Treatment for airway cast obstruction
11225651 · 2022-01-18
Assignee
Inventors
Cpc classification
C12Y304/21068
CHEMISTRY; METALLURGY
C12Y304/21073
CHEMISTRY; METALLURGY
C12N9/6462
CHEMISTRY; METALLURGY
C12N9/6459
CHEMISTRY; METALLURGY
A61K9/0078
HUMAN NECESSITIES
International classification
A61K9/00
HUMAN NECESSITIES
Abstract
The present invention is directed to methods of treatment of airway obstruction associated with fibrin-containing cast formation by administering a fibrinolytic agent.
Claims
1. A method to increase the survival of a subject having been exposed to sulfur mustard (SM) or an analog thereof, wherein exposure to the SM or analog thereof results in fibrin-containing cast formation in the airways of the subject, comprising administering to the airway of the subject a fibrinolytic agent, wherein survival of the subject is increased.
2. The method of claim 1, wherein the fibrinolytic agent is selected from the group consisting of tissue plasminogen activator (tPA), a tPA analog, urokinase plasminogen activator (uPA) and a uPA analog.
3. The method of claim 2, wherein the fibrinolytic agent is tPA.
4. The method of claim 3, wherein the tPA is administered in a dose amount of about 0.1 mg/kg/dose to about 1.0 mg/kg/dose.
5. The method of claim 3, wherein the tPA is administered in a dose amount of about 0.4 mg/kg/dose to about 0.8 mg/kg/dose.
6. The method of claim 1, wherein the fibrinolytic agent is administered to the airway of the subject by a delivery method selected from the group consisting of inhalation, nebulization, aerosolization, intratracheal delivery, bronchoscopy and intubation.
7. The method of claim 1, wherein the step of administering comprising administering to the subject an initial dose of the fibrinolytic agent followed by administering an additional dose of the fibrinolytic agent to the subject.
8. The method of claim 1, wherein the step of administering the fibrinolytic agent is conducted immediately after exposure of the subject to the SM or analog thereof.
9. The method of claim 1, wherein the step of administering the fibrinolytic agent is conducted within about 0 hours to about 14 days after exposure of the subject to the SM or analog thereof.
10. The method of claim 1, wherein the step of administering the fibrinolytic agent comprises administering an initial dose of the fibrinolytic agent following exposure of the subject to the SM or analog thereof and administering at least one additional dose of the fibrinolytic agent after the administration of the initial dose of the fibrinolytic agent.
11. The method of claim 10, wherein the step of administering the at least one additional dose is conducted about 30 minutes to about 60 minutes after the administration of the initial dose.
12. The method of claim 11, wherein the step of administering the at least one additional dose is repeated.
13. The method of claim 12, wherein the step of administering the at least one additional dose is repeated about every 4 hours to about every 6 hours.
14. The method of claim 1, wherein the subject is human.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
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DETAILED DESCRIPTION
(33) This invention generally relates to methods for treating airway obstruction in a subject and/or to reduce airway obstruction in a subject as well as for increasing the survival of a subject having airway obstruction. The invention includes the administration of a fibrinolytic agent.
(34) The airway obstruction may result from the formation of fibrin-containing casts in the airways, which can include airspaces, of the subject, including but not limited to the trachea, bronchi and more distal airways.
(35) The formation of the fibrin-containing casts may be the result of the subject having been exposed to a toxic inhaled chemical (TIC). In one aspect, the exposure to the TIC results in fibrin-containing cast formation in the airways and/or airspaces of the subject. In still another aspect, the fibrin-containing cast formation in the airways and/or airspaces of the subject results in the formation of plastic bronchitis.
(36) The toxic inhaled chemical, can be sulfur mustard (SM) also referred to herein as HD (“Hun Stuff Distilled”) or authentic sulfur mustard, sulfur mustard analogs (such as CEES), Bhopal agent and other agents that are known to cause fibrin casts. The TIC can be formulated into a vapor form as well as into an aerosol form, a gas form, particulate forms and combination thereof. In a preferred embodiment, the TIC is formulated into a vapor form. In one aspect, the aerosol form of the TIC could be formulated into an ethanolic solution.
(37) The fibrinolytic agent may be any fibrinolytic compound. The fibrinolytic compound can be a plasminogen activator. In one aspect, the plasminogen activator can be a tissue plasminogen activator (tPA) such as tenectaplase (TNK) or a recombinant tPA (such as rh-TPA), a tPA analog, a urokinase plasminogen activator (uPA), a uPa analog and combinations thereof. An analog of tPA or uPA can include a portion of the tPA or uPA, such as a single chain uPa or tPA. In a preferred embodiment, the fibrinolytic agent is tPA.
(38) Over the past three decades, tPA has become accepted therapy for a spectrum of intravascular events, including acute myocardial infarction (1993, N Engl J Med 329:1615-1622), thrombotic stroke (Lansberg, M. G. et al., 2012, Chest 141:e601S-6365), acute pulmonary embolism and severe deep vein thromboses (Kearon, C. et al., 2012, Chest 141:e419S-4945). It also can be useful in removing clots from blocked catheters and grafts (arteriovenous shunts) (Hilleman, D. et al., 2011, Pharmacotherapy 31:1031-1040), treatment of unusual acute thrombotic events (Garcia, A. et al., 2011, J Pediatr Surg 46:2021-2024), treatment of empyemas and pleural effusions (Rahman, N. M. et al., 2011, N Engl J Med 365:518-526), and to limit amputation in frostbite (Johnson, A. R. et al., 2011, Foot Ankle Spec 4:344-348). tPA also can be useful in animal models for treating a variety of extravascular fibrin deposition disorders including prevention of intra-abdominal adhesions 9 van Goor, H. et al., 1996, Eur Surg Res 28:287-294), and clearance of airways after burns and smoke inhalation (Enkhbaatar, P. et al., 2004, Shock 22:70-75). In addition, it has been reported anecdotally as beneficial therapy for patients with plastic bronchitis after Fontan procedure in congenital heart disease (Do, T. et al., 2009, Pediatr Cardiol 30:352-355; Costello, J. M. et al., 2002, Pediatrics 109:e67; Wakeham, M. K. et al., 2005, Pediatr Crit Care Med 6:76-78).
