Compositions and methods for treating pleural space infections and hemothorax

Abstract

Compositions and methods for increasing intrapleural fibrinolysis and inhibiting, treating, and / or preventing pleural space infections and / or hemothorax are disclosed.

Description

[0001] COMPOSITIONS AND METHODS FOR TREATING PLEURAL SPACE INFECTIONS AND HEMOTHORAX

[0002] This application claims priority under 35 U.S.C. §119(e) to U.S. Provisional Patent Application No. 63 / 535,132, filed August 29, 2023. The foregoing application is incorporated by reference herein.

[0003] This invention was made with government support under Grant No. UM1- HL120877, awarded by the National Institutes of Health. The government has certain rights in the invention.

[0004] FIELD OF THE INVENTION

[0005] The present invention relates to the field of medicine. More specifically, the invention provides compositions and methods for increasing intrapleural fibrinolysis, particularly following traumatic or non-traumatic hemothorax, and for inhibiting, treating, and / or preventing pleural space infections.

[0006] BACKGROUND OF THE INVENTION

[0007] Several publications and patent documents are cited throughout the specification in order to describe the state of the art to which this invention pertains. Each of these citations is incorporated herein by reference as though set forth in full.

[0008] Approximately 80,000 patients develop pleural space (also known as pleural cavity) infections annually in the US and UK combined (Davies, et al. (2010) Thorax 65(Suppl 2):ii41 -53). Hallmarks of pleural space infection are the infiltration of neutrophils that release their contents including neutrophil extracellular traps and toxic granule proteases like neutrophil elastase, and the formation of fibrin loculations that render drainage with thoracentesis or tube thoracostomy inadequate (Twaddel, et al. (2021) Chest 160(5): 1645-1655; Alegre, et al. (2000) Respiration 67(4):426-32; Komissarov, et al. (2018) Am. J. Physiol. Lung Cell Mol. Physiol., 314(5):L757-L768). As a result, intrapleural lytic therapy with tissue-plasminogen activator (tPA) and DNase has become the standard of care since the publication of Second Multicenter Intrapleural Sepsis Trial (MIST-2) (Rahman, et al. (2011) N. Engl. J. Med., 365(6):518-26). Despite these advances, failure rates of intrapleural fibrinolytics are 20% or more, frequently necessitating invasive surgery (Rahman, et al. (2011) N. Engl. J. Med., 365(6):518-26; Piccolo et al. (2015) J. Thorac. Dis., 7(6):999-1008). Further, completion of the standard tPA / DNase protocol requires six doses over three days (once every 12 hours), and there is currently no reliable method to predict which patients will have a successful response (Piccolo et al. (2015) J. Thorac. Dis., 7(6):999-1008). Thus, if there were alternative methods that have improved success rates, or an objective prediction method for who is likely to fail intrapleural lytic therapy, this would provide patients definitive therapy sooner, improve outcomes, and reduce hospital length of stay.

[0009] Similarly, in patients suffering traumatic injury to the chest, up to 38% will develop a hemothorax (Zeiler et al. (2020) Clin. Pulm. Med., 27(1): 1-12; Al-Koudmani et al. (2012) J. Cardiothorac. Surg., 7:35). Despite standard drainage using tube thoracostomy, 5-30% of those patients will develop a retained hemothorax, defined as the presence of undrained blood 72 hours after chest tube placement (Holsen et al. (2019) Ann. Phrmacother., 53(10) 1060-1066). Patients with residual hemothorax are at significantly increased risk of empyema, lung entrapment and fibrothorax, generally requiring an additional procedure for removal of the retained blood in the chest, including thoracotomy, video-assisted thoracoscopic surgery, or administration of intrapleural fibrinolytic agents. The use of alteplase as an intrapleural fibrinolytic agent is increasingly being used in many trauma centers because it avoids the need for an additional surgical procedure requiring general anesthesia, and intrapleural administration of this drug has been shown to have a very low risk of bleeding. There is no uniformly accepted dose, though most patients receive a bolus intrapleural dose of 10-100 mg of alteplase per day for up to three days (Holsen et al. (2019) Ann. Phrmacother., 53(10): 1060-1066). Based on data from six retrospective reviews and one meta-analysis, the success of this approach ranges from 67-85% (Stiles et al. (2014) Am. J. Surg. 207(6):960-963; Ben-Or et al. (2011) Ann. Thorac. Surg. 91(3):860-864; Abu-Daff et al. (2013) BMJ Open. 3(2):e001887; Thommi et al. (2007) Am. J. Ther. 14(4):341 -345; Skeete et al. (2004) J. Trauma. 57(6): 1178-1183; Heimes et al. (2017) J. Thorac. Dis. 9(5): 1310-1316).

[0010] An improved method of intrapleural fibrinolysis to reduce this 15-33% failure rate and enhance the success rate following a single dose would reduce the need for additional surgical procedures, reduce morbidity and length of hospital stay, and improve outcomes. SUMMARY OF THE INVENTION

[0011] In accordance with the present invention, methods of increasing intrapleural fibrinolysis in a subject in need thereof are provided. Methods of treating, inhibiting, and / or preventing a pleural space infection in a subject in need thereof are also provided. Methods of treating, inhibiting, and / or preventing hemothorax in a subject in need thereof are also provided. In certain embodiments, the methods comprise administering plasminogen and / or an elastase inhibitor to the subject. In certain embodiments, the plasminogen and / or an elastase inhibitor are administered intrapleurally to the subject. In certain embodiments, methods further comprise administering (e.g., intrapleurally) a plasminogen activator (e.g., urokinase plasminogen activator (uPA), tissue-plasminogen activator (tPA), or streptokinase) to the subject, optionally with DNase. In certain embodiments, methods further comprise administering (e.g., intrapleurally) tissueplasminogen activator (tPA) to the subject, optionally with DNase. In certain embodiments, the plasminogen is human plasminogen, particularly human-tvmh (Ryplazim®). In certain embodiments, the elastase inhibitor is an inhibitor of neutrophil elastase. In certain embodiments, the tPA is human tPA, particularly alteplase or tenecteplase. In certain embodiments, wherein the DNase is human deoxyribonuclease I, particularly dornase alfa. In certain embodiments, the subject to be treated was previously unsuccessfully treated with intrapleural lytic therapy with tPA and DNase. In certain embodiments, the subject has a neutrophil elastase activity in pleural fluid greater than in plasma. In certain embodiments, the subject has reduced plasminogen in pleural fluid compared to plasma. In certain embodiments, the method further comprises measuring the neutrophil elastase activity in the pleural fluid and, optionally, plasma of the subject. In certain embodiments, the method further comprises measuring the amount of plasminogen in the pleural fluid and, optionally, plasma of the subject.

[0012] In accordance with another aspect of the instant invention, kits for practicing the methods of the instant invention are provided. In certain embodiments, the kit comprises tissue-plasminogen activator (tPA) and a plasminogen and / or an elastase inhibitor. In certain embodiments, the kit further comprises DNase. In certain embodiments, the components of the kit (e.g., tissue-plasminogen activator (tPA) and a plasminogen and / or an elastase inhibitor) are lyophilized. In certain embodiments, the components of the kit are contained within containers or vials. In certain embodiments, the kit comprises a container comprising lyophilized tissue-plasminogen activator (tPA) and lyophilized plasminogen. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 provides a consolidated standards for reporting trials diagram for the parent trial. The study samples were obtained from comparing intrapleural fibrinolysis with early surgery for initial treatment of complex pleural infections. DNAse = deoxyribonuclease; tPA = tissue plasminogen activator; VATS = video-assisted thoracoscopic surgery.

