George Quick

Antidote-mediated control of an anticoagulant aptamer in vivo

Patient safety and treatment outcome could be improved if physicians could rapidly control the activity of therapeutic agents in their patients. Antidote control is the safest way to regulate drug activity, because unlike rapidly clearing drugs, control of the drug activity is independent of underlying patient physiology and co-morbidities. Until recently, however, there was no general method to discover antidote-controlled drugs. Here we demonstrate that the activity and side effects of a specific class of drugs, called aptamers, can be controlled by matched antidotes in vivo. The drug, an anticoagulant aptamer, systemically induces anticoagulation in pigs and inhibits thrombosis in murine models. The antidote rapidly reverses anticoagulation engendered by the drug, and prevents drug-induced bleeding in surgically challenged animals. These results demonstrate that rationally designed drug-antidote pairs can be generated to provide control over drug activities in animals.

Development of universal antidotes to control aptamer activity.

With an ever increasing number of people taking numerous medications, the need to safely administer drugs and limit unintended side effects has never been greater. Antidote control remains the most direct means to counteract acute side effects of drugs, but, unfortunately, it has been challenging and cost prohibitive to generate antidotes for most therapeutic agents. Here we describe the development of a set of antidote molecules that are capable of counteracting the effects of an entire class of therapeutic agents based upon aptamers. These universal antidotes exploit the fact that, when systemically administered, aptamers are the only free extracellular oligonucleotides found in circulation. We show that protein- and polymer-based molecules that capture oligonucleotides can reverse the activity of several aptamers in vitro and counteract aptamer activity in vivo. The availability of universal antidotes to control the activity of any aptamer suggests that aptamers may be a particularly safe class of therapeutics.

Increasing tidal volumes and pulmonary overdistention adversely affect pulmonary vascular mechanics and cardiac output in a pediatric swine model

OBJECTIVES In a pediatric swine model, the effects of increasing tidal volumes and the subsequent development of pulmonary overdistention on cardiopulmonary interactions were studied. The objective was to test the hypothesis that increasing tidal volumes adversely affect pulmonary vascular mechanics and cardiac output. An additional goal was to determine whether the effects of pulmonary overdistention are dependent on delivered tidal volume and/or positive end-expiratory pressure (PEEP, end-expiratory lung volume). DESIGN Prospective, randomized, controlled laboratory trial. SETTING University research laboratory. SUBJECTS Eleven 4- to 6-wk-old swine, weighing 8 to 12 kg. INTERVENTIONS Piglets with normal lungs were anesthetized, intubated, and paralyzed. After median sternotomy, pressure transducers were placed in the right ventricle, pulmonary artery, and left atrium. An ultrasonic flow probe was placed around the pulmonary artery. MEASUREMENTS AND MAIN RESULTS The swine were ventilated and data were collected with delivered tidal volumes of 10, 15, 20, and 25 mL/kg and PEEP settings of 5 and 10 cm H2O in a random order. Pulmonary overdistention was defined as a decrease in dynamic compliance of > or =20% when compared with a compliance measured at a baseline tidal volume of 10 mL/kg. At this baseline tidal volume, airway pressure-volume curves did not demonstrate pulmonary overdistention. Tidal volumes and airway pressures were measured by a pneumotachometer and the Pediatric Pulmonary Function Workstation. Inspiratory time (0.75 sec), FIO2 (0.3), and minute ventilation were held constant. We evaluated the pulmonary vascular and cardiac effects of the various tidal volume and PEEP settings by measuring pulmonary vascular resistance, pulmonary characteristic impedance, and cardiac output. When compared with a tidal volume of 10 mL/kg, a tidal volume of 20 mL/kg resulted in a significant decrease in dynamic compliance from 10.5 +/- 0.9 to 8.4 +/- 0.6 mL/cm H2O (p = .02) at a constant PEEP of 5 cm H2O. The decrease in dynamic compliance of 20% indicated the presence of pulmonary overdistention by definition. As the tidal volume was increased from 10 to 20 mL/kg, pulmonary vascular resistance (1351 +/- 94 vs. 2266 +/- 233 dyne x sec/cm5; p = .004) and characteristic impedance (167 +/- 12 vs. 219 +/- 22 dyne x sec/cm5; p = .02) significantly increased, while cardiac output significantly decreased (951 +/- 61 vs. 708 +/- 48 mL/min; p = .001). Each of these effects of pulmonary overdistention were further magnified when the tidal volume was increased to 25 mL/kg. The tidal volume-induced alterations in pulmonary vascular mechanics, characteristic impedance, and cardiac output occurred to a greater degree when the PEEP was increased to 10 cm H2O. Pulmonary vascular resistance and characteristic impedance were significantly increased and cardiac output significantly decreased for all tidal volumes studied at a PEEP of 10 cm H2O as compared with 5 cm H2O. CONCLUSIONS Increasing tidal volumes, increasing PEEP levels, and the development of pulmonary overdistention had detrimental effects on the cardiovascular system by increasing pulmonary vascular resistance and characteristic impedance while significantly decreasing cardiac output. Delivered tidal volumes of >15 mL/kg should be utilized cautiously. Careful monitoring of respiratory mechanics and cardiac function, especially in neonatal and pediatric patients, is warranted.