(39) tPA is also currently used as first-line therapy in several clot-associated diseases such as stroke (Barreto, A. D. et al., 2012, Stroke 43:770-775; Kablau, M. et al., 2012, Int J Stroke) and myocardial infarction (Fitchett, D. H. et al., 2011, Can J Cardiol 27 Suppl A: 5402-412), and can improve survival with these life-threatening entities. In regards to plastic bronchitis, no placebo-controlled clinical trials have been done to evaluate the effects of tPA. For this reason, it is unknown if tPA can truly alter mortality and/or morbidity measures associated with plastic bronchitis. Furthermore, the proper dose required for tPA efficacy has not yet been assessed in vivo.
(40) The fibrinolytic agent may be administered to the subject by any airway or intra-airway delivery method. Such methods are known in the art and include but are not limited to administration by inhalation, nebulization, aerolization and/or intratracheal delivery. Nebulized tPA, nor any other form of airway-delivered tPA or fibrinolytic agent, is not believed to have been known to have been used for treatment of any TIC. Another possible mode of administration is to intubate a subject (for example a subject on a ventilator) and attach a nebulizer to the endotracheal tube (ETT) for delivery of the fibrinolytic agent. Other modes of administration can be via direct administration into the airways, such as by bronchoscopy or intubation or bronchoscopy thru a newly placed ETT. In a preferred embodiment the fibrinolytic agent is administered to the subject by intratracheal delivery.
(41) In another embodiment, the subject is administered a dose of the fibrinolytic agent. The subject may be administered an initial dose (i.e. loading dose) followed by a second or additional dose at a later time point at the onset of therapy (referred to as loading regimen). In still a further embodiment, the subject may be administered an initial dose followed by at least one additional dose of the fibrinolytic agent.
(42) In one embodiment, the initial dose is administered following exposure of the subject to a TIC. In another embodiment, the initial dose is administered immediately after an initial exposure of the subject to the TIC. In still another embodiment, the initial dose can be administered within about 1 hour, about 2 hours, about 3 hours, about 4, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 12 hours, about 14 hour, about 16 hours, about 20 hours, about 24 hours, about 48 hours, about 72 hours, about 4 days, about 8 days, about 12 days about, about 14 days or times in between, after an initial exposure of the subject to a TIC.
(43) The additional dose or at least one additional dose can be administered at least 30 minutes after the administration of the initial dose, at least one hour after the administration of the initial dose, at least two hours after the administration of the initial dose, at least 5 hours after the administration of the initial dose, at least 24 hours after the initial dose, at least 48 hours after administration of the initial dose or any time in between. Preferably, the additional or second dose is administered about 30 minutes to one hour after administration of the initial dose.
(44) The dosing regimen of administering an initial dose followed by at least one additional dose at a later time point at the onset of therapy (i.e. the loading regimen), may be repeated as often as necessary. The loading regimen can be repeated about every 3 hours, about every 4 hours, about every 8 hours, about every 16 hours, about every 20 hours, about every 24 hours, about every 36 hours, about every 72 hours, about every 5 days, about every 10 days, about every 20 days, about every 30 days, about every 45 days, about every 6 months or about every year. Preferably, the repeated dosing regimen is repeated about every four hours to about every six hours.
(45) To determine if re-dosing is needed following the initial loading regimen, oxygen saturation levels may be determined in the subject. If the oxygen saturation levels in the subject drop below an unacceptable level, such as 85-90%, the re-dosing loading regimen may be initiated again (one dose followed by a second dose at a later time point). It is possible that the subject may only need to be administered a single additional dose instead of the loading regimen. The re-dosing can occur about every 3 hours, about every 4 hours, about every 8 hours, about every 16 hours, about every 20 hours, about every 24 hours, about every 36 hours, about every 72 hours, or about every 5 days, about every 10 days, about every 20 days, about every 30 days, about every 45 days, about every 6 months or about every year or until no more oxygenation changes occur. The repeated dosing may be dependent upon the oxygenation decline due to plastic bronchitis, which induces cast formation. Repeated dosing can be done on a scheduled basis or can be done on an as needed basis (i.e. ‘pro re nata’ (PRN) basis).