[0014] Figures 2A-2E show that pleural fluid from patients with empyema showed increased neutrophil elastase activity, reduced plasminogen concentration, and multiple plasminogen degradation fragments. Figures 2A-2B: Box-and- whisker plots showing amidolytic assay of elastase activity (Fig. 2A) and enzyme-linked immunosorbent assay (ELISA) for plasminogen concentration (Fig. 2B) performed on pleural fluid and plasma samples from 10 patients with pleural space infections. All samples were run in duplicate with individual patient means graphed as scatter dots and group medians with or without interquartile range and range shown. Figs. 2C-2E: Western blotting for plasminogen Kringle domains 1 through 3 was performed on Glu-plasminogen incubated with increasing concentrations of human neutrophil elastase (Fig. 2C), Glu-plasminogen incubated in the supernatant from purified neutrophils activated by phorbol myristate acetate with or without elastase inhibitor (Fig. 2D), or pleural fluid from 10 patients with pleural space infections (Fig. 2E). *P < .05 using two-sided Wilcoxon signed-rank test. El = elastase inhibitor (N-(methoxysuccinyl)-Ala-Ala-Pro-Val (SEQ ID NO: 1)- chloromethyl ketone); HNE = human neutrophil elastase; PLG = plasminogen; PM = protein marker; PMA = phorbol 12-myristate 13-acetate.

[0015] Figures 3 A and 3B provide graphs showing that PALI activity is low despite high PAI-1 antigen levels in pleural fluid from patients with infected pleural space disease. Fig. 3A (left): Total PALI antigen levels (inclusive of latent or inactive PALI) and the amount of active PALI were measured via ELISA and a capture-based ELISA-like assay, respectively, in the pleural fluid of patients with infected pleural space disease obtained before intervention. Fig. 3A (right): The amount of PALI that is active (i.e., capable of inhibiting tPA) relative to the total PALI antigen present is shown. Fig. 3B: The PALI activity over time (before intervention, day 1 after intervention, and day 2 after intervention) is compared between the tPA and deoxyribonuclease treatment group and the VATS treatment group. *P < .05, Mann-Whitney U test.aPleural fluid was available only for five of the six total patients in the tPA and deoxyribonuclease treatment group for PALI assays because of lack of enough sample remaining after all other assays were performed. PAI-1 = plasminogen activator inhibitor 1; tPA = tissue plasminogen activator; VATS = video-assisted thoracoscopic surgery.

[0016] Figures 4A and 4B show that plasminogen supplementation to pleural fluid from patients with empyema enables clot lysis in vitro. Turbidity clot lysis assays were performed by adding pleural fluid to human fibrinogen and human a-thrombin in 10 mM CaCl with either 33 pM tissue plasminogen activator (tPA) (Fig. 4A) or 330 pM tPA (Fig. 4B). Absorbance at 405 nM was measured every minute for 360 minutes. Mean absorbance is plotted as solid line with SEM shown as the bounding dotted lines. PLG = plasminogen; tPA = tissue plasminogen activator.

[0017] DETAILED DESCRIPTION OF THE INVENTION tPA stimulates fibrinolysis by cleaving plasminogen into its active protease plasmin, which then dissolves fibrin that makes up clots and pleural loculations (Cesarman-Maus, et al. (2005) Br. J. Haematol., 129(3):307-21). However, in the presence of neutrophil elastase, plasminogen is cleaved in several locations, which ultimately renders tPA ineffective at generating a lytic response because of a lack of full- length plasminogen (Barrett, et al. (2017) J. Trauma Acute Care Surg., 83(6): 1053-1061). To date, the explanation in the literature for why multiple repeat doses of tPA and deoxyribonuclease are required for successful treatment of complex pleural collections and why, despite these repeat doses, a significant failure rate persists, has been the observation that high antigen levels of tPA’s native inhibitor, plasminogen activator inhibitor 1 (PAI-1), are present in pleural fluid of these patients (Bedawi et al. (2023) Am. J. Respir. Crit. Care Med., 207(6):731-739). However, PAI-1 is known to be conformationally labile with a functional half-life of 1 to 2 hours before spontaneous conversion to the latent form of PAI-1 that is incapable of inhibiting tPA (Berkenpas, et al. (1995) EMBO J., 14(13):2969-2977). Thus, PAI-1 antigen levels do not necessarily reflect PAI-1 activity (i.e., its ability to inhibit tPA). Additionally, PAI-l ’s bait region that is directly responsible for inhibiting tPA is known to be cleaved by neutrophil elastase, rendering PAI-1 inactive (Wu et al. (1995) Blood 86(3): 1056-1061), setting up the potential for further discordance between PAI-1 antigen measurements and PAI-1 activity against tPA. Thus, although it is now established that PAI-1 antigen levels are high in pleural fluid of complex infected pleural collections, it is not clear how much of this is capable of tPA inhibition. It is also not clear how much full-length plasminogen is present in such a highly inflamed extravascular environment for tPA to activate and generate fibrinolysis.

[0018] As such, it was hypothesized that the pleural fluid from patients following traumatic chest injury and from patients with complicated parapneumonic effusions and empyema would have higher elastase activity levels and lower plasminogen levels relative to their circulating plasma, that PAI-1 activity of the pleural fluid would be fractionally low relative to PAI-1 antigen levels, and that their pleural fluid would have minimal fibrinolytic potential that could be rescued with plasminogen supplementation.

[0019] Herein, pleural fluid was collected from thoracostomy tubes prior to intrapleural lytic therapy in patients (n = 10) with pleural pH <7.2. It was determined that pleural fluid elastase activities were > 4-fold higher on median (p = 0.02) and plasminogen levels > 3-fold lower on median (p = 0.04) when compared to their corresponding plasma levels. On Western blot analysis, large quantities of plasminogen degradation fragments were observed that were consistent with those observed when plasminogen was incubated with activated neutrophil supernatant or purified neutrophil elastase. Plasminogen activator inhibitor 1 (PAI-1), the native tPA inhibitor, showed high antigen levels before the intervention, but the overwhelming majority of this PAI-1 (82%) was not active (P = .003), and all PAI-1 activity was lost by day 2 after the intervention in patients receiving intrapleural tPA and deoxyribonuclease. Finally, using turbidity clot lysis assays, 9 of 10 patients’ pleural fluid were found to be unable to generate a significant fibrinolytic response when challenged with tPA and that plasminogen supplementation rescued fibrinolysis in all patients in response to tPA.

[0020] In accordance with the instant invention, methods of increasing intrapleural fibrinolysis in a subject are provided. In certain embodiments, the method comprises administering plasminogen and / or an elastase inhibitor to the subject. In certain embodiments, the method comprises administering plasminogen to the subject. In certain embodiments, the method comprises administering an elastase inhibitor to the subject. In certain embodiments, the method further comprises administering a plasminogen activator. In certain embodiments, the method further comprises administering a DNase. The components (e.g., plasminogen and / or an elastase inhibitor) may be administered intrapleurally to the subject (e.g., by direct injection into the pleural space, by intrapleural catheter, by chest tube, etc.). In certain embodiments, the plasminogen and / or an elastase inhibitor are administered to the subject with intrapleural fibrinolytic therapy (e.g., before, after, and / or at the same time as the intrapleural fibrinolytic therapy). In certain embodiments, the intrapleural fibrinolytic therapy comprises administration of a plasminogen activator (e.g., tissue-plasminogen activator (tPA)), optionally with DNase. In certain embodiments, the method comprises administering plasminogen and / or an elastase inhibitor at least at the same time as the plasminogen activator (e.g., tPA).

