Designing inhaled protein therapeutics for topical lung delivery: what are the next steps?

Figures & data

Table 1. Inhaled protein therapeutics in clinical development for topical lung applications.

Protein therapeutic		Clinical development				Inhalation system
Name(s)	Molecular characteristics	Companies/Sponsors	Stage	Therapeutic indication	Dosing regimen	Formulation	Inhaler	Immunogenicity (ADAs)	Reference/Clinical trial # (NCT)* /Patent # (EP/WO)
Marketed
Dornase alfa	Deoxyribonuclease I (37 kDa)	Roche	Marketed	Cystic fibrosis	2.5 mg/d	C = 1 mg/mL Exc: sodium chloride, calcium chloride dehydrate (pH 6.3)	Jet or mesh nebulizers	ADAs in <5% of patients (n = 643)	Pulmozyme® SmPC (a)
Under clinical evaluation
ALX-0171/VR465	Trivalent Nanobody™ (42 kDa) targets RSV F-protein	Ablynx	Phase IIb ongoing	RSV infection	3 different doses Once daily x 3 d	C = 50 mg/mL Exc: sodium chloride, phosphate buffer (among others)	Mesh nebulizer (customized Vectura FOX-Flamingo® system, Vectura Group plc, Chippenham, UK)	ADAs in 23% of patients (n = 30)	NCT02979431 (b,c)
VR942/UCB4144/CDP7766	Anti-IL-13 mAb fragment	UCB Pharma	Phase I completed (2016)	Asthma	0.5–20 mg, single- or repeated dose (10 d)	N/A	DPI	No clinical evidence of immunogenicity	NCT02473939 (d)
Alpha-1 antitrypsin (Kamada-API)	Lung serine protease inhibitor (52 kDa)	Kamada	Phase II/III (Eur) completed (2015)	Alpha-1 antitrypsin deficiency	N/A	C = 20 mg/mL	Mesh nebulizer: customized PARI eFlow®, PARI GmbH, Starnberg, Germany	N/A	NCT01217671 (e)
			Phase II (US) completed (2015)	Alpha-1 antitrypsin deficiency	80–160 mg/d	N/A	Mesh nebulizer: customized PARI eFlow®	N/A	NCT02001688
Alpha-1 antitrypsin (Alpha-1 HC)	Lung serine protease inhibitor (52 kDa)	Grifols Therapeutics	Phase II completed (2013)	Cystic fibrosis	100–200 mg/d x  21 d	C = 50 mg/mL Exc: PBS, polysorbate	Nebulizer	N/A	NCT01684410
GSK2862277	Anti-TNF-R1 mAb VH domain (13 kDa)	GSK	Phase II terminated (2018)	Acute lung injury	26 mg, single dose	Lyophilizate, reconstitution before nebulization Exc (Phase I trial): sucrose, glycine, sodium dihydrogen phosphate, polysorbate 80	Mesh nebulizer (PARI eFlow® with s30 mesh)	ADA in 6% of healthy donors (n = 100)	NCT02221037 (f,g)
AIR DNase™/Alidornase alfa/PRX-110	Actin-inhibition-resistant deoxyribonuclease I (37 kDa)	Protalix BioTherapeutics	Phase I competed (2016)	Cystic fibrosis	1.25 – 5 mg/d x  5 d	N/A	N/A	N/A	NCT02605590
			Phase II ongoing	Cystic fibrosis	2.5 mg/d x 28 d	N/A	Mesh nebulizer (Philips I-neb AAD®, Koninklijke Philips N.V., Amsterdam, Netherlands)	N/A	NCT02722122
Alteplase	Tissue plasminogen activator (tPA) (70 kDa)	University of Michigan, Genentech	Phase II ongoing	Plastic bronchitis	5 mg every 6 h  x 3 d	Exc (Activase®, Genentech Inc., South San Francisco, CA, USA): arginine, phosphoric acid, polysorbate 80	Nebulizer	N/A	NCT02315898