(46) The dose amount may be in a range having a lower endpoint from about 0.10 mg/kg/dose, about 0.15 mg/kg/dose, about 0.20 mg/kg/dose, about 0.25 mg/kg/dose, about 0.30 mg/kg/dose, about 0.35 mg/kg/dose, about 0.40 mg/kg/dose, about 0.45 mg/kg/dose or about 0.50 mg/kg/dose. In addition, the effective dose may be in a range having an upper endpoint of about 1.0 mg/kg/dose, about 0.95 mg/kg/dose, about 0.90 mg/kg/dose, about 0.85 mg/kg/dose, about 0.80 mg/kg/dose, about 0.75 mg/kg/dose, about 0.70 mg/kg/dose, about 0.65 mg/kg/dose, about 0.60 mg/kg/dose or about 0.55 mg/kg/dose. In a preferred embodiment, the dose is about 0.70 mg/kg/dose. For tPA, the dose amount can be about 0.1 mg/kg/dose to about 1.0 mg/kg/dose with a preferred amount of about 0.4 mg/kg/dose to about 0.8 mg/kg/dose. The dose amount can be weight adjusted for the subject. The dose amount can be adjusted based on the administration route. A direct administration routes, such as by bronchoscopy or intubation, the tPA dose amount may range in an amount of about 0.1 mg/kg/dose to about 1.0 mg.Math.kg/dose. The dose amount that can be administered can also be the equivalent milligram amount of the fibrinolytic agent required to deliver an equivalent plasminogen activator activity as compared to a 0.1 mg/kg/dose to a 1.0 mg/kg/dose of tPA, specifically rh-tPA (Genetech). Plasminogen activator activity can be used to detect the activities of tPA, uPA and analogs thereof in vitro. Such activity can be determined using known plasminogen activator activity assays, including fluorescent-based assays. The assays can use native fibrin in the bottom of a multi-well plate, or they can be used in solution working with fluorescent substrates for plasmin that serve as a surrogate for fibrin. One such substrate is D-ala-leu-lys-7-amido-4-methylcoumarin. The ‘activity’ can be detected as long as the incubation contains this substrate (D-ala-leu-lys-7-amido-4-methylcoumarin) and a plasminogen (such as rat plasminogen or human plasminogen). The assay is relatively linear over a broad range of tPA or uPA activities. The activity can be inhibited by a relatively specific inhibitor such as PAI-1 (plasminogen activator inhibitor-1) or a nonspecific protease inhibitor such as leupeptin.
(47) In still another embodiment, the subject is a mammal, including but not limited to a human, dog, and cat. In yet another embodiment, the subject is a human child.
(48) In still another embodiment, the fibrinolytic agent may be provided in a kit and/or a container. The kit can include a fibrinolytic agent such as those disclosed herein, including tPA, a tPA analog, uPA and a uPA analog as well as including a device for administering the fibrinolytic agent to the airways, including the airspaces of the subject. The device for administering the fibrinolytic agent to the airways are well known in the art and can include an atomizer, a nebulizer, a catheter, a bronchoscope, endotracheal tube, tracheostomy tube or a laryngeal mask airway, or any combination of these modes.
(49) In yet another embodiment, the fibrinolytic agent and a pharmaceutically acceptable carrier may be administered to the subject. Such carriers are well known to those in the art.
(50) In still yet another embodiment, the fibrinolytic agent may be administered to subject followed by administration of an anticoagulant such as heparin or Tissue Factor Pathway Inhibitor (TFPI) at a time point following the administration of the fibrinolytic agent.
(51) As disclosed in the Example section below, the Inventors have found that optimal intratracheal tPA treatment for SM and an SM analog (CEES) induced severe plastic bronchitis in rats eliminated mortality in this almost uniformly fatal disease model, and that tPA also greatly improved other morbidity outcome measures often associated with plastic bronchitis. The Inventors found that a tPA dose of 0.7 mg/kg/dose delivered intratracheally, via a two-dose regimen given 1 hour apart, followed by repeated treatments during the 48 hour interval, resulted in 0% mortality by 48 hours after plastic bronchitis induction with SM and an SM-analog inhalation, compared to 90% mortality with no treatment, and 100% mortality in both PBS and isoflurane controls. Furthermore, improved morbidity with tPA was evidenced by normalization of plastic bronchitis-associated hypoxemia, hypercarbia, and acidosis, as well as by dose-dependent improvements in both respiratory distress and airway fibrin casts (i.e. Main and Dependent Bronchi composite cast scores) after treatment. Using a systematic approach, the Inventors have demonstrated the first in vivo dosimetric assessment of intratracheal tPA efficacy on survival and morbidity in plastic bronchitis, induced by inhalation of a sulfur mustard analog inhalation as well as following inhalation of authentic SM (referred to herein as “SM”).
(52) Previously, it has been reported that plastic bronchitis can result from acute inhalation of toxic chemicals such as a sulfur mustard analog (for example CEES), via a mechanism involving damage to the bronchial circulation, with resultant leakage of plasma contents into airways (Veress, L. A. et al., 2010, Am J Respir Crit Care Med 182:1352-1361). The Inventors have shown that fibrin-containing casts occluded conducting airway generations 3-15, the region of bronchial artery distribution, and that indeed, the entire bronchial circulation had greatly increased permeability. Disclosed herein, it is shown that the resulting airway obstruction from casts leads to impaired gas exchange and tissue oxygenation, respiratory distress, and often death. In addition, even when treatment is delayed for several hours after injury the Inventors demonstrate that tPA can reverse airway fibrin deposition, resulting in improved gas exchange and tissue oxygenation, eliminating respiratory distress and death as demonstrated in a relevant animal model of toxic chemical inhalation disclosed herein. This is a considerable advance relative to previously reported findings, as it supports that, not only do airway obstructive lesions stain positively by fibrin, but, further, that the airway obstructive lesions are responsible for the gas exchange abnormalities, clinical respiratory distress and mortality in this model. Thus, fibrin is not just immunologically detectable, but, more importantly, fibrin is structurally responsible for the disease pathophysiology in this model for this disorder.