[0021] In accordance with the instant invention, methods of treating, inhibiting, and / or preventing a pleural space infection in a subject are provided. In certain embodiments, the subject has empyema, such as parapneumonic empyema. In certain embodiments, the subject has pleural effusion, such as parapneumonic pleural effusion or complex pleural effusion. In certain embodiments, the method comprises administering plasminogen and / or an elastase inhibitor to the subject. In certain embodiments, the method comprises administering plasminogen to the subject. In certain embodiments, the method comprises administering an elastase inhibitor to the subject. In certain embodiments, the method further comprises administering a plasminogen activator. In certain embodiments, the method further comprises administering a DNase. The components (e.g., plasminogen and / or an elastase inhibitor) may be administered intrapleurally to the subject (e.g., by direct injection into the pleural space, by intrapleural catheter, by chest tube, etc.). In certain embodiments, the plasminogen and / or an elastase inhibitor are administered to the subject with intrapleural fibrinolytic therapy (e.g., before, after, and / or at the same time as the intrapleural fibrinolytic therapy). In certain embodiments, the intrapleural fibrinolytic therapy comprises administration of a plasminogen activator (e.g., tissue-plasminogen activator (tPA)), optionally with DNase. In certain embodiments, the method comprises administering plasminogen and / or an elastase inhibitor at least at the same time as a plasminogen activator (e.g., tPA).

[0022] In accordance with the instant invention, methods of treating, inhibiting, and / or preventing hemothorax in a subject are provided. In certain embodiments, the hemothorax is a retained hemothorax. In certain embodiments, the hemothorax is a non- traumatic hemothorax. In certain embodiments, the hemothorax is a traumatic hemothorax (e.g., caused by a traumatic injury, particularly to the chest). In certain embodiments, the hemothorax is a retained traumatic hemothorax. In certain embodiments, the method comprises administering plasminogen and / or an elastase inhibitor to the subject. In certain embodiments, the method comprises administering plasminogen to the subject. In certain embodiments, the method comprises administering an elastase inhibitor to the subject. In certain embodiments, the method further comprises administering a plasminogen activator. In certain embodiments, the method further comprises administering a DNase. The components (e.g., plasminogen and / or an elastase inhibitor) may be administered intrapleurally to the subject (e.g., by direct injection into the pleural space, by intrapleural catheter, by chest tube, etc.). In certain embodiments, the plasminogen and / or an elastase inhibitor are administered to the subject with intrapleural fibrinolytic therapy (e.g., before, after, and / or at the same time as the intrapleural fibrinolytic therapy). In certain embodiments, the intrapleural fibrinolytic therapy comprises administration of a plasminogen activator (e.g., tissue-plasminogen activator (tPA)), optionally with DNase. In certain embodiments, the method comprises administering plasminogen and / or an elastase inhibitor at least at the same time as a plasminogen activator (e.g., tPA).

[0023] The plasminogen of the instant invention may be human plasminogen. The amino acid and nucleotide sequences of plasminogen (e.g., human plasminogen) are known in the art (see, e.g., Gene ID: 5340; GenBank Accession Nos. NM_000301.5 and NP 000292.1). In certain embodiments, the plasminogen is a recombinant plasminogen, particularly recombinant human plasminogen. In certain embodiment, the plasminogen is isolated or purified from blood or plasma, particularly human blood or plasma. In certain embodiments, the plasminogen is Ryplazim® (plasminogen, human-tvmh; Kedrion BioPharma, Fort Lee, NJ). An example of the amino acid sequence of the human plasminogen (precursor) is:

[0024] 1 MEHKEVVLLL LLFLKSGQGE PLDDYVNTQG ASLFSVTKKQ LGAGS IEECA AKCEEDEE FT 61 CRAFQYHSKE QQCVIMAENR KSS I I IRMRD VVLFEKKVYL SECKTGNGKN YRGTMSKTKN 121 GITCQKWSST S PHRPRFS PA THPSEGLEEN YCRNPDNDPQ GPWCYTTDPE KRYDYCDI LE 181 CEEECMHCSG ENYDGKI SKT MSGLECQAWD SQS PHAHGYI PSKFPNKNLK KNYCRNPDRE 241 LRPWCFTTDP NKRWELCDI P RCTTPPPSSG PTYQCLKGTG ENYRGNVAVT VSGHTCQHWS 301 AQTPHTHNRT PENFPCKNLD ENYCRNPDGK RAPWCHTTNS QVRWEYCKI P SCDSS PVSTE 361 QLAPTAPPEL TPVVQDCYHG DGQSYRGTSS TTTTGKKCQS WSSMTPHRHQ KTPENYPNAG 421 LTMNYCRNPD ADKGPWCFTT DPSVRWEYCN LKKCSGTEAS VVAPPPVVLL PDVETPSEED 481 CMFGNGKGYR GKRATTVTGT PCQDWAAQE P HRHS I FTPET NPRAGLEKNY CRNPDGDVGG 541 PWCYTTNPRK LYDYCDVPQC AAPS FDCGKP QVE PKKCPGR VVGGCVAHPH SWPWQVSLRT 601 RFGMHFCGGT LIS PEWVLTA AHCLEKS PRP SSYKVI LGAH QEVNLE PHVQ E IEVSRLFLE 661 PTRKDIALLK LSS PAVITDK VI PACLPS PN YVVADRTECF ITGWGETQGT FGAGLLKEAQ 721 LPVIENKVCN RYE FLNGRVQ STELCAGHLA GGTDSCQGDS GGPLVCFEKD KYI LQGVTSW 781 GLGCARPNKP GVYVRVSRFV TWIEGVMRNN ( SEQ ID NO : 2 )

[0025] Amino acids 1-19 of SEQ ID NO: 2 is a signal peptide. Mature human plasminogen may comprise amino acids 20-810 of SEQ ID NO: 2. In certain embodiments, the human plasminogen has at least 90%, 95%, 97%, 99%, or 100% homology or identity with SEQ ID NO: 2. In certain embodiments, the human plasminogen has at least 90%, 95%, 97%, 99%, or 100% homology or identity with amino acids 20-810 of SEQ ID NO: 2. The elastase inhibitor of the instant invention may be human elastase inhibitor. In certain embodiments, the elastase inhibitor is an inhibitor of neutrophil elastase. Elastase inhibitors may be small molecules, nucleic acid molecules (e.g., inhibitory nucleic acid molecules such as siRNA, antisense molecules, shRNA, microRNA, etc.), or proteins (e.g., anti-elastase antibodies or antigen-binding fragments thereof, elastase substrates, etc.). In certain embodiments, the elastase inhibitor is a small molecule. Elastase inhibitors are known in the art. Examples of elastase inhibitors include, without limitation: sivelestat (e.g., Elaspol® (sivelestat sodium hydrate)), elastatinal, alpha-1 proteinase inhibitor (e.g., Glassia®, Prolastin®-C, Aralast®, Zemaira®, Trypsone™, and Respreeza™), alvelestat (MPH966; Mereo BioPharma), and N-(methoxysuccinyl)-Ala- Ala-Pro-Val (SEQ ID NO: l)-cholorom ethyl ketone. In certain embodiments, the alpha- 1 proteinase inhibitor is isolated or purified from blood or plasma, particularly human blood or plasma. Polypeptide elastase inhibitors are also known in the art (Ahmad et al. (2020) Front. Pharmacol. 11 :688; incorporated herein by reference for disclosed elastase inhibitors).