ALX-009: lactoferrin (+hypothiocyanite)	Antibacterial glycoprotein (80 kDa)	Alaxia Pharma	Phase I ongoing	Cystic fibrosis	Single/multiple administrations	C = 4 mg/mL	Nebulizer	N/A	NCT02598999
Molgramostim	Recombinant human GM-CSF (14 kDa)	University of Giessen	Phase II ongoing	ARDS	150–450 µg/d x 3 d	N/A	N/A	N/A	NCT02595060
		Savara Pharmaceuticals	Phase II/Phase III ongoing	APAP	300 µg/d x 24 w continuously or intermittently	N/A	Mesh nebulizer (investigational PARI eFlow®)	N/A	NCT02702180 NCT03482752
			Phase IIa ongoing	NTM infections	300 µg/d x 24 w	N/A	Mesh nebulizer (investigational PARI eFlow®)	N/A	NCT03421743
Sargramostim (Leukine®, Sanofi-Aventis U.S. LLC, Bridgewater, NJ, USA)	Recombinant human GM-CSF (14 kDa)	Milton S. Hershey Medical Center	Phase I ongoing	Pediatric ARDS	25–250 µg/m^2 x 7 d	Exc (Leukine®): mannitol, sucrose, tromethamine ±benzylalcohol	Nebulizer	N/A	NCT02601365 Leukine® PI
		Children’s Hospital Medical Center, (Cincinnati), Genzyme	Phase II completed (2014)	aPAP	250 µg/w**	Exc (Leukine®): mannitol, sucrose, tromethamine ±benzylalcohol	Nebulizer	N/A	NCT01511068 Leukine® PI
		Children’s Hospital Medical Center (Cincinnati)	Phase I ongoing	aPAP	125–250 µg, single dose	Exc (Leukine®): mannitol, sucrose, tromethamine ±benzylalcohol	N/A	N/A	NCT03006146 Leukine® PI
		IRCCS Policlinico S. Matteo	Phase II/III (status unknown)	aPAP	250 µg/d**	Exc (Leukine®): mannitol, sucrose, tromethamine ±benzylalcohol	N/A	N/A	NCT00901511 Leukine® PI
		Barbara Ann Karmanos Cancer Institute	Phase II completed (2010)	Lung metastases/cancer	250 µg bid**	Exc (Leukine®): mannitol, sucrose, tromethamine ±benzylalcohol	N/A	N/A	NCT00514215 Leukine® PI
		Children’s Oncology Group	Phase II completed (2013)	Osteosarcoma with lung spreading	240–1750 µg bid**	Exc (Leukine®): mannitol, sucrose, tromethamine ±benzylalcohol	N/A	N/A	NCT00066365 Arndt 2010 Leukine® PI
GM-CSF	Recombinant human GM-CSF (14 kDa)	Niigata University Medical & Dental Hospital	Phase II completed (2017)	aPAP	125 µg bid**	N/A	N/A	N/A	NCT02835742
		Peking Union Medical College Hospital	Phase II (status unknown)	aPAP	150 µg bid**	N/A	Nebulizer	N/A	NCT02243228
		Dai Huaping	Phase II ongoing	aPAP	150 µg bid**	N/A	N/A	N/A	NCT03316651
SNG001	Interferon beta-1a (22 kDa)	Synairgen	Phase II completed (2012)	Asthma	6 mIU/d x 14 d	Neutral pH, no mannitol, no human serum albumin	Mesh nebulizer (Philips I-neb®, Koninklijke Philips N.V., Amsterdam, Netherlands)	N/A	NCT01126177 (h)
		Synairgen, AstraZeneca	Phase II completed (2016)	Asthma	24 μg/d x 14 d	C = 48 µg/mL	Mesh nebulizer (Philips I-neb®)	N/A	NCT02491684