(53) Development of airway obstructive lesions containing fibrin in conducting airways has also been noted with authentic SM, regardless of route of entry, both in humans as well as in various animal models using either neat or ethanolic SM vapor to produce injury (Willems, J. L., 1989, Ann Med Milit Belg 3S:1-61). Since the early 1900's, human victims of mustard gas injury have been reported to contain ‘fibrinous pseudomembranes’ within their airways, at times resulting in death by suffocation from complete blockage of bronchial or tracheal passages by these ‘pseudomembranes’ (Eisenmenger, W. et al., 1991, J Forensic Sci 36:1688-1698). More recent reports of human SM exposures from the Iran-Iraq war (1984-1986) have given detailed descriptions of the deleterious effects of these airway casts, reporting death in 80% of patients needing intubation for respiratory insufficiency due to severe airway obstruction (Eisenmenger, W. et al., 1991, J Forensic Sci 36:1688-1698). These human reports correlate with the findings presented here, where a nose only inhalation model of SM-analogs in rats was used, with ‘pseudomembranes’ further defined as fibrin-containing casts of plastic bronchitis. In regards to other animal models, when ethanolic SM vapor was delivered to glass catheter-intubated rats, the same type of injury in the conducting airways occurred, with fibrin-containing casts present in the bronchi and bronchioles (Gao, X. et al., 2011, Toxicol Pathol 39:1056-1064). In the porcine SM model, where high dose SM was delivered as a neat vapor via nasal inhalation, gas exchange abnormalities mirroring these findings were noted, but more importantly, airway obstructive lesions caused mortality in 40% of the pigs in less than 6 hours from initial SM exposure (Fairhall, S. J. et al., 2010, Inhal Toxicol 22:1135-1143). Thus, an injury affecting the conducting airways has been reported in both humans and animal models of SM inhalation, found in the SM analog-inhalation model disclosed herein.
(54) The selectivity for injury of the conducting airways with SM or CEES, particularly to the areas of bronchial (systemic) circulation, is of interest. While SM is more reactive and toxic than the SM analog, they both tend to cause injury very near their point of entry, with dissipation down the airways. It is believed that the more proximal conducting airways are injured because of a) a heightened bronchial circulation susceptibility to mustard injury, b) the highly reactive nature of the agent as it dissipates while traversing down the airways, and c) the size of particles created by either the SM analog aerosol or SM vapor is suitable for distribution in the affected airways. With respect to particle size in this disclosed model, the count median diameter for the SM analog-ethanolic aerosol is 0.6 micron, and the mass median diameter of the aerosol is 3.87 microns. This small particle size assures that the particles can reach all lung compartments, including both distal airways and alveoli. Certainly, a small amount of the SM analog-ethanolic particles can agglomerate and become deposited more proximally into the uppermost airways, as previous reported in a publication showing extensive nasal injury after SM analog inhalation (O'Neill, H. C. et al., 2011, Am J Respir Cell Mol Biol 45:323-331). Nevertheless, a pulmonary injury selective to the proximal conducting airways is the dominating feature of acute mustard inhalation, despite the ability of particles to reach and deposit within alveolar surfaces.
(55) Disclosed herein, intratracheal administration of tPA completely eliminated SM and an SM analog exposure-related mortality from plastic bronchitis, even when rescue treatment was delayed almost 6 hours after injury, and yet was uniformly effective, provided that two consecutive doses were given once dosing was initiated. This effect despite delay of treatment is of important clinical relevance, because patients with plastic bronchitis often do not present early in their clinical course, but rather present to an acute care facility when their cast obstruction is severe enough to cause significant morbidity (such as respiratory distress and hypoxemia). Moreover, victims of sulfur mustard poisoning are often unaware that they have been exposed until several hours later, when skin blisters and respiratory symptoms arise as their airways begin to form obstructive bronchial casts. Thus, emergency medical care can be significantly delayed, and tPA treatment still be extremely effective as ‘late rescue’ therapy for any underlying cause of plastic bronchitis.
(56) Dose-related improvements in multiple parameters related to CEES-inhalation injury after tPA treatment were noted. First, tissue oxygen delivery, as measured via pulse oximetry at 12 hours, was improved in a dose-dependent fashion. Second, dose-related reduction in percent airway obstruction by airway casts as assessed morphometrically during airway microdissection was detected. This effect was most profound with the highest tPA dose (0.7 mg/kg) used, and no bleeding side effects were observed. Third, dose-related reduction in clinical symptoms of respiratory distress was recognized, confirming the likely relationship between tissue oxygenation, airway obstruction by casts, and clinical respiratory distress. In addition, the Inventors also found a greatly beneficial effect of tPA treatment on pulmonary gas exchange and tissue acidosis. All tPA doses tested resulted in significant improvements of SM and an SM analog-associated gas exchange abnormalities (as reflected by normalization of p.sub.aO.sub.2, p.sub.aCO.sub.2, and arterial blood pH), while plasma lactate showed normalization only with the highest (0.7 mg/kg) dose. These non-uniform dose-related morbidity measure findings are most likely due to the varying severity of inadequate tissue oxygen delivery seen with lower tPA doses, corresponding to a lesser efficacy of cast lysis, and thus a diminished capacity for respiratory compensation at higher degrees of airway obstruction. Therefore, it is most likely that a threshold level of airway obstruction from casts exists, below which normal ventilation and arterial oxygenation is still maintained, while tissue perfusion is already compromised.
(57) The following examples are provided for the purpose of illustration and are not intended to limit the scope of the present invention. Any variations which occur to the skilled artisan are intended to fall within the scope of the present invention. Each publication, sequence or other reference disclosed below and elsewhere herein is incorporated herein by reference in its entirety, to the extent that there is no inconsistency with the present disclosure.