[0026] Plasminogen activators are known in the art. In certain embodiments, the plasminogen activator is human. Examples of plasminogen activators include, without limitation: urokinase plasminogen activator (uPA), tissue-plasminogen activator (tPA), and streptokinase. In certain embodiments, the plasminogen activator is resistant to inhibition by PAI-1. In certain embodiments, tissue-plasminogen activator (tPA) may be human tPA. The amino acid and nucleotide sequences of tPA (e.g., human tPA) are known in the art (see, e.g., Gene ID: 5327; GenBank Accession Nos. NM_000930.5 and NP 000921.1). In certain embodiments, the tPA is a recombinant tPA, particularly recombinant human tPA. In certain embodiment, the tPA is isolated or purified from blood or plasma, particularly human blood or plasma. In certain embodiments, the tPA is alteplase (e.g., Activase®) or Tenecteplase (TNKase®). An example of the amino acid sequence of human tPA (preproprotein) is:

[0027] 1 MDAMKRGLCC VLLLCGAVFV SPSQEIHARF RRGARSYQVI CRDEKTQMIY QQHQSWLRPV

[0028] 61 LRSNRVEYCW CNSGRAQCHS VPVKSCSEPR CFNGGTCQQA LYFSDFVCQC PEGFAGKCCE 121 IDTRATCYED QGISYRGTWS TAESGAECTN WNSSALAQKP YSGRRPDAIR LGLGNHNYCR 181 NPDRDSKPWC YVFKAGKYSS EFCSTPACSE GNSDCYFGNG SAYRGTHSLT ESGASCLPWN 241 SMILIGKVYT AQNPSAQALG LGKHNYCRNP DGDAKPWCHV LKNRRLTWEY CDVPSCSTCG 301 LRQYSQPQFR IKGGLFADIA SHPWQAAI FA KHRRSPGERF LCGGILISSC WILSAAHCFQ 361 ERFPPHHLTV ILGRTYRVVP GEEEQKFEVE KYIVHKEFDD DTYDNDIALL QLKSDSSRCA 421 QESSVVRTVC LPPADLQLPD WTECELSGYG KHEALSPFYS ERLKEAHVRL YPSSRCTSQH 481 LLNRTVTDNM LCAGDTRSGG PQANLHDACQ GDSGGPLVCL NDGRMTLVGI ISWGLGCGQK 541 DVPGVYTKVT NYLDWIRDNM RP ( SEQ ID NO : 3 ) Mature human tPA may comprise amino acids 36-562 of SEQ ID NO: 3. In certain embodiments, the human tPA has at least 90%, 95%, 97%, 99%, or 100% homology or identity with SEQ ID NO: 3. In certain embodiments, the human tPA has at least 90%, 95%, 97%, 99%, or 100% homology or identity with amino acids 36-562 of SEQ ID NO: 3.

[0029] The DNase of the instant invention may be human DNase, particularly human deoxyribonuclease I. The amino acid and nucleotide sequences of DNase (e.g., human DNase) are known in the art (see, e.g., Gene ID: 1773; GenBank Accession Nos. NM_001351825.2 and NP_001338754.1). In certain embodiments, the DNase is a recombinant DNase, particularly recombinant human DNase. In certain embodiments, the DNase is dornase alfa (e.g., Pulmozyme®). An example of the amino acid sequence of human DNase (precursor) is:

[0030] 1 MRGMKLLGAL LALAALLQGA VSLKIAAFNI QTFGETKMSN ATLVSYIVQI LSRYDIALVQ

[0031] 61 EVRDSHLTAV GKLLDNLNQD APDTYHYVVS E PLGRNSYKE RYLFVYRPDQ VSAVDSYYYD 121 DGCE PCGNDT FNRE PAIVRF FSRFTEVRE F AIVPLHAAPG DAVAE IDALY DVYLDVQEKW 181 GLEDVMLMGD FNAGCSYVRP SQWSS IRLWT S PTFQWLI PD SADTTATPTH CAYDRIVVAG 241 MLLRGAVVPD SALPFNFQAA YGLSDQLAQA I SDHYPVEVM LK ( SEQ ID NO : 4 )

[0032] Mature human DNase may comprise amino acids 23-282 of SEQ ID NO: 4. In certain embodiments, the human DNase has at least 90%, 95%, 97%, 99%, or 100% homology or identity with SEQ ID NO: 4. In certain embodiments, the human DNase has at least 90%, 95%, 97%, 99%, or 100% homology or identity with amino acids 23-282 of SEQ ID NO: 4.

[0033] In certain embodiments, the subject to be treated with the methods of the instant invention (e.g., with a plasminogen and / or an elastase inhibitor) has previously received intrapleural lytic therapy with tPA and DNase. In certain embodiments, the subject to be treated with the methods of the instant invention (e.g., with a plasminogen and / or an elastase inhibitor) has previously been unsuccessfully treated with intrapleural lytic therapy with tPA and DNase. Intrapleural lytic therapy with tPA and DNase typically requires six doses over three days. In certain embodiments, the subject to be treated with the methods of the instant invention (e.g., with a plasminogen and / or an elastase inhibitor) has previously been unsuccessfully surgically treated for an infection in pleural cavity or unsuccessfully treated surgical with a washout of the pleural space and / or hemothorax.

[0034] In certain embodiments, the subject to be treated with the methods of the instant invention (e.g., with a plasminogen and / or an elastase inhibitor) has a neutrophil elastase activity in their pleural fluid greater than in their plasma (e.g., at least 1-fold greater, at least 2-fold greater, at least 3 -fold greater, at least 4-fold greater, at least 5 -fold greater or more). In certain embodiments, the subject to be treated with the methods of the instant invention (e.g., with a plasminogen and / or an elastase inhibitor) has reduced plasminogen in their pleural fluid compared to their plasma (e.g., at least a 1-fold reduction, at least a 2 -fold reduction, at least a 3 -fold reduction, at least a 4-fold reduction, or more). The methods of the instant invention may further comprise (e.g., prior to the administration of a plasminogen and / or an elastase inhibitor) determining and / or measuring 1) the neutrophil elastase activity in the pleural fluid and plasma of the subject, wherein the therapy of the instant invention (e.g., a plasminogen and / or an elastase inhibitor) is administered when the subject has a neutrophil elastase activity in their pleural fluid greater than in their plasma; and / or 2) the amount of plasminogen in the pleural fluid and plasma of the subject, wherein the therapy of the instant invention (e.g., a plasminogen and / or an elastase inhibitor) is administered when the subject has reduced plasminogen in their pleural fluid compared to their plasma. The methods of the instant invention may further comprise (e.g., prior to the administration of a plasminogen and / or an elastase inhibitor) determining and / or measuring 1) the neutrophil elastase activity in the pleural fluid of the subject, wherein the therapy of the instant invention (e.g., a plasminogen and / or an elastase inhibitor) is administered when the subject has a neutrophil elastase activity in their pleural fluid greater than a standard amount (e.g., the normal amount in pleural fluid or in the plasma in healthy individuals (e.g., without an infection in the pleural cavity)); and / or 2) the amount of plasminogen in the pleural fluid of the subject, wherein the therapy of the instant invention (e.g., a plasminogen and / or an elastase inhibitor) is administered when the subject has reduced plasminogen in their pleural fluid compared to a standard amount (e.g., the normal amount in pleural fluid or in the plasma in healthy individuals (e.g., without an infection in the pleural cavity)).

