure. A T1-mapping sequence was performed during tidal respiration before and 5 minutes after the administration of a gadolinium-chelate contrast agent. T1-mapping values were compared between the baseline and heart failure scans. The percentage of fibrosis of heart failure myocardial tissue was compared with similar left ventricular tissue from control animals using trichrome blue histologic analysis. RESULTS: In the study cohort, differences were found between the baseline and heart failure T1-mapping values before the administration of contrast agent (960 ± 96 and 726 ± 94 ms, respectively; p = 0.02) and after contrast agent administration (546 ± 180 and 300 ± 171 ms, respectively; p = 0.005). The animals with heart failure also had a difference histologically in the percentage of myocardial collagen compared with tissue from healthy control animals (control, 5.4% ± 1.0%; heart failure, 9.4% ± 1.6%; p < 0.001). CONCLUSION: The proposed T1-mapping technique can quantify diffuse myocardial changes associated with heart failure without the use of a contrast agent and without breath-holding. These T1 changes appear to be associated with increases in the percentage of myocardial collagen that in this study were not detected by traditional myocardial delayed enhancement imaging. T1 mapping may be a useful technique for detecting early but clinically significant myocardial fibrosis.

A porcine model of complement-mediated infusion reactions to drug carrier nanosystems and other medicines.

Intravenous administration of low (milligram) doses of nanoparticulate materials in pigs can lead to acute cardiopulmonary, hemodynamic, hematological, biochemical and dermatological changes within minutes, mimicking the human infusion (or anaphylactoid) reactions to many state-of-the-art (nano)medicines and biologicals. Because of the causal role of complement (C) activation, the phenomenon was called C activation-related pseudoallergy (CARPA). This review summarizes the available information on porcine CARPA caused by different liposomes and polymers. It provides methodical details of the model and addresses the quantitation, sensitivity, specificity, reproducibility and variability of symptoms caused by different reactogenic drugs. We describe a unique feature of the model: the rise of tachyphylaxis (self-induced tolerance) as a function of structural properties of reactogenic agents. For drugs that cause tachyphylactic CARPA, such as liposomal doxorubicin (Doxil), the review recapitulates a recently reported method of desensitization, which may prevent this, as well as many similar hypersensitivity reactions. In explaining the underlying mechanism of tachyphylactic CARPA, a new theory on "double hit" is outlined, wherein the pulmonary intravascular macrophages (PIM cells) of pigs give aggravated response to simultaneous stimulation of their anaphylatoxin and other surface receptors (e.g., toll-like, PAMP, DAMP or mannose) that recognize vesicle surface molecular patterns. The porcine CARPA model might provide unique advantages in studying the mechanism of severe hypersensitivity reactions in man to i.v. drugs, as well as in identifying drugs and drug carriers that may cause such reactions.

Prevention of infusion reactions to PEGylated liposomal doxorubicin via tachyphylaxis induction by placebo vesicles: a porcine model.

PEGylated liposomal doxorubicin (Doxil) has been used in cancer chemotherapy for 16 years. Clinical experience shows that it can cause mild-to-severe hypersensitivity (infusion) reactions, which are manifestations of complement (C) activation-related pseudoallergy (CARPA). Although in most cases CARPA is inconsequential, a main symptom, cardiopulmonary distress, may be life threatening in hypersensitive individuals. To date, the prevention of Doxil-induced CARPA is based on premedication and a slow infusion protocol. The present study suggests desensitization by Doxil-like empty liposomes, called placebo Doxil (Doxebo), as an alternative strategy, which is based on the tachyphylactic nature of Doxil reactions. Doxebo-induced tolerance to Doxil was shown to develop within minutes and to be specific to Doxil-like PEGylated liposomes. The procedure of desensitization involves slow, low-dose pre-infusion of Doxebo before Doxil treatment which minimizes the ensuing physiological changes or keeps them subclinical. Although the mechanism of tolerance induction is not yet clear, the effector arm of C response is unlikely to be affected, as the vascular reactivity of desensitized pigs to zymosan remains intact. Desensitization with empty vesicles represents a novel approach for reducing the risk of anaphylactic reactions to drug carrier liposomes. The underlying immediate, most likely passive silencing of an innate immune response may represent a novel mechanism of tolerance induction which may work for other reactogenic nanosystems as well.

Liposome-induced complement activation and related cardiopulmonary distress in pigs: factors promoting reactogenicity of Doxil and AmBisome.

Hypersensitivity reactions to liposomal drugs, often observed with Doxil and AmBisome, can arise from activation of the complement (C) system by phospholipid bilayers. To understand the mechanism of this adverse immune reaction called C activation-related pseudoallergy (CARPA), we analyzed the relationship among liposome features, C activation in human serum in vitro, and liposome-induced cardiovascular distress in pigs, a model for human CARPA. Among the structural variables (surface charge, presence of saturated, unsaturated, and PEGylated phospholipids, and cisplatin vs. doxorubicin inside liposomes), high negative surface charge and the presence of doxorubicin were significant contributors to reactogenicity both in vitro and in vivo. Morphological analysis suggested that the effect of doxorubicin might be indirect, via distorting the sphericity of liposomes and, if leaked, causing aggregation. The parallelism among C activation, cardiopulmonary reactions in pigs, and high rate of hypersensitivity reactions to Doxil and AmBisome in humans strengthens the utility of the applied tests in predicting the risk of CARPA. FROM THE CLINICAL EDITOR: The authors studied complement activation-related pseudoallergy (CARPA) in a porcine model and demonstrate that high negative surface charge and drug effects leading to distortion of liposome sphericity might be the most critical factors leading to CARPA. The applied tests might be used to predict CARPA in humans.