EXAMPLES
Example 1
(58) This example demonstrates that intratracheal administration of tPA diminishes airway fibrin-containing casts while improving clinical respiratory distress, pulmonary gas exchange, tissue oxygenation, and oxygen utilization in the disclosed model of severe chemically-induced plastic bronchitis. In addition, mortality, which was associated with hypoxemia and clinical respiratory distress, was eliminated.
(59) In this example, adult rats exposed to sulfur mustard analog (CEES, 2-chloroethyl ethylsulfide; a sulfur mustard, abbreviated as SM for this example) were treated with intratracheal tPA (0.15-0.7 mg/kg, 5.5 hours and 6.5 hours after SM exposure), compared to controls (no treatment, isoflurane, and placebo). Respiratory distress and pulse oximetry were assessed (for 12 hours or 48 hours), and arterial blood gases were obtained at study termination (12 hours). Airway microdissection on fixed lungs was done to assess airway obstruction by casts. Optimal intratracheal tPA treatment (0.7 mg/kg) completely eliminated mortality (0% at 48 hours) and greatly improved morbidity in this nearly uniformly fatal disease model disclosed herein (90-100% mortality at 48 hour). tPA normalized plastic bronchitis-associated hypoxemia, hypercarbia, and lactic acidosis, and improved respiratory distress (i.e. decreased clinical distress scores) while decreasing airway fibrin casts.
(60) Methods Used in Example 1:
(61) Chemicals
(62) 2-chloroethyl ethyl sulfide (CEES) was obtained from TCI America (Portland, Oreg.). Tissue plasminogen activator (tPA) was purchased from Genentech (Roche, San Francisco, Calif. and as disclosed in U.S. Pat. No. 5,612,029 incorporated herein by reference). All other chemicals were purchased from Sigma-Aldrich Chemical Co. (St. Louis, Mo.) unless otherwise indicated.
(63) Animal Care
(64) The Institutional Animal Care and Use Committee (IACUC) of National Jewish Health (NJH) center approved this study. Adult male (300-350 g) Sprague-Dawley rats (Harlan Co., Indianapolis, Ind.) were used.
(65) Inhalation Exposure to CEES
(66) CEES (10%) inhalation exposure was conducted as previously described (Veress, L. A. et al. 2010, Am. J. Respir. Crit. Care Med. 182:1352-1361). 10% CEES in ethanol was used exclusively in these experiments, in order to produce the desired level of injury. Briefly, anesthetized rats were placed in a nose-only inhalation system (CH Technologies, Westwood, N.J.), and were delivered the aerosolized CEES for 15 minutes, then removed from polycarbonate tubes, and observed until fully recovered from anesthesia.
(67) Respiratory Distress Clinical Scoring
(68) The respiratory distress clinical scoring criteria used were developed by the inventors and are specific for plastic bronchitis-induced respiratory distress in this rat model of CEES chemical inhalation. Respiratory quality, wheezing/stridor and activity depression were assessed (see Table 1) and agreed upon by 2 separate laboratory workers, and the individual scores (0 to 3) were added to obtain a cumulative score (max of 9). A score of 10 was given to rats that died prior to the conclusion of the experiment.
(69) TABLE-US-00001 TABLE 1 RESPIRATORY CLINICAL DISTRESS SCORE CRITERIA RESPIRATORY SCORE QUALITY STRIDOR ACTIVITY 0 Normal Normal Normal 1 Mild abdominal Stridor with Mildly depressed breathing with activity only, activity tachypnea (>100) mild 2 Moderate abdominal Stridor at Moderately depressed breathing, possibly rest, mild to activity (movement with mild gasping moderate with stimulation) 3 Severe abdominal Stridor at Obtunded, no move- breathing, severe rest, severe ment with stimulation; gasping, and low or severe agitation respiratory rate (<60) with stimulation
Euthanasia Criteria
(70) Animals were euthanized if oxygen saturation <70%, and respiratory distress score of 7 or greater. Euthanasia was performed using pentobarbital overdose (Sleepaway, Fort Dodge Animal Health, Fort Dodge, Iowa) (Veress, L. A. et al. 2010, Am. J. Respir. Crit. Care Med. 182:1352-1361). Animals were continuously monitored throughout experiments, and were euthanized prior to completion of experiment if institutional IACUC-approved euthanasia criteria were met. Euthanasia criteria were met if a rat had both: 1) oxygen saturation <70% (by a small animal pulse oximeter), and 2) respiratory clinical distress score >7. Animals were assessed for meeting euthanasia criteria by 2 observers, and if disagreement occurred, a third was asked for consultation.
(71) Lung Fixation
(72) Animals were euthanized at 12 hours after exposure as per experimental design, or if required prior to study termination. Tracheas were cannulated, and lungs were fixed at 20 cm H.sub.2O with 4% paraformaldehyde in phosphate-buffered saline (PBS) for 30 minutes, and then surgically removed.