[0035] In accordance with another aspect of the instant invention, kits are provided. The kits may be used to practice the methods of the instant invention. In certain embodiments, the kit comprises plasminogen and / or an elastase inhibitor. In certain embodiments, the kit further comprises tissue-plasminogen activator (tPA) and / or DNase. In certain embodiments, the kit comprises plasminogen and tPA. In certain embodiments, the kit comprises plasminogen, tPA, and DNase. In certain embodiments, the kit comprises an elastase inhibitor and tPA. In certain embodiments, the kit comprises an elastase inhibitor, tPA, and DNase. In certain embodiments, the components of the kit are contained within a container or vial. One or more of the components of the kit may be contained in a composition with a carrier, particularly a pharmaceutically acceptable carrier, within the container or vial. In certain embodiments, one or more of the components of the kit is lyophilized and contained within a container or vial. The kits may further comprise buffers, solutions, diluents, and / or carriers (e.g., pharmaceutically acceptable carriers) in one or more containers or vials. When one or more components is lyophilized, the kit may comprise one or more reconstitution buffers for the lyophilized component.

[0036] In certain embodiments, the kit comprises lyophilized tPA and lyophilized plasminogen in one container or vial. In certain embodiments, the kit comprises lyophilized tPA and lyophilized elastase inhibitor in one container or vial. In certain embodiments, the kits comprise DNase, optionally lyophilized, in a separate container or vial. In certain embodiments, the kit comprises lyophilized tPA, lyophilized plasminogen, and lyophilized DNase in one container or vial. In certain embodiments, the kit comprises lyophilized tPA, lyophilized elastase inhibitor, and lyophilized DNase in one container or vial.

[0037] The compounds of the instant invention will generally be administered to a patient as a pharmaceutical preparation. The term “patient” as used herein refers to human or animal subjects. These compounds may be employed therapeutically, under the guidance of a physician.

[0038] The pharmaceutical preparation comprising the compounds of the invention may be conveniently formulated for administration with an acceptable medium such as water, buffered saline, ethanol, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol and the like), dimethyl sulfoxide (DMSO), oils, detergents, suspending agents or suitable mixtures thereof. The concentration of the compounds in the chosen medium may be varied and the medium may be chosen based on the desired route of administration of the pharmaceutical preparation. Except insofar as any conventional media or agent is incompatible with the compounds to be administered, its use in the pharmaceutical preparation is contemplated.

[0039] The dose and dosage regimen of the compounds according to the invention that is suitable for administration to a particular patient may be determined by a physician considering the patient's age, sex, weight, general medical condition, and the specific condition and severity thereof for which the compound is being administered. The physician may also consider the route of administration of the compound, the pharmaceutical carrier with which the compounds may be combined, and the compounds’ biological activity.

[0040] Selection of a suitable pharmaceutical composition will also depend upon the mode of administration chosen. For example, the compositions of the invention may be administered by direct injection, intrapleural catheter, or chest tube. In this instance, a pharmaceutical composition comprises the components dispersed in a medium that is compatible with the site of administration (e.g., pleural cavity).

[0041] Compositions of the instant invention may be administered by any method. In certain embodiments, the compositions of the instant invention are administered intrapleurally. In certain embodiments, the methods of administration include but are not limited to intrapleural administration. In a particular embodiment, the compositions are administered by direct injection. In a particular embodiment, the compositions are administered by a catheter or tube (e.g., chest tube). Pharmaceutical compositions for injection are known in the art. If injection is selected as a method for administering the composition, steps should be taken to ensure that sufficient amounts of the components reach their target cells, tissues, and / or compartments to exert a biological effect.

[0042] Pharmaceutical compositions containing compounds of the present invention as the active ingredient in intimate admixture with a pharmaceutical carrier can be prepared according to conventional pharmaceutical compounding techniques. The carrier may take a wide variety of forms depending on the form of preparation desired for administration. Injectable suspensions may be prepared, in which case appropriate liquid carriers, suspending agents and the like may be employed.

[0043] Compositions of the invention may be formulated in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form, as used herein, refers to a physically discrete unit of the pharmaceutical composition appropriate for the patient undergoing treatment. Each dosage should contain a quantity of active ingredient calculated to produce the desired effect in association with the selected pharmaceutical carrier. Procedures for determining the appropriate dosage unit are well known to those skilled in the art. For example, the appropriate dosage unit for the administration of the composition may be determined by evaluating the toxicity of the composition in animal models. Various concentrations of the components in composition may be administered to mice or other mammals, and the minimal and maximal dosages may be determined based on the beneficial results and side effects observed as a result of the treatment. A pharmaceutical preparation of the invention may be formulated in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form, as used herein, refers to a physically discrete unit of the pharmaceutical preparation appropriate for the patient undergoing treatment. Each dosage should contain a quantity of active ingredient calculated to produce the desired effect in association with the selected pharmaceutical carrier. Procedures for determining the appropriate dosage unit are well known to those skilled in the art. Dosage units may be proportionately increased or decreased based on the weight of the patient. Appropriate concentrations for alleviation of a particular pathological condition may be determined by dosage concentration curve calculations, as known in the art.

[0044] The compositions comprising the compounds of the instant invention may be administered at appropriate intervals, if needed, until the pathological symptoms are reduced or alleviated, after which the dosage may be reduced to a maintenance level or treatment may be stopped. The appropriate interval in a particular case would normally depend on the condition of the patient.

[0045] Definitions

[0046] The following definitions are provided to facilitate an understanding of the present invention.

[0047] The singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise.

[0048] With respect to protein, the term “isolated protein” is sometimes used herein. This term may refer to a protein produced by expression of an isolated nucleic acid molecule of the invention. Alternatively, this term may refer to a protein which has been sufficiently separated from other proteins with which it would naturally be associated (e.g., so as to exist in “substantially pure” form). “Isolated” is not meant to exclude artificial or synthetic mixtures with other compounds or materials, or the presence of impurities that do not interfere with the fundamental activity, and that may be present, for example, due to incomplete purification, or the addition of stabilizers.

[0049] The term “substantially pure” refers to a preparation comprising at least 50-60% by weight the compound of interest (e.g., nucleic acid, oligonucleotide, protein, etc.), particularly at least 75% by weight, or at least 90-99% or more by weight of the compound of interest. Purity may be measured by methods appropriate for the compound of interest (e.g. chromatographic methods, agarose or polyacrylamide gel electrophoresis, HPLC analysis, and the like).

[0050] “Pharmaceutically acceptable” indicates approval by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans.

[0051] A “carrier” refers to, for example, a diluent, adjuvant, preservative (e.g., Thimersol, benzyl alcohol), anti-oxidant (e.g., ascorbic acid, sodium metabisulfite), solubilizer (e.g., polysorbate 80), emulsifier, buffer (e.g., Tris HC1, acetate, phosphate), antimicrobial, bulking substance (e.g., lactose, mannitol), excipient, auxiliary agent or vehicle with which an active agent of the present invention is administered. Pharmaceutically acceptable carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin. Water or aqueous saline solutions and aqueous dextrose and glycerol solutions are preferably employed as carriers, particularly for injectable solutions. Suitable pharmaceutical carriers are described in “Remington's Pharmaceutical Sciences” by E.W. Martin (Mack Publishing Co., Easton, PA); Gennaro, A. R., Remington: The Science and Practice of Pharmacy, (Lippincott, Williams and Wilkins); Liberman, et al., Eds., Pharmaceutical Dosage Forms, Marcel Decker, New York, N.Y.; and Kibbe, et al., Eds., Handbook of Pharmaceutical Excipients, American Pharmaceutical Association, Washington.