AZD1402/ PRS-060	Anticalin protein (~18 kDa) Target: IL-4Rα	Pieris Pharmaceuticals, AstraZeneca	Phase I ongoing	Asthma	Single dose	N/A	Nebulizer	N/A	NCT03384290 (i)
DAS181 (Fludase®, Ansun BioPharma Inc., San Diego, CA, USA)	Recombinant neuraminidase (46 kDa)	Ansun Biopharma	Phase II	Influenza virus infection	10 mg/d x 3 d	N/A (named F02)	DPI	N/A	NCT01037205 EP2841092A1
		National Institute of Allergy and Infectious Diseases (NIAID)	Phase I completed (2011)	Influenza virus infection	20–30 mg single dose or 20 mg/d x 10 d	C = 70% (w/w) Exc: histidine, trehalose, citric acid, acetate buffer (named F03)	DPI	IgA or IgG in 88% of patients (n = 18)	NCT01173224 (j)
			Phase I completed (2012)	Influenza virus infection	20 mg/d x 3 d	C = 65% (w/w) Exc: histidine, trehalose, MgSO4, citric acid, acetate buffer (named F04)	DPI		NCT01651494 (j)
DAS181 (Paradase®, Ansun BioPharma Inc., San Diego, CA, USA)	Recombinant neuraminidase (46 kDa)	Ansun BioPharma	Phase II completed (2017)	Parainfluenza virus infection	DPI: 10 mg/d x 7 d Nebulizer: 3.2–4.5 mg/d x 7 d (maximum total dose = 28.9 mg)	DPI: F02 (undefined) Nebulization: F02 dissolved in 10 mL normal saline	DPI (Teva Cyclohaler®, Teva Pharmaceutical Industries Ltd., Petah Tikva, Israel) Nebulizer	N/A	NCT01924793 (k)
			Phase II completed (2016)	Parainfluenza virus infection	4.5 mg/d x 10 d	F02 (undefined)	DPI	N/A	NCT01644877
CSJ117	Anti-TSLP mAb fragment (46 kDa)	Novartis	Phase I ongoing	Asthma	3 different doses 1 dose/d x 12 w	Exc: from among leucine, trileucine, HCl, trehalose, mannitol. Buffers: from among histidine and glycine	N/A	N/A	NCT03138811 WO2017042701A1
AER-001/ BAY 16–9996/Pitrakinra/Aerovant®, Aerovance Inc., Berkeley, CA, USA	rhIL-4 (14 kDa) IL-13R and IL-4R antagonist	Aerovance, Bayer	Phase II completed (2006)	Asthma	60 mg bid x 28 d	N/A	Jet nebulizer (PARI LC® Plus, PARI GmbH, Starnberg, Germany)	N/A	NCT00535431
		Aerovance, Bayer	Phase II completed (2010)	Asthma	1–10 mg bid	N/A	DPI	N/A	NCT00801853 (l)
rhIL-2	Recombinant cytokine 15 kDa	MD Anderson Cancer Center	Phase I/II ongoing	Sarcomas, lung metastases	1 mg/d x 21 d	C = 0.33 mg/mL Exc (Proleukin®, Novartis Pharma, Basel, Switzerland): mannitol, sodium laurylsulphate, phosphate buffer	Nebulizer	N/A	NCT01590069
Stopped/Inactive
IFN-γ 1b	Interferon gamma-1b	InterMune, NIAID	Inactive	MAC infections, cystic fibrosis	500–1000 µg x 3/w	N/A	N/A	N/A	NCT00043355 NCT00021567 NCT00043316
Omalizumab	Anti-IgE IgG1 (150 kDa)	Genentech/Novartis	Stopped	Asthma	1–10 mg/d x 8 w	N/A	Jet nebulizer (PARI IS-2)	IgG in 5% patients (n = 22)	(m)
Alpha-1 antitrypsin	Lung serine protease inhibitor (52 kDa)	Kamada	Phase II completed (2008)	Cystic fibrosis	80 mg/kg x 7–28 d	N/A	Mesh nebulizer (customized PARI eFlow®)	N/A	NCT00499837