(73) Airway Cast Scoring
(74) Cast scoring was developed based on previously described microdissection techniques done by our group (Veress, L. A. et al. 2010, Am. J. Respir. Crit. Care Med. 182:1352-1361). Fixed lung was separated into five lobes by cross-sectioning each lobar bronchus at site of take-off from central airway bronchus (
(75) TABLE-US-00002 TABLE 2 Weighted Cast Score Calculation Raw Lobar % volume of Weighted Cast score lobe/total lung lobar score RUL score × (10%/100) = weighted RUL score RML score × (14%/100) = weighted RML score RLL score × (28.5%/100) = weighted RLL score RA score × (11%/100) = weighted RA score LL score × (36.5%/100) = weighted LL score Sum of individual Total weighted weighted scores = lung cast score RUL = right upper lobe; RML = right middle lobe; RLL = right lower lobe; RA = right accessory lobe; LL = left lobe
Noninvasive Oxygen Saturation Measurements
(76) A small animal oximeter (Starr Life Sciences, Oakmont, Pa.) in unanesthetized rats was used with XL CollarClip Sensor. An average value was recorded every five seconds using the oximeter software, and 3 of these readings were averaged to give a final value.
(77) Arterial Blood Gas Measurements (ABG)
(78) Arterial blood from descending aorta was collected (via 21 gauge butterfly catheter) and 100 μl was immediately placed into a pre-calibrated test card (EPOC-BGEM Test Card,) and analyzed using the EPOC-Vet Blood Analysis system (Epocal Inc., Ottawa, ON, Canada) at room temperature. Values for arterial pH, p.sub.aCO.sub.2, p.sub.aO.sub.2, bicarbonate, and lactate were assessed and reported.
(79) Statistical Analysis
(80) Prism 5.01 software (GraphPad, La Jolla, Calif.) was used, with one-way analysis of variance (ANOVA) plus Tukey's post-hoc analysis or Kruskal-Wallis plus Dunn's post-hoc analysis, depending on distribution of data. A p value <0.05 was significant.
(81) Results
(82) Optimal tPA Treatment Regimen Improves Survival
(83) Survival over 48 hours after high level CEES (10%) inhalation in rats with and without intratracheal tPA (0.7 mg/kg) was measured. Three control groups were also evaluated. These included: 1) rats given no drug but exposed to CEES (NT), 2) rats receiving no drug treatment, exposed to CEES and given isoflurane anesthesia (Iso), and 3) rats given CEES exposure, isoflurane, and intratracheally delivered diluent for tPA (PBS, placebo). High level CEES exposure caused high mortality at 48 h in all control groups, including 90% mortality with NT, 100% with Iso, and 100% with PBS (
(84) Initial tPA dosing was delayed until 5.5 hours after exposure. This dosing time was based on previous observations that: 1) casts begin to form in airways at 4 hours (Veress, L. A. et al. 2010, Am. J. Respir. Crit. Care Med. 182:1352-1361), and 2) oxygen saturations decreased below 90% at 5 hours after exposure (
(85) Oxygen Saturation Improves with tPA Treatment
(86) A dose-effect of intratracheal tPA on oxygen saturation (SpO.sub.2) in rats with plastic bronchitis after CEES inhalation was determined. Four tPA doses (0.15 mg/kg, 0.3 mg/kg, 0.5 mg/kg and 0.7 mg/kg) were tested and given as a one-time two-dose regimen at 5.5 hours and 6.5 hours, followed by pulse oximetry at 12 hours. A dose-dependent improvement was detected in SpO.sub.2 with tPA treatment as compared to all three controls tested (
(87) TABLE-US-00003 TABLE 3 Oxygen Saturation with CEES Inhalation Plus tPA vs. Controls CEES + Pre-exposure treatment (%)* 5 h (%)* 8 h (%)* 10 h (%)* 12 h (%)* Mortality NT 94.1 ± 0.4 88.1 ± 1.6 77.6 ± 1.7 78.9 ± 2.4 78.4 ± 3.2 12.50% n = 16 n = 16 n = 15 n = 14 n = 14 Iso 93.5 ± 0.6 88.4 ± 2.4 79.0 ± 4.8 82.8 ± 3.8 84.3 ± 2.7 30% n = 10 n = 10 n = 9 n = 8 n = 7 PBS 92.4 ± 1.2 83.1 ± 1.7 81.1 ± 3.1 75.2 ± 11.6 71.6 ± 0 75% n = 4 n = 4 n = 4 n = 2 n = 1 0.15 mg/mL 93.5 ± 0.5 85.9 ± 0.8 79.0 ± 3.1 75.6 ± 3.8 76.5 ± 2.6 0% tPA n = 6 n = 6 n = 6 n = 6 n = 6 0.3 mg/mL 95 ± 0.4 90.1 ± 1.3 85.5 ± 2.3 84.7 ± 2.7 88.6 ± 0.9 0% tPA n = 6 n = 6 n = 6 n = 6 n = 6 0.5 mg/mL 94.4 ± 0.6 89.2 ± 2.2 82.1 ± 2.4 80.5 ± 2.5 86.4 ± 1.3 0% tPA n = 6 n = 6 n = 6 n = 6 n = 6 0.7 mg/mL 93.4 ± 0.3 89.3 ± 1.2 88.0 ± 0.9 90.1 ± 1.0 91.4 ± 0.9 0% tPA n = 16 n = 16 n = 16 n = 16 n = 16 *Values shown are mean ± SEM NT = no treatment control (CEES alone) Iso = isoflurane control PBS = phosphate buffered saline control
Reduction of Airway Obstruction by Fibrin Casts with tPA Treatment
(88) The effects of tPA on fibrin casts within airways was assets. The inventors developed a quantitative scoring system of total airway obstruction by casts (