[0052] The term “treat” as used herein refers to any type of treatment that imparts a benefit to a patient afflicted with a disease, including improvement in the condition of the patient (e.g., in one or more symptoms), delay in the progression of the condition, etc.

[0053] As used herein, the term “prevent” refers to the prophylactic treatment of a subject who is at risk of developing a condition resulting in a decrease in the probability that the subject will develop the condition.

[0054] A “therapeutically effective amount” of a compound or a pharmaceutical composition refers to an amount effective to prevent, inhibit, treat, or lessen the symptoms of a particular disorder or disease.

[0055] As used herein, the term “subject” refers to an animal, particularly a mammal, particularly a human.

[0056] As used herein, the term “small molecule” refers to a substance or compound that has a relatively low molecular weight (e.g., less than 4,000, less than 2,000, particularly less than 1 kDa or 800 Da). Typically, small molecules are organic, but are not proteins, polypeptides, or nucleic acids, though they may be amino acids or dipeptides. As used herein, a “kit” refers to a packaged combination, optionally including reagents and other products and / or components for practicing methods using the elements of the combination. In certain embodiments, a kit comprises one or more containers or vials comprising one or more of the above mentioned components of the instant invention. In certain embodiments, the kit may comprise a container or vial comprising a carrier, buffer, diluent, and / or reconstitution buffer. The kit may also comprise means for administering the components of the kit (e.g., needles and / or catheters). The kit may also comprise instruction material.

[0057] The following example is provided to illustrate various embodiments of the present invention. The example is illustrative and is not intended to limit the invention in any way.

[0058] EXAMPLE

[0059] Traumatic hemothorax management remains unstandardized, although the goal of all management approaches is to prevent retained hemothorax and its associated complications (e.g., trapped lung, empyema, etc.). Strategies such as intrathoracic irrigation via tube thoracostomy have been tried with some success (Kugler, et al., (2017) J. Trauma Acute Care Surg., 83(6): 1136-1141), but once a retained hemothorax has developed the treatment options remain limited. While those patients who are operative candidates may benefit from operative drainage, many polytrauma patients are poor operative candidates, where retained hemothoraces in these non-operative candidates have recently been treated using intra-pleural Tissue Plasminogen Activator (tPA) (Dennis, B.M. (2017) J. Trauma Acute Care Surg., 82(4):728-732) in a manner similar to treatment of loculated parapneumonic effusions. However, intrapleural tPA alone has not been shown to be effective without the addition of DNase (Rahman, et al., (2011) N. Engl. J. Med., 365(6):518-26), at least in part because neutrophil extracellular traps (NETs) comprised of neutrophil DNA are thought to be interwoven within the clot structure. tPA by definition cannot work to lyse fibrin clot (retained hemothorax) without the presence of its target zymogen plasminogen, where tPA cleaves plasminogen to plasmin, the active protease that cleaves fibrin polymers into soluble fibrin degradation products. Human neutrophil elastase in concentrations relevant to human physiology can rapidly cleave plasminogen to inactive fragments, depleting the plasminogen stores and rendering it unavailable for generation of plasmin by tPA to cleave fibrin clots (Scapini, et al. (2002) J. Immunol., 168(11):5798-804; Barrett, et al. (2017) J. Trauma Acute Care Surg., 83(6): 1053-1061). When patients have parapneumonic effusions or hemothoraces, they have large numbers of neutrophils present in the intra-pleural cavity (Light, R.W. (1997) Eur. Respir. J., 10(2): 476-81; DiVietro, et al., (2015) Clin. Med. Insights Case Rep., 8: 71-6). Neutrophil elastase is released in response to numerous inflammatory molecules, including IL-8 (Scapini, et al. (2002) J. Immunol., 168(11):5798-804) and complement fragment C5a, both of which are present at high levels in clotted blood such as that seen in hemothorax as well as in infection (Gardner, et al. (2004) Biochem. Biophys. Res. Commun, 321(2):306-12; Schnabel, et al. (2010) Blood 115(26):5289-99). Thus, in retained hemothorax it is likely that human neutrophil elastase released by activated neutrophils exhausts much of the plasminogen in the pleural cavity, making thrombolysis with tPA less effective. The same may also be true of parapneumonic effusions, which are known to contain large numbers of neutrophils and high levels of human neutrophil elastase (Segura, et al. (1998) Am. J. Respir. Crit. Care Med., 157(5 Pt 1): 1565-72) that could potentially degrade much of the plasminogen associated with the fibrin loculations and would explain why benefits from tPA alone were not seen

[0060] (Rahman, et al., (2011) N. Engl. J. Med., 365(6):518-26).

[0061] Methods

[0062] Study Design and Methods

[0063] A scientific investigation was performed to test the overall hypothesis that inflammatory plasminogen degradation, not high PAI-1 activity, is responsible for failed intrapleural fibrinolysis in complex pleural space collections. Pleural fluid was obtained via a pleural catheter or thoracostomy tube from adult inpatients (n = 10) with infected pleural collections, as well as concordant circulating plasma in 3.2% sodium citrate. The samples were obtained as part of a randomized trial evaluating intrapleural fibrinolytic therapy with tPA and deoxyribonuclease vs early video-assisted thoracoscopic surgical (VATS) intervention as the initial treatment for complex pleural space infection (ClinicalTrials.gov Identifier: NCT03583931). The trial profile is shown in Figure 1. The trial was terminated early because of the CO VID-19 pandemic, and thus the results have not been reported because of lack of adequate sample size to compare clinical outcomes between the two interventions and groups. Inclusion criteria were adults (aged > 18 years) with a pleural effusion who underwent chest tube placement with pleural pH of < 7.2 and required ICU admission. Exclusion criteria included existing malignancy or malignant cells from initial pleural fluid sample, end-stage liver disease (Child-Pugh class B or higher), coagulopathy, inability to tolerate surgical procedure, frank purulence from chest tube (i.e., needed surgery regardless), recent surgery of abdomen or thorax that precluded the use of tPA, or baseline neurologic impairment that required a proxy for consent. The pleural fluid and plasma samples were collected before the intervention and on days 1, 2, and 3 after the intervention with written informed consent and institutional review board approval (Identifier: COMIRB #17-0857). Although the trial enrolled 11 patients, no pleural fluid sample was available before the intervention for one patient, so that patient was excluded from the present study and analysis. Patient samples were aliquoted after collection and stored immediately at -80°C.

[0064] Pleural Fluid and Plasma Assays for Elastase Activity and Plasminogen Antigen Content Elastase activity of pleural fluid and plasma was measured via an amidolytic assay according to the Beer-Lambert law by measuring changes in absorption at a 400-nm wavelength over 1 hour in the presence of 1 mM N-Methoxysuccinyl-Ala-Ala-Pro-Val (SEQ ID NO 1) p-nitroanilide (extinction coefficient, 12,300 M^cm'1) dissolved in pH 7.5 100 mM Tris buffer (Castillo, et al. (1979) Anal. Biochem., 99(l):53-64). Plasminogen antigen levels of pleural fluid and plasma were measured by enzyme-linked immunosorbent assay (ELISA) according to manufacturer instructions (LSBio). The elastase activity and plasminogen antigen levels of pleural fluid then were compared with their corresponding plasma levels to adjudicate high or low activity or antigen levels relative to what circulates in the intravascular space where endogenous fibrinolysis normally occurs.