Abbreviations: ADA, anti-drug antibody; mAb, monoclonal antibody; Fab, antigen-binding fragment; RSV, respiratory syncytial virus; DPI, dry powder inhaler; mg/d, milligrams per day; PBS, phosphate buffer saline; TNF-R1, tumor necrosis factor-receptor 1; ARDS, acute respiratory distress syndrome; w, weeks; aPAP, autoimmune pulmonary alveolar proteinosis; BID, bis in die; NTM, nontuberculous Mycobacterium; C, protein concentration in the final product; Exc, excipients; GM-CSF, granulocyte-macrophage colony-stimulating factor; MAC, Mycobacterium avium complex; NIAID, National Institute of Allergy and Infectious Diseases; SmPC, summary of product characteristics; PI: prescribing information.

* Clinical trial numbers refer to the latest clinical trials registered at clinicaltrials.gov

** Detailed dosing regimens are detailed in the corresponding clinical trial description (see clinicaltrials.gov)

N/A: not available

Methodology: clinical trials involving inhaled protein therapeutics for topical lung applications were identified using the clinicaltrials.gov database. The following research filters were applied: study type: interventional studies; conditions: respiratory tract (lung and bronchial) diseases.

(a) Fuchs HJ et al. N. Engl. J. Med. (1994).

(b) Van Heeke G et al. Pharmacol. Ther. (2017).

(c) http://www.ablynx.com/uploads/data/files/ablynx_alx-0171_first-in-infant study results_webcast presentation.pdf.

(d) Burgess G et al. ATS 2017 Poster (2017).

(e) http://www.kamada.com/news_item.php?ID=285.

(f) https://www.gsk-clinicalstudyregister.com/files2/gsk-116343-protocol-redact.pdf.

(g) Cordy JC et al. Clin Exp Immunol. (2015).

(h) Djukanović R et al. Am. J. Respir. Crit. Care Med. (2014).

(i) http://www.jefferies.com/CMSFiles/Jefferies.com/files/Pieris Pharmaceuticals Inc.pdf.

(j) Zenilman JM et al. Antivir. Res. (2015).

(k) Salvatore M et al. Biol. Blood Marrow Transplant. (2016).

(l) Longphre MV et al. Prog Respir Res. (2010).

(m) Fahy J. et al. Am. J. Respir. Crit. Care Med. (1999).

Table 2. Desirable characteristics for protein inhalers, and trends in nebulization devices.

Inhaler technology	Precise, reproducible dosing	Compatibility with high doses	Minimal drug loss, and high lung deposition	Maintenance of protein integrity	Limitations/remarks	Reference
Marketed nebulizers
Jet nebulizers					Device performance affected by the drug solution’s physicochemical properties (viscosity, surface tension, etc.). Lung deposition can be enhanced with breath-synchronized or breath-actuated devices (jet- and mesh nebulizers).	(a,b)
Ultrasonic nebulizers
Mesh nebulizers
Soft mist inhalers					Relatively unsuitable with dispersed systems
Emerging/investigational devices with at least one feasibility study reported for proteins
Aradigm AERx®, Aradigm Corporation, Hayward, CA, USA (slow-velocity mist)					Aerosolization profile affected by the drug solution’s properties	(c)
Surface acoustic wave (SAW) devices	Not available				Lack of in vivo data	(c,d)
Legend

Trends are based on our best knowledge of literature reports.

NB: The Staccato™, Alexza Pharmaceuticals, Mountain View, CA, USA technology was not included in this table, given its incompatibility with peptides and proteins.

(a) Respaud R et al. Expert Opin. Drug Deliv. (2015).

(b) Hertel SP et al. Adv. Drug Deliv. Rev. (2015).

(c) Carvalho TC et al. J. Pharm. Pharmacol. (2016).

(d) Cortez-Jugo C et al. Biomicrofluidics. (2015).

Figure 1. The development of nebulized protein formulations: several key questions have yet to be addressed