(89) Respiratory Clinical Scoring Improvement after tPA Administration
(90) The inventors developed a clinical respiratory distress scoring system, based upon signs of distress in rats due to plastic bronchitis, including: 1) respiratory quality (i.e. work of breathing), 2) level of activity, and 3) degree of stridor (Table 1). The three higher tPA doses tested, but not 0.15 mg/kg, significantly improved respiratory clinical scores at 12 hours versus controls (p=0.004;
(91) TABLE-US-00004 TABLE 4 Respiratory Distress Scores with CEES Inhalation, Plus tPA vs. Controls Signifi- CEES + Pre- cance.sup.# (Pre treatment exposure 5 h 8 h 10 h 12 h to 12 h) NT 0 1.5 ± 4.0 ± 5.3 ± 0.8 5.7 ± 0.8 p < 0.0001 0.2 0.7 Iso 0 2.3 ± 5.3 ± 6.3 ± 2.0 6.3 ± 1.9 p = 0.0009 0.9 1.5 PBS 0 2.3 ± 3.5 ± 7.3 ± 1.5 8.8 ± 1.2 p < 0.0001 0.9 0.9 0.15 mg/ 0 1.6 ± 2.8 ± 4.0 ± 0.8 4.0 ± 0.9 p = 0.008 mL tPA 0.2 0.6 0.3 mg/ 0 1.2 ± 2 ± 0.8 ± 0.3 1.5 ± 0.4 ns mL tPA 0.2 0.3 0.5 mg/ 0 1.7 ± 4.3 ± 2.3 ± 0.6 2.0 ± 0.7 ns mL tPA 1.2 0.7 0.7 mg/ 0 1.5 ± 2.0 ± 1.5 ± 0.3 2.3 ± 0.6 ns mL tPA 0.2 0.4 *Values shown are mean ± SEM .sup.#Statistical analysis by ANOVA, with Tukey's post-hoc analysis ns = not significant NT = no treatment control (CEES alone) Iso = isoflurane control PBS = phosphate buffered saline control
tPA Normalizes Arterial Blood Gas (ABG) Abnormalities
(92) ABGs were obtained 12 hours after CEES exposure in rats receiving escalating tPA doses (0.15-0.7 mg/kg) at 5.5 hours and 6.5 hours, and compared these results to those from an untreated CEES-exposed group (NT), as well as an unexposed naïve group (at an altitude of 5292 ft, barometric pressure 624 mmHg,
(93) A dose-dependent improvement in blood lactate levels with tPA was also detected (
Example 2
(94) This example demonstrates that tissue plasminogen activator (tPA) increases survival thus preventing death following sulfur mustard (SM) vapor exposure.
(95) Methods Used in Example 2:
(96) Sprague-Dawley rats (250 g), intubated, under anesthesia, inhaled a fatal dose of SM ethanolic vapor (3.8 mg/kg), resulting in death by 12 hours with no treatment. Pulse oximetry (pOx) measurements were obtained every one hour, with concurrent scoring of respiratory distress. Treatment group received tPA intratracheally (0.7 mg/kg in 50 μl, 2 doses q 1 h, n=9) under isoflurane anesthesia when pOx was below 85% (at 5 h). Two control groups were also evaluated [placebo (PBS-diluent n=8) and untreated but SM-exposed (NT, n=8)]. Re-dosing was performed when pOx decreased <85% again. Arterial blood gases (ABGs) were obtained at euthanasia, and cast scoring (airway obstruction scoring system) was performed on fixed lungs by microdissection.
(97) Results
(98) By 12 hours, mortality from SM exposure alone (NT) was 100% (median survival: 10 hour), and with placebo-treatment was 75% (median survival: 8 hours). Rescue treatment with tPA resulted in 0% mortality, as all animals survived. Moreover, pOx at euthanasia in tPA-treated animals was significantly greater versus controls (mean±SEM: 92.3±0.8% with tPA; 63.0±5.1% with NT; 65.1±4.9% with placebo). ABG measurements at euthanasia were also greatly improved, showing recue from SM-induced respiratory failure and ventilation defects (p.sub.aCO.sub.2, mean±SEM, mmHg: 57±4 with tPA; 113±4 with NT; 112±8 with placebo), as well as rescue from SM-induced arterial acidosis (mean pH: 7.32±0.02 with tPA; 7.12±0.01 with NT; 7.09±0.04 with placebo). Serum bicarbonate was also significantly improved. Moreover, tPA treatment resulted in a greatly reduced airway fibrin cast burden, and thus prevented airflow obstruction.
(99) High level SM inhalation causes significant mortality due to progressive respiratory failure from airway obstructing fibrin casts. Airway administration of tPA 5 hours after exposure prevented death due to SM inhalation in the rat. Moreover, tPA normalized both oxygenation and ventilation defects seen due to high level SM inhalation, thereby rescuing from respiratory distress and failure. Intra-airway tPA should be considered as a life-saving rescue therapy for human patients after a significant SM inhalation exposure incident.
Example 3
(100) This example demonstrates that the administration of intratracheal tPA over a 48 hour period after inhaling a lethal quantity of sulfur mustard SM (3.8 mg/kg) diminishes airway fibrin-containing casts while improving clinical respiratory distress, pulmonary gas exchange, tissue oxygenation, and oxygen utilization. In addition, mortality, which was associated with hypoxemia and clinical respiratory distress, was eliminated.