[0065] Western Blot Analysis of Inflammatory Plasminogen Degradation

[0066] Human neutrophils were purified from healthy volunteer whole blood using density gradient centrifugation (Barret, et al. (2018) Clin. Exp. Immunol., 194(1): 103- 117), activation or degranulation was stimulated with 500 nM phorbol 12-myristate 13- acetate (Sigma- Aldrich), and supernatant was collected via serial centrifugation followed by coincubation with a physiologic level (180 pg / mL (Keragala, et al. (2021) Blood 137(21):2881 -2889)) of human Glu-plasminogen (ProLytix) for 6 hours at 37°C. Inhibition of elastase in phorbol 12-myristate 13 -acetate-activated neutrophil supernatant was achieved using 1 mM N-(methoxysuccinyl)-Ala-Ala-Pro-Val (SEQ ID NO: 1)- chloromethyl ketone. Purified human neutrophil elastase was obtained commercially (Bio-Rad) for plasminogen coincubation in a similar manner as for activated neutrophil supernatant. Western blot analysis of coincubation experiment supernatant and patient pleural fluid obtained before the intervention was performed under reducing conditions according to a method (Towbin, et al. (1979) Proc. Natl. Acad. Sci., 76(9):4350-4354) using mouse antihuman plasminogen and angiostatin that recognizes the N-terminal Kringle domains 1 through 3 (Clone GMA 013; Millipore) of plasminogen with fluorescent secondary antibody labeling on a LI-COR Odyssey® CLx.

[0067] Turbidity Clot Lysis Assays

[0068] Turbidity assays to measure fibrinolytic potential of pleural fluid were performed by mixing pleural fluid with 5 nM purified human a-thrombin (ProLytix), 100 mg / dL purified human fibrinogen (ProLytix), either 33 pM or 330 pM recombinant human tPA (Sigma- Aldrich), and without or with 62.5 mg / mL purified human Glu-plasminogen (ProLytix) and measuring absorption at 405 nm wavelength (Barrett, et al. (2017) J. Trauma Acute Care Surg., 83(6): 1053-1061.).

[0069] Measurement of PAI-1 Total Antigen and PAI-1 Activity of Pleural Fluid

[0070] Pleural fluid samples obtained before the intervention were measured for total PAI-1 antigen levels using an ELISA and following the manufacturer’s instructions (Innovative Research). The PALI activity then was measured in these samples, in addition to pleural fluid samples obtained on days 1 and 2 after the intervention, using a commercially available assay that works similarly to an ELISA, but rather than using a capture antibody to detect total antigen, it uses an immobilized urokinase-plasminogen activator that reacts and complexes with active PALI such that only active PALI is captured and detected by the assay, with one unit of PALI activity equating to 1.34 ng of active PALI antigen (Innovative Research).

[0071] Statistical Analysis

[0072] Statistical analysis was performed using GraphPad Prism version 9.4.1 software (GraphPad Software, Inc.). Results are reported as median (interquartile range) or number (percentage) as appropriate, with pairwise comparisons between pleural fluid and corresponding plasma elastase activities and plasminogen levels and between pleural fluid PAI-1 antigen and corresponding activity levels, made via nonparametric testing using Wilcoxon matched-pairs signed-rank tests. To evaluate the impact of time on PAI-1 activity within treatment groups, a mixed model was performed that uses a compound symmetry covariance matrix that is fit using restricted maximum likelihood with Giesser- Greenhouse correction, whereas comparisons of PAI-1 activity between the two intervention groups (tPA plus deoxyribonuclease vs VATS) were performed using Mann- Whitney U tests. All data were analyzed on an intention-to-treat basis, because all samples were collected as part of each patient’s initial intervention (tPA plus deoxyribonuclease or VATS) and the two patients in whom lytic therapy failed who subsequently underwent surgery did so after completion of all 3 days of lytic therapy (when subsequent samples no longer were collected). An a value was set at .05.

[0073] Results

[0074] Of the 10 included patients, the median age was 39 years (interquartile range, 33.5-56.5 years), seven patients (70%) were male, nine patients (90%) identified as White, and three patients (30%) identified as Hispanic (Table 1). The median duration of symptoms before admission was 7 days (interquartile range, 2-13.3 days). Compared with the respective plasma, pleural fluid showed a median more than fourfold higher neutrophil elastase activity (P = .02) and median more than threefold reduction in plasminogen antigen (P = .04) (Figs. 2A and 2B). Of note, the two patients in whom intrapleural lytic therapy failed who required trial crossover to the surgical group (i.e., after completing 3 days of lytic therapy and showing no improvement, which then required surgery to treat the complex pleural space infection) showed the lowest pleural plasminogen antigen levels. Western blot analysis of pleural fluid demonstrated large quantities of plasminogen cleavage fragments in all patients that were distinct from intact Glu-plasminogen or active plasmin. These fragments closely mirrored those seen after coincubation of Glu-plasminogen with activated human neutrophil supernatant or purified human neutrophil elastase (Figs. 2C-2E).

[0075] TABLE 1: Patient baseline characteristics. Data are presented as No. (%) or median (interquartile range) unless otherwise indicated.

[0076] When total PAI-1 antigen levels in the pleural fluid obtained before the intervention was measured, high levels were found similar to those reported (Bedawi, et al. (2023) Am. J. Respir. Crit. Care Med., 207(6):731-739) (Fig. 3 A). However, on measuring PAI-1 activity in the pleural fluid obtained before the intervention, the majority (82%) of the total PAI-1 antigen detected was not active (P = .003) (Fig. 3A). PAI-1 activity then was measured at the subsequent time points after intervention for both intervention groups (tPA plus deoxyribonuclease and VATS), where type III tests of fixed effects demonstrated a significant impact of time on PAI-1 activity in both the tPA plus deoxyribonuclease group (F = 2.238; Pr > F = 0.045) and the VATS group (F = 4.286; Pr > F = 0.043). In patients receiving intrapleural tPA plus DNAse therapy, virtually all PAI-1 activity was lost by day 2 after the intervention (Fig. 3B), consistent with depletion of all active PAI-1 by administration of tPA. In contrast, PAI-1 activity over time in the VATS group remained detectable at all time points, initially rising on day 1 after the intervention and then falling, on day 2 after the intervention, to just less than the baseline before the intervention (Fig. 3B), consistent with the lack of tPA administration in this group. When comparing the PAI-1 activity between the tPA plus deoxyribonuclease and VATS groups at the measured time points, a significant difference was present on day 2 after the intervention (P = .016).

[0077] Using turbidity assays that measure fibrinolytic activity, the pleural fluid of only one patient (10%) was able to demonstrate a lytic response in the absence of supplemental plasminogen, whereas the remaining nine patients (90%) required plasminogen supplementation to the pleural fluid to generate fibrinolysis (Figs. 4A-4B). Two patients (20%) were able to generate a complete fibrinolytic response at just 33 pM tPA in the presence of supplemental plasminogen, seven patients (70%) generated a complete fibrinolytic response at 330 pM tPA in the presence of supplemental plasminogen, and one patient (10%) demonstrated a partial response in the presence of 330 pM tPA and supplemental plasminogen (i.e., showed measurable fibrinolysis, but did not reach the transition midpoint of fibrinolysis at the 6-hour assay end point).