Soluble Complement Receptor-1 Protects Heart, Lung, and Cardiac Myofilament Function From Cardiopulmonary Bypass Damage

BACKGROUND Host defense system activation occurs with cardiopulmonary bypass (CPB) and is thought to contribute to the pathophysiological consequences of CPB. Complement inhibition effects on the post-CPB syndrome were tested with soluble complement receptor-1 (sCR1). METHODS AND RESULTS Twenty neonatal pigs (weight 1.8 to 2.8 kg) were randomized to control and sCR1-treated groups. LV pressure and volume, left atrial pressure, pulmonary artery pressure and flow, and respiratory system compliance and resistance were measured. Preload recruitable stroke work, isovolumic diastolic relaxation time constant (tau), and pulmonary vascular resistance were determined. Pre-CPB measures were not statistically significantly different between the 2 groups. After CPB, preload recruitable stroke work was significantly higher in the sCR1 group (n=5, 46.8+/-3.2x10(3) vs n=6, 34.3+/-3.7x10(3) erg/cm(3), P=0.042); tau was significantly lower in the sCR1 group (26.4+/-1.5, 42.4+/-6. 6 ms, P=0.003); pulmonary vascular resistance was significantly lower in the sCR1 group (5860+/-1360 vs 12 170+/-1200 dyn. s/cm(5), P=0.009); arterial PO(2) in 100% FIO(2) was significantly higher in the sCR1 group (406+/-63 vs 148+/-33 mm Hg, P=0.01); lung compliance and airway resistance did not differ significantly. The post-CPB Hill coefficient of atrial myocardium was higher in the sCR1 group (2.88+/-0.29 vs 1.88+/-0.16, P=0.023). CONCLUSIONS sCR1 meaningfully moderates the post-CPB syndrome, supporting the hypothesis that complement activation contributes to this syndrome.

Heliox does not affect gas exchange during high-frequency oscillatory ventilation if tidal volume is held constant.

ObjectiveTo compare gas exchange with heliox and oxygen-enriched air during high-frequency oscillatory ventilation, while controlling for tidal volume, in a pediatric swine model of acute lung injury. We hypothesized that when tidal volume delivery is held constant, heliox does not alter gas exchange. DesignRandomized, crossover trial. SettingUniversity animal research laboratory. SubjectsTen swine (4.4–5.4 kg). InterventionsAcute lung injury (A-a gradient of >300 mm Hg) was created using repeated saline lavage during conventional mechanical ventilation. The animals were then administered high-frequency oscillatory ventilation and ventilated with 60% oxygen/40% helium and 60% oxygen/40% nitrogen in a randomized, crossover trial. When changing gas mixtures within each animal, mean airway pressure (Paw = 16.8 ± 0.3 cm H2O) and frequency (10 Hz) were held constant. Oscillation amplitude (&Dgr;P) was adjusted to maintain constant tidal volume delivery as measured by respiratory inductive plethysmography. Next, the animals were ventilated with 40% oxygen/60% helium and 40% oxygen/60% nitrogen in a randomized crossover trial, again controlling for tidal volume. Measurements and Main ResultsGas exchange was assessed by arterial blood gas analysis after ventilation with each gas mixture. We demonstrated no significant difference in Paco2 or Pao2 between the heliox and oxygen-enriched air with either the 40% or 60% oxygen mixtures. The oscillation amplitude required to achieve the same tidal volume delivery was significantly less with heliox. ConclusionsWe conclude that if tidal volume delivery is maintained constant, heliox does not alter gas exchange when compared with oxygen-enriched air. However, to achieve the same tidal volume delivery, a lower oscillation amplitude is required with heliox. The clinical benefit of heliox administration during high-frequency oscillatory ventilation has yet to be determined. Possible advantages of heliox include improved ventilation of larger patients when approaching the power limitations of the Sensormedics 3100A oscillator and a potential reduction in the oscillation amplitude delivered to the more proximal gas exchange units.

Right ventricular injury in young swine: effects of catecholamines on right ventricular function and pulmonary vascular mechanics.