(101) Procedural design for Example 3 is shown in
(102) Pulse oximetry values (estimated arterial oxygen saturation) in each group (i.e. SM+no treatment (NT, or SM alone, n=12); SM+placebo (PBS, n=13); or SM+tPA treatment (n=9)) was determined at the time of randomization. None were statistically different from the other (median of ˜81%) (
(103) The survival of rats in each group noted above over the 48 hour study period was determined. Survival curves for rats exposed to SM (3.8 mg/kg) and given: 1) SM alone (referred to as Sm alone (n=12), 2) SM+PBS (referred to as SM/placebo (n=13)), or 3) SM+tPA treatment (referred to as SM/tPA (n=9)) is shown in
(104) The mean±SEM pulse oximetry values for the rats was tracked in the three treatment groups defined above from the time of randomization as shown in
(105) The individual pulse oximetry results from the rats treated with tPA after randomization (at the time of initial decrease of pOx to <85%), and receipt of the initial induction dose (show as time=0 on this graph) is shown in
(106) The clinical scores at euthanasia was determined (elective or at 48 hour study termination) in all study groups as shown in
(107) The respiratory quality scores at euthanasia in all study groups was determined as shown in
(108) The total clinical distress scores was determined to trend over time×48 h after SM exposure in all three groups. Placebo treatment and non-treatment groups resulted in a sharp increase in clinical distress, with a maximum distress score of 9 in alive rats. tPA treated animals stabilized after receiving their induction tPA dose, and their distress plateaued at a minimal score of ˜2 (out of 10) for the entire 48 hour experiment, with the receipt of maintenance tPA therapy every 4 hours (data not shown).
(109) The arterial pH in naïve animals and all study groups at euthanasia was determined as shown in
(110) The arterial carbon dioxide tension (p.sub.aCO.sub.2, mm Hg) in naïve animals (air breathing controls) and all study groups at euthanasia was also determined as shown in
(111) The blood lactate in naïve animals (air breathing) and all study groups at euthanasia after exposure to 3.8 mg/kg SM was detected as shown in
(112) The hematocrit values for naïve animals (air breathing) and the three SM-exposed groups (no treatment-NT, placebo treatment-PBS or the tPA treatment) was determined as shown in
(113) Airway obstruction scores by fibrin casts in the three groups after mustard exposure were determined. Casts were revealed by airway microdissection after lung fixation (at euthanasia or 48 hours) of all major central lobar bronchi (5 lobes). These scores are obtained after microdissection of the trachea and all five lobes by morphometric study to quantitate the percent airway occlusion of each of the five main lobar bronchi by fibrin casts. These methods are described in Veress, L. A., et al. American Journal of Respiratory Cell and Molecular Biology 2013 48:4, 439-447. A score of 7 indicates 100% occlusion of all 5 main bronchi. The percent airway occlusion by cast was measured, then converted to a nominal score, weighted per ratio of lobar volume to total lung, then added for a composite score. SM-exposed (no treatment-NT) and placebo (PBS)-treated rats had scores exceeding 50% airway occlusion overall, while airway occlusion of main lobar bronchi in tPA-treated rats was negligible (score 4=50-65% total airway luminal occlusion). p<0.0005, ANOVA, Tukey's post-hoc analysis. Results are shown in
Example 4
(114) This example demonstrates the administration of a tPA analog, Reteplase (RETAVASE®), to animals who have been exposed to sulfur mustard (SM) vapor and that all indicators of airway obstruction, impaired gas exchange, oxygen delivery, and survival were improved in rPA treated animals as compared to untreated or vehicle-treated controls.
(115) Methods
(116) For retevase studies, rats were exposed to authentic sulfur mustard (SM) at Aberdeen Proving Grounds (Aberdeen, Md.; USAMRICD). The exposure was to SM in an ethanolic vapor at a dose of 3.8 mg/kg inhaled (identical to doses used in tPA efficacy studies at the same site). Following exposures, four doses of rPA was given at 5 and 6 hours post exposure, then again at 10 and 11 hours post exposure. Each dose was 50 micro liters of agent given intratracheally, in liquid form, or vehicle (diluent for rPA). For the active agent (rPA), two concentrations were given, 0.05 units/kg and 0.10 units/kg of rPA. Animals were monitored continuously throughout a 12 hour study. Euthanasia criteria were utilized exactly as applied in the above tPA experiments. Animals surviving to 12 hours after SM exposure were then electively euthanized at the end of the 12 hour study period.
(117) Results
(118) The effect on survival with retevase (rPA) treatment as compared to various controls is shown in
(119)
(120)
(121) The arterial blood pH values for rPA treated rats versus those of various control groups is shown in
(122) The arterial blood carbon dioxide content of rats treated with rPA versus those of 3 control groups is shown in
(123)
(124) The arterial blood oxygen content (partial pressure of oxygen; p.sub.aO.sub.2) of anesthetized animals at euthanasia is shown in
(125)
(126) It was noted that some animals in the retevase-treated groups, especially at higher doses, showed immediate respiratory distress following dosing with the rPA preparation. It was noted that these retevase (rPA) solutions appeared to be viscous and adhesive (‘sticky’), especially at higher concentrations, and thus these solutions may have contributed to airway obstruction and respiratory distress, either due to direct mass effect and/or chemosensitivity. This may explain why rats treated with the lowest dose of rPA appeared to fare better than those treated with higher doses for some parameters.
(127) The foregoing examples of the present invention have been presented for purposes of illustration and description. Furthermore, these examples are not intended to limit the invention to the form disclosed herein. Consequently, variations and modifications commensurate with the teachings of the description of the invention, and the skill or knowledge of the relevant art, are within the scope of the present invention. The specific embodiments described in the examples provided herein are intended to further explain the best mode known for practicing the invention and to enable others skilled in the art to utilize the invention in such, or other, embodiments and with various modifications required by the particular applications or uses of the present invention. It is intended that the appended claims be construed to include alternative embodiments to the extent permitted by the prior art.
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