[0078] The results presented herein demonstrated that pleural fluid from patients with pleural space infection have an insufficient amount of functional plasminogen to generate a robust fibrinolytic response to tPA. Neutrophil elastase activity was elevated in the patients’ pleural fluid samples, and degradation patterns observed on Western blotting for plasminogen of this pleural fluid were consistent with inflammatory protease degradation of plasminogen to nonfunctional fragments. Although pleural fluid demonstrated high PAI-1 antigen levels before the intervention, most of this (82%) was not active, and thus not capable of inhibiting tPA. Additionally, in the six patients who were randomized to treatment with tPA and deoxyribonuclease, only two of them showed detectable PAI-1 activity after the first day of fibrinolytic therapy, and after the second day of fibrinolytic therapy, no PAI-1 activity was detectable in these patients. In contrast, the four patients who were randomized to surgery (VATS) as the initial intervention showed increased levels of PAI- 1 activity on day 1 after the intervention that fell, on day 2 after the intervention, to slightly less than the baseline before surgery, supporting the relationship between tPA administration and depletion of PAI- 1 activity. These findings directly challenge the current dogma that multiple repeat doses of tPA and deoxyribonuclease are necessary because of high PAI-1 levels, because tPA and deoxyribonuclease therapy would be expected to resolve patients’ complex collections by day 2, given the absence of detectable PAI-1 activity in the pleural fluid at that time.

[0079] When the fibrinolytic potential of the patients’ pleural fluid samples before the intervention was examined, only one patient was capable of a fibrinolytic response to tPA challenge in the presence of fibrin clot. On supplementation of plasminogen to these same pleural fluid samples, fibrinolysis was rescued, with complete and rapid lysis in 9 of the 10 patient samples and a partial response in the remaining one. These findings further challenge the idea that high PAI-1 levels are responsible for the need to give multiple repeat doses of fibrinolytic therapy and for failure of fibrinolytic therapy, because plasminogen supplementation would not be able to overcome inhibition of tPA by PAI-1 by definition because plasminogen activation to plasmin requires active tPA (or another plasminogen activator) to be present. It is also worth noting that the concentrations of tPA used in the present experiments were multiple orders of magnitude lower than what is administered into the pleural space during intrapleural fibrinolytic therapy when estimating based on the standard 10-mg dose of tPA given six times (Rahman, et al. (2011) N. Engl. J. Med., 365(6):518-526) and a 2.5-L average pleural effusion volume (Ibitoye, et al. (2018) Ultrasonography 37(3):254-260), which further challenges the notion that PAI-1 is the major contributor to intrapleural fibrinolytic failure.

[0080] When taken together, these observations indicate that the need for multiple large doses of tPA relative to the small pleural space in the Second Multicenter Intrapleural Sepsis Trial and the significant failure rate of resolving fibrin loculations despite very high tPA concentrations may be related to the low levels of tPA’s substrate zymogen (plasminogen). The observed plasminogen deficiency itself seems to be related to the highly inflammatory proteases released by neutrophils, specifically elastase, in inflamed and infected environments (Alegre, et al. (2000) Respiration 67(4):426-432) that degrade plasminogen and render it incapable of participating in fibrinolysis in response to tPA (Barrett, et al. (2017) J. Trauma Acute Care Surg., 83(6): 1053-1061). These observations are an important translational finding, because a first-ever US Food and Drug Administration-approved purified human plasminogen is available (Ryplazim; Kedrion BioPharma) that was approved in 2023 for use in type I plasminogen deficiency and could be investigated via clinical trial as a novel approach to include as part of intrapleural fibrinolytic therapy. In addition, intrapleural elastase inhibitors may also aid in preventing development of a blunted fibrinolytic response.

[0081] In summary, pleural fluid from infected pleural cavities have low fibrinolytic potential because of neutrophilic inflammatory degradation of plasminogen. Although PAI-1 antigen levels in this pleural fluid are high, as reported previously, the PAI-1 has limited activity that is depleted fully by day 2 after tPA administration. Thus, PAI-1 does not adequately explain why intrapleural fibrinolytic therapy requires multiple repeat administrations and, despite that, sometimes may fail. Intrapleural plasminogen supplementation when administering tPA and deoxyribonuclease represents a novel strategy to improve the speed and success rates of intrapleural lytic therapy for complex pleural space collections. Particularly, plasminogen supplementation represents a novel strategy to improve the speed and success rates of intrapleural lytic therapy, particularly for complex pleural space collections. While certain of the preferred embodiments of the present invention have been described and specifically exemplified above, it is not intended that the invention be limited to such embodiments. Various modifications may be made thereto without departing from the scope and spirit of the present invention, as set forth in the following claims.

Claims

WHAT IS CLAIMED IS1. A method of increasing intrapleural fibrinolysis in a subject, said method comprising administering plasminogen and / or an elastase inhibitor to the subject.

2. A method of treating, inhibiting, and / or preventing a pleural space infection in a subject, said method comprising administering plasminogen and / or an elastase inhibitor to the subject.

3. The method of claim 2, wherein said subject has a hemothorax, empyema and / or pleural effusion.

4. A method of treating, inhibiting, and / or preventing hemothorax in a subject, said method comprising administering plasminogen and / or an elastase inhibitor to the subject.

5. The method of any one of claims 1-4, comprising administering plasminogen to the subject.

6. The method of any one of claims 1-4, comprising administering an elastase inhibitor to the subject.

7. The method of any one of claims 1-4, further comprising administering a plasminogen activator to the subject.

8. The method of claim 7, wherein said plasminogen activator is tissue-plasminogen activator (tPA).

9. The method of claim 7, further comprising administering DNase to the subject.

10. The method of any one of claims 1-4, further comprising administering DNase to the subject.

11. The method of any one of claims 1-10, wherein said plasminogen and / or an elastase inhibitor are administered intrapleurally to the subject.

12. The method of any one of claims 1-11, wherein said plasminogen is human plasminogen.

13. The method of any one of claims 1-12, wherein said plasminogen is human-tvmh(Ryplazim®).

14. The method of any one of claims 1-13, wherein said elastase inhibitor is an inhibitor of neutrophil elastase.

15. The method of any one of claims 1-14, wherein said elastase inhibitor is selected from the group consisting of sivelestat, elastatinal, alpha-1 proteinase inhibitor, alvelestat, polypeptide elastase inhibitors, and N-(methoxysuccinyl)-Ala-Ala-Pro-Val (SEQ ID NO: 1)-cholorom ethyl ketone.

16. The method of claim 8, wherein said tPA is human tPA.

17. The method of claim 8, wherein said tPA is alteplase or Tenecteplase.

18. The method of claim 10, wherein said DNase is human deoxyribonuclease I.

19. The method of claim 10, wherein said DNase is domase alfa.

20. The method of any one of claims 1-19, wherein said subject was previously unsuccessfully treated with intrapleural lytic therapy with plasminogen activator and DNase.

21. The method of any one of claims 1-20, wherein said subject has a neutrophil elastase activity in pleural fluid greater than in plasma.

22. The method of any one of claims 1-21, wherein said subject has reduced plasminogen in pleural fluid compared to plasma.

23. The method of any one of claims 1-22, further comprising measuring the neutrophil elastase activity in the pleural fluid and, optionally, plasma of the subject.

24. The method of any one of claims 1-23, further comprising measuring the amount of plasminogen in the pleural fluid and, optionally, plasma of the subject.

25. A kit comprising a plasminogen activator and a plasminogen and / or an elastase inhibitor.

26. The kit of claim 25, wherein said plasminogen activator is tissue-plasminogen activator (tPA).

27. The kit of claim 25 or 26, further comprising DNase.

28. The kit of any one of claims 25-27, wherein said tissue-plasminogen activator (tPA) and a plasminogen and / or an elastase inhibitor are lyophilized.

29. The kit of any one of claims 25-28, wherein said kit comprises a container comprising lyophilized plasminogen activator and lyophilized plasminogen.

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