Metered dose inhaler containing aprotinin a protease inhibitor

Metered dose inhaler containing aprotinin, a protease inhibitor, as a drug against Influenza Oleg Zhirnov, N. Bokova, E. Isaeva, I. Vorobjeva, O. Saphonova, N. Malyshev D. I. Ivanovsky Institute of Virology, Moscow, Russia Infectious clinics N 1, Moscow, Russia

Influenza virus HA PB 1, PB 2, PA NEP M 2 NP M 1 NA 100 nm

Influenza virus proteolytic activation Non-active Virus Active virus Host proteases Uncleaved HA 0 (75 k. D) Cleaved HA 1(55 k. D) + HA 2(20 k. D)

Signal peptide Signalase HA 1(55 k. D) RBC Cleavage Site S C B R Cleavage S IAP Fusion peptide S S Anchor peptide HA 0 (75 k. D) HA 2 (20 k. D)

Summary 1. Receptor protein HA locates as spike on the virus surface. 2. HA is cleaved by host respiratory protease: HA 0 (75 k. D) → HA 1 (55 k. D) + HA 2 (20 k. D). 3. This HA cleavage activates virus infectivity. 4. The HA cleavage promotes infection spread.

Host proteases ( Influenza activating proteases ) Serine proteases: Transmembrane protease, serine 2 ( TMPRSS 2 ) Transmembrane protease, serine 4 ( TMPRSS 4 ) Human airway trypsin-like protease ( HAT ) Matriptase Location: Nasal, Tracheal, Bronchial, Lung (alveolocytes type II), Intestinal epithelium

Suggestion Suppression of Flu proteolytic activation with the help of specific protease inhibitor will reduce virus multiplication and provide therapeutic efficacy.

Rational Decision Aprotinin has been selected for this aim as the officinal compound already approved for human usage.

APROTININ STRUCTURE ( 58 a. a. polypeptide from bovine lungs ) 15 C Y P T G P K A R C G K S A E D C M 50 I G Y V R N R K A P C N F E A G L C G F C D T PR R Lys Met I Lys 15 R Y F N T A Q K C L G A N Primary N C Tertiary

Clinical Forms of Aprotinin Usage Systemic injection form Local aerosol form ( Aerus TM ) Intravenous infusion and injection Nasal and oropharyngeal infusions and inhalations 25, 000 – 900, 000 КIU/day 800 – 3, 000 КIU/day

Trachea-bronchial epithelium infected with human (a, c) and avian (b, d) Influenza Virus ( ciliated, secretory, basal cells + mucus blanket ) Matrosovich et al. 2004

Aprotinin inhibits flu virus HA 0 cleavage in Trachea-bronchial epithelium - + Apr Мol. Wt. HA 0 HA 1+NA 75 к. Д HA 2 20 к. Д 50 к. Д 8 5 10 Infectivity (pfu/ml) Zhirnov et al. 2011 Antiviral Chemother 21: 169 -174

Virus titer (lg pfu) Aprotinin inhibits replication of Influenza Virus H 1 N 1 pdm in Trachea-bronchial epithelium 10 8 6 Control 4 + APR 2 Hours post infection Zhirnov et al. 2011

Summary: 1. Aprotinin inhibits influenza virus НА 0 → HA 1+HA 2 cleavage and suppresses virus replication. 2. A second target of aprotinin is inhibition of inflammation in infection locus.