Acute right ventricular (RV) injury is commonly encountered in infants and children after cardiac surgery. Empiric medical therapy for these patients results from a paucity of data on which to base medical management and the absence of animal models that allow rigorous laboratory testing. Specifically, exogenous catecholamines have unclear effects on the injured right ventricle and pulmonary vasculature in the young. Ten anesthetized piglets (9–12 kg) were instrumented with epicardial transducers, micromanometers, and a pulmonary artery flow probe. RV injury was induced with a cryoablation probe. Dopamine at 10 μg/kg/min, dobutamine at 10 μg/kg/min, and epinephrine (EP) at 0.1 μg/kg/min were infused in a random order. RV contractility was evaluated using preload recruitable stroke work. Diastolic function was described by the end-diastolic pressure-volume relation, peak negative derivative of the pressure waveform, and peak filling rate. In addition to routine hemodynamic measurements, Fourier transformation of the pressure and flow waveforms allowed calculation of input resistance, characteristic impedance, RV total hydraulic power, and transpulmonary vascular efficiency. Cryoablation led to a stable reproducible injury, decreased preload recruitable stroke work, and impaired diastolic function as measured by all three indices. Infusion of each catecholamine improved preload recruitable stroke work and peak negative derivative of the pressure waveform. Dobutamine and EP both decreased indices of pulmonary vascular impedance, whereas EP was the only inotrope that significantly improved transpulmonary vascular efficiency. Although all three inotropes improved systolic and diastolic RV function, only EP decreased input resistance, decreased pulmonary vascular resistance, and increased transpulmonary vascular efficiency.

Optimizing liquid ventilation as a lung protection strategy for neonatal cardiopulmonary bypass: full functional residual capacity dosing is more effective than half functional residual capacity dosing.

OBJECTIVE To evaluate and compare the protective effects of two different perflubron doses on hemodynamics and lung function in a neonatal animal model of cardiopulmonary bypass-induced lung injury. DESIGN Prospective, randomized, controlled study. SETTING Animal laboratory of the Department of Surgery, Duke University Medical Center. SUBJECTS Twenty-one neonatal swine. INTERVENTIONS One-wk-old swine (2.2-3.2 kg) were randomized to receive cardiopulmonary bypass with full functional residual capacity perflubron (n = 7), cardiopulmonary bypass with half functional residual capacity perflubron (n = 7), or cardiopulmonary bypass alone (n = 7). This last group served as control animals, receiving cardiopulmonary bypass with conventional ventilation. Liquid lung ventilation animals received perflubron via the endotracheal tube at either full functional residual capacity (16-20 mL/kg) or half functional residual capacity (10 mL/kg) before the initiation of cardiopulmonary bypass. Each animal was placed on nonpulsatile cardiopulmonary bypass and cooled to a nasopharyngeal temperature of 18 degrees C (64.4 degrees F). Low-flow cardiopulmonary bypass (35 mL/kg/min) was instituted for 90 mins. The blood flow rate was then returned to 100 mL/kg/min. The animals were warmed to 36 degrees C (96.8 degrees F) and separated from cardiopulmonary bypass. Data were obtained at 30, 60, and 90 mins after separation from cardiopulmonary bypass. MEASUREMENTS AND MAIN RESULTS Cardiopulmonary bypass without liquid lung ventilation resulted in a significant decrease in cardiac output and oxygen delivery and a significant increase in pulmonary vascular resistance in the post-bypass period. Full functional residual capacity liquid lung ventilation administered before bypass resulted in no change in cardiac output and oxygen delivery after bypass. Full functional residual capacity liquid lung ventilation resulted in lower pulmonary vascular resistance after bypass compared with both control and half functional residual capacity liquid lung ventilation animals. CONCLUSIONS These data suggest that liquid lung ventilation dosing at full functional residual capacity before bypass is more effective than half functional residual capacity in minimizing the lung injury associated with neonatal cardiopulmonary bypass. Full functional residual capacity dosing may optimize alveolar distention and lung volume, as well as improve oxygen delivery compared with half functional residual capacity dosing.