The effect of lipid emulsion infusion on postmortem ropivacaine concentrations in swine: endeavoring to comprehend a soldier's death.

BACKGROUND: Lipid emulsion (20%) is advocated as a rescue drug for local anesthetic toxicity. No study has measured the impact of lipid emulsion therapy on postmortem local anesthetic serum levels. METHODS: We anesthetized Yorkshire swine (n = 11) and standard monitors were placed. The swine received 1.5 mg/kg/min IV ropivacaine until death (asystole). Blood samples were drawn before infusion (baseline) and at 5-minute intervals during the infusion for measurement of blood gases and free, bound, and total serum ropivacaine concentrations via high-performance liquid chromatography. Five swine received ropivacaine only, and 6 swine received ropivacaine plus a single bolus dose of 20% lipid emulsion (1 mg/kg) when the mean arterial blood pressure reached 50 mm Hg. Ropivacaine infusions were terminated at asystole and no resuscitation was initiated. Total ropivacaine dose and time to death were recorded. The swine were cooled (mean temperature, 25.5°C ± 0.8°C at 6 hours postmortem) to reflect morgue conditions. Serum samples were drawn at asystole, 1, 3, and 6 hours postmortem for analysis. Additionally, a craniotomy and laparotomy were performed at those times to remove 1.5 to 3 g each of brain, lung, liver, kidney, and muscle for analysis. RESULTS: Analysis of the postmortem serum ropivacaine concentrations in the control and the lipid-treated animals indicated that both the total (bound and not bound to proteins) and free (not bound to proteins) ropivacaine concentrations were significantly higher in the lipid-treated animals (P = 0.0094 and P = 0.0063, respectively). Furthermore, time had a significant effect on increasing the postmortem free ropivacaine concentrations (P = 0.0095). The lipid group had a statistically significant earlier onset of death (asystole) compared with the control group (P = 0.0274). Tissue analysis indicated that the ropivacaine concentration significantly decreased postmortem in the lung, kidney, and brain tissues of the lipid-treated animals (P = 0.0168, P = 0.0073, and P = 0.0018, respectively). Tissue drug concentrations in the control animals remained unchanged after death. CONCLUSIONS: Our data show that postmortem blood samples in swine that experience local anesthetic cardiovascular collapse and are treated with lipid emulsions will result in measurements that cannot be directly extrapolated to premortem drug concentrations.

In vitro and in vivo complement activation and related anaphylactic effects associated with polyethylenimine and polyethylenimine-graft-poly(ethylene glycol) block copolymers.

Complement activation by polymeric gene and drug delivery systems has been overlooked in the past. As more reports appear in the literature concerning immunogenicity of polymers and their impact on gene expression patterns, it is important to address possible immune side effects of polymers, namely complement activation. Therefore, in this study the activity of low and high molecular weight poly(ethylene imine) and two PEGylated derivatives to induce complement activation were investigated in human serum. These in vitro results revealed that PEI 25 kDa caused significant and concentration dependent complement activation, whereas none of the other polymers induced such effects at their IC(50) concentrations determined by MTT-assays. To verify these in vitro results, additionally, studies were carried out in a swine model after intravenous administration, showing complement activation-related pseudoallergy (CARPA), reflected in symptoms of transient cardiopulmonary distress. Injections of PEI 25 kDa or PEI(25k)-PEG(2k)(10) at a dose of 0.05 and 0.1 mg/kg caused strong reactivity, while PEI 5 kDa and with PEI(25k)-PEG(20k)(1) were also reactogenic at 0.1 mg/kg. It was found that PEI 25 kDa caused both self- and cross-tolerance, whereas the PEG-PEIs were neither self- nor cross-reactively tachyphylactic. As a result of this study, it was shown that PEGylation of polycations with PEG of 20 kDa or higher molecular weight may be favorable. However, potential safety concerns in the development of PEI-based polymeric carriers for drugs and nucleic acids and their translation from bench to bedside need to be taken into consideration for human application.

Animal models of complement-mediated hypersensitivity reactions to liposomes and other lipid-based nanoparticles.

Intravenous injection of some liposomal drugs, diagnostic agents, micelles and other lipid-based nanoparticles can cause acute hypersensitivity reactions (HSRs) in a high percentage (up to 45%) of patients, with hemodynamic, respiratory and cutaneous manifestations. The phenomenon can be explained with activation of the complement (C) system on the surface of lipid particles, leading to anaphylatoxin (C5a and C3a) liberation and subsequent release reactions of mast cells, basophils and possibly other inflammatory cells in blood. These reactions can be reproduced and studied in pigs, dogs and rats, animal models which differ from each other in sensitivity and spectrum of symptoms. In the most sensitive pig model, a few miligrams of liposome (phospholipid) can cause anaphylactoid shock, characterized by pulmonary hypertension, systemic hypotension, decreased cardiac output and major cardiac arrhythmias. Pigs also display cutaneous symptoms, such as flushing and rash. The sensitivity of dogs to hemodynamic changes is close to that of pigs, but unlike pigs, dogs also react to micellar lipids (such as Cremophor EL) and their response includes pronounced blood cell and vegetative neural changes (e.g., leukopenia followed by leukocytosis, thrombocytopenia, fluid excretions). Rats are relatively insensitive inasmuch as hypotension, their most prominent response to liposomes, is induced only by one or two orders of magnitude higher phospholipid doses (based on body weight) compared to the reactogenic dose in pigs and dogs. It is suggested that the porcine and dog models are applicable for measuring and predicting the (pseudo)allergic activity of particulate "nanodrugs".