Aprotinin targets Anti-inflammatory effects of aprotinin References 1. Plasmin a. Suppresses fibrinolysis and, prevents hemorrhage but does not prevent dissolution of blood clots in small vessels decreasing blood stasis. b. Suppresses kallikrein cleavage and super production of bradykinin c. Inhibits plasmin-dependent transmigration of bacteria across respiratory epithelium e. Down regulates plasmin-induced proinflammatory activation during infectious pneumonia f. Normalizes sodium homeostasis by inhibiting plasmin dependent activation of extracellular Na channel (ENa. C). [Ray and Marsh 1997] [Wachtfogel et al. , 1993] [Attali et al. , 2008] [Akaike et al. , 1989] [Passero et al. , 2010] 2. Kallikrein 3. Monocyte and granulocyte adhesion molecules 4. Nitric oxide synthase (NOS) a. Suppresses proinflammatory cytokine production. b. Prevents kininogen cleavage and decreases generation of kinins. c. Preserves capillary permeability d. Attenuates early fibrinolytic events e. Attenuates kinin-dependent extravasation (transplasmosis) of serum proteases a. Prevents the expression of pro-inflammatory adhesive integrins (e. g. , CD 11 b) and decreases neutrophil degranulation and extravasation at inflammatory sites d. Down regulates release of TNF-α, IL-6, IL-8, elastase c. Elevates secretion of anti-inflammatory IL-10 [Poullis et al. , 2000] [Wachtfogel et al. , 1993; 1995] [Yayama et al. , 2003] Reduces concentration of oxide radicals (NO, 0 2 - ) and their deleterious effects on tissues at infection locus [Bruda et al. , 1998] [Hill and Robinson, 1997] [Harig et al. , 1999] [Gilliand et al. , 1999] [Asimakopoulos et al. , 2001] [Pruefer et al. , 2003] 5. Prostasin/epithelial sodium channel (ENa. C) a. Suppresses activation of protease-activated ENa. C in respiratory epithelium and normalizes mucus viscosity and water balance in airway surface fluid (ASL). b. Enhances mucociliary clearance. [Planes and Caughery, 2007] [Coote et al. , 2009] 6. Complement components 3 a, 5 a a. Attenuates plasmin-dependent complement generation b. Decreases activation of complement due to reduced formation of C 3 a and C 5 a [Huber-lung et al. , 2006 ] [Amara et al. , 2008] [Sarma et al. , 2006] a. Prevents thrombin-induced platelet dysfunction and over aggregation and decreases vascular thrombosis b. Preserves platelet glycoprotein receptor (GPIa, GPIIb/IIIa) function. [Khan et al. , 2005] [Poullis et al. , 2000] 7. Platelet proteaseactivated receptor (PAR-1)

Duration of symptoms in Flu patients treated with aprotinin mini-inhaler Ch e st p ain rh ino rrh ea So re th ro at He ad ac he W ea kn es s Fe ve r Co ug h Duration (Days) Ingavirin Aprotinin Zhirnov & Malyshev, 2014

Viral loads in naso-pharyngeal washes in H 1 N 1 pdm patients treated with aprotinin MDI Number of washes (%) Ingavirin (Control) Aprotinin 106 -104 103 -101 <101 Viral genomes in ml of nasopharyngeal wash

Clinical Benefits of Aerosolized Aprotinin Against Influenza 1. About two-fold reduction in duration of headache, throat pain, hoarseness, fever, earlier discharge from hospital. 2. Aprotinin inhalations were well tolerated by patients and no adverse reactions were observed.

Aprotinin inhibits vicious cycle in Flu pathogenesis Virus activation Non-active virus Aprotinin (АERUS) Proteases Primarily Infection locus Active virus Infection Inflammation Aprotinin (АERUS) Proteases

Benefits of Aprotinin usage against Flu 1. Aprotinin is a polyvalent drug effective against many strains of Influenza A, B, and C viruses. 2. Aprotinin, as a host-targeted antiviral, will not select resistance for influenza viruses. 3. Aprotinin has a binary therapeutic effect: (а) inhibits virus replication; (b) reduces inflammation in infection locus. 4. A local aerosol inhalation form of aprotinin is recommended against Influenza.

Zhirnov O. P. , Klenk H. D. , Wright P. F. «Aprotinin and similar protease inhibitors as drugs against influenza» Antiviral Research v. 92: 27 -36 (2011) Zhirnov O. P. , Malyshev N. A. «New in the treatment of influenza: a propellant metered dose inhaler containing aprotinin» Treating Doctor N 1: 57 -62 (2014) (Russian). zhirnov@inbox. ru

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Victory Day May 5, 1945 !!! My father, Peter T. Zhirnov a soldier of a 2 -nd World War, is dedicated