A Novel Inhaled Organic Nitrate That Affects Pulmonary Vascular Tone in a Piglet Model of Hypoxia-Induced Pulmonary Hypertension

Persistent pulmonary hypertension of the newborn is characterized by elevated pulmonary vascular resistance after birth leading to right-to-left shunting and systemic arterial hypoxemia. Inhaled nitric oxide (NO) is effective in reducing the need for extracorporeal membrane oxygenation, but it has potential toxicities, especially in an oxygen-rich environment. A number of other NO-based molecules have been given by inhalation, but their structure–function relationships have not been established. Recent studies have raised the idea that toxic and beneficial properties can be separated. We synthesized a novel organic nitrate [ethyl nitrate (ENO2)], tested it in vitro, and administered it to hypoxic piglets. ENO2 lowered pulmonary artery pressure and raised the Po2 in arterial blood but did not alter systemic vascular resistance or methemoglobin levels. In addition, we tested the effect of ENO2 in the presence of the thiol glutathione, both in vivo and in vitro, and found its action to be enhanced. Although ENO2 is less potent than inhaled NO on a dose-equivalency basis, pretreatment of hypoxic animals with glutathione, which may be depleted in injured lungs, led to a markedly enhanced effect (largely mitigating the difference in potency). These results suggest that ENO2 may hold promise as a safe alternative to NO, particularly in hypoxemic conditions characterized by thiol depletion.

Liquid lung ventilation reduces neutrophil sequestration in a neonatal swine model of cardiopulmonary bypass.

ObjectiveLiquid lung ventilation has been demonstrated to improve cardiorespiratory function after cardiopulmonary bypass. We hypothesized that liquid lung ventilation (LLV) would decrease the pulmonary inflammatory response after cardiopulmonary bypass (CPB). DesignProspective, randomized, experimental, controlled, nonblinded study. SettingAnimal research laboratory at a university setting. SubjectsA total of 24 neonatal piglets. InterventionsAfter intubation with a cuffed endotracheal tube, swine were conventionally ventilated. After surgical cannulation, each piglet was placed on conventional nonpulsatile CPB and cooled to 18°C (64.4°F). Subsequently, the animals were exposed to 90 mins of low-flow CPB (35 mL/kg/min). Animals were rewarmed to 37°C (98.6°F), removed from CPB, and ventilated for 90 min. Ten animals received conventional gas ventilation only (control), seven received initiation of LLV before CPB (prevention), and seven received initiation of LLV during the rewarming phase of CPB (treatment). After the animals were killed, the lungs were removed en bloc. The left lobe was dissected and formalin-fixed at 20 cm H2O overnight, followed by paraffin embedding. Sections were taken from the paraffin-embedded lungs. Neutrophil accumulation and lung injury were assessed by histochemical staining with leukocyte esterase and morphometrics, respectively. One hundred microscopic images were digitized from each tissue sample for lung morphometrics, and neutrophil counts were obtained from every fifth image. Measurements and Main Results Lung tissue sections showed a significantly lower number of neutrophils per alveolar area in the prevention and treatment groups than in the control group (control 681 ± 65, prevention 380 ± 49, treatment 412 ± 101 neutrophils per alveolar area [cells/mm2];p < .05 for both prevention and treatment compared with control). There were no differences in lung injury as assessed with morphometrics or hemodynamic measurements between any of the three groups. ConclusionsThe data suggest that LLV reduces the CPB-induced neutrophil sequestration in the pulmonary parenchyma independent of its effects on the circulatory physiology or evidence of early lung injury.

Reversal of Flow through Chronic Coronary Collateral Vessels

The purpose of this study was to determine whether blood flow through chronic collateral vessels may reverse its direction to supply acutely ischemic myocardium. Ameroid constrictors were placed on the circumflex coronary artery (CCA) of 12 dogs to promote collateral flow (CQ) from the left anterior descending (LAD) to the CCA. Twelve weeks later, myocardial blood flow (MBF) was determined (ml/g/min) using radioactive tracer microspheres. Control LAD subepi-cardial (EPI) and subendocardial (ENDO) flows were 1.17 ± 0.11 and 1.00 ± 0.10 (Mean ± SEM), respectively. CCA EPI and ENDO flows were 1.25 ± 0.12 and 1.12 ± 0.20, respectively. The LAD was then occluded, and MBF to the CCA bed decreased by an average of 0.31 ± 0.03 ml/g/min (P < 0.005). This decrease in MBF quantitated the amount of CQ from the LAD to the CCA bed. An aortocoronary bypass graft was anastomosed to the distal CCA and MBF to the LAD bed immediately increased by an average of 0.30 ± 0.02 ml/g/min (P < 0.005). This increase in MBF represented reversed CQ from the CCA to the acutely ischemic LAD bed. It had a normal transmural distribution (ENDO/EPI = 0.9). Four hours later, total reversed CQ to the LAD bed remained unchanged, but was redistributed toward the EPI (ENDO/EPI = 0.6). These data document that chronic collateral vessels are capable of immediate and sustained conduction of CQ in a retrograde direction and suggest that these collateral vessels may play a role in limiting the degree and transmural extent of ischemic injury when a perioperative myocardial infarction occurs in the vascular bed of a nonbypassed coronary artery.