Any question?
Help needed?

31 August 2021

Repurposing of Plasminogen: An Orphan Medicinal Product Suitable for SARS-CoV-2 Inhalable Therapeutic

Alfatestlab is proud to report here below the summary of  very interesting paper published in October 2020 by our customer Kedrion in which are reported some data obtained in Alfatestlab.[1]

The SARS-CoV-2 infection is associated with pulmonary coagulopathy, which determines the deposition of fibrin in the air spaces and lung parenchyma. The resulting lung lesions compromise patient pulmonary function and increase mortality, or end in permanent lung damage for those who have recovered from the COVID-19 disease. Therefore, local pulmonary fibrinolysis can be efficacious in degrading pre-existing fibrin clots and reducing the conversion of lung lesions into lasting scars. Plasminogen is considered a key player in fibrinolysis processes, and in view of a bench-to-bedside translation, XXXX and al. have focused on the aerosolization of an orphan medicinal product (OMP) for ligneous conjunctivitis: human plasminogen (PLG-OMP) eye drops. As such, the sterile and preservative-free solution guarantees the pharmaceutical quality of GMP production and meets the Ph. Eur. requirements of liquid preparations for nebulization. PLG-OMP aerosolization was evaluated both from technological and stability viewpoints, after being submitted to either jet or ultrasonic nebulization. This is the first study that focuses on the technological and biochemical aspects of aerosolized plasminogen, which could affect both treatment efficacy and clinical dosage delivery. Increasing evidence for the need of local fibrinolytic therapy could merge with the availability of PLG-OMP as an easy handling solution, readily aerosolizable for a fast translation into an extended clinical efficacy assessment in COVID-19 patient.

Indeed, the administration by inhalation route permits direct delivery of the drug to the site of action, thus favoring the rapid onset of therapeutic effects. Pulmonary administration allows for the achievement of high local drug dosages, limiting systemic toxicity and side effects, which may occur with parenteral administration [16]. Similarly, the loco-regional administration of plasminogen could provide targeted, non-generalized action, which would not be achievable by the parenteral route. Administration by inhalation is strictly related to the use of medical devices, such as nebulizers, pressured inhaler, etc. Among these, the simplest way to introduce a drug into clinical practice is to aerosolize it starting from its aqueous solution by using continuous nebulization. Presently, there are three main nebulization technologies, i.e., pneumatic (jet nebulization), ultrasonic, and using a microperforated vibrating membrane. Each device is designed to produce aerosols with controlled droplet size distributions to reach different segments of the respiratory tract. However, every nebulization technique causes shear stress and heat, which can affect drug physiochemical properties, thus precluding its therapeutic action [17].

It is thus mandatory to evaluate the nebulization of the drug dosage form in order to establish its suitability to nebulization, both in terms of aerosol droplet size distribution and drug stability upon nebulization. Among the three technologies, the vibrating mesh is the most recent and more expensive, less aggressive towards labile drugs, typically with a medium size mesh of 5 µm, and still not widespread. Jet nebulizers are considered the gold standard for delivering drugs to hospitalized patients in clinical settings, yet mechanical shear forces can damage the delivered drugs. As for ultrasonic nebulizers, they are often associated with high drug degradation due to both the shear stress at the air-liquid interface and the cavitation mechanism, as well as the dissipation of energy in the form of heat. This latter technique is considered deleterious for protein drugs, which are subject to thermal unfolding and denaturation [17,18].

Since different nebulization technologies could provide diverse harmful stresses and aerosol outputs, it is necessary to collect preclinical technological information on the appropriate choice of nebulizer as a function of drug product and therapy [19].

In this work, jet and ultrasonic nebulization were evaluated for the pulmonary delivery of plasminogen by using a pharmaceutical grade homogeneous aqueous solution of the plasma derived protein. Presently, pharmaceutical grade plasma-derived plasminogen (PLG) is available as eye drops (Kedrion S.p.A.) and currently used as an orphan medicinal product (OMP), according to Italian law 648/96, for the treatment of ligneous conjunctivitis. The conceptualization of the research considers that the requirements of the European Pharmacopoeia (Ph. Eur.) 10th edition for inhaled solutions are similar to those for eye drops (solutions) and that the dosage form prepared according to GMP standards guarantees the safety of pharmaceutical quality for a ready translation to clinic.

Droplet Size Distribution 

To determine the droplet size distribution of the aerosols, laser diffraction measurements were performed in Alfatestlab. As displayed in Figure 3, the aerosols obtained for both PLG-OMP and iPLG-OMP have particle size distribution (PSD) superimposable on that obtained for the NaCl 0.9% reference solution, mostly irrespective of the applied nebulization technique. Considering that the physical properties of PLG-OMP and iPLG-OMP are similar to those of the physiological saline solution, the results obtained by laser diffraction confirm what was expected. No significant difference was observed between PLG-OMP and iPLG-OMP when submitted to the same nebulization setting (Table 3).

However, it should be noted that the aerosol produced by US nebulization is not homogeneous over time, as observed by monitoring the Transmission value of detector 0 during the entire nebulisation period (data not reported). The results evidenced a heterogeneous polymodal size distribution, displaying droplets with median diameter values differing by two orders of magnitude. This behaviour is recorded for both the reference physiological solution and the samples, this being the limit of device performance. The wide polydispersion obtained with US nebulization is confirmed by the high SPAN value (Table 3).

Concerning the jet nebulizations, the generated aerosols have a narrow particle size distribution, with small SPAN values and median diameters analogous to those obtained for the NaCl reference solution. Additionally, these parameters fall within the range indicated by the device manufacturer. It is also worth noting that the percentages of droplets below 10 µm and 5 µm are strictly dependent on nebulization settings and not on the nebulised solution sample, since both PLG-OMP and iPLG-OMP share similar physical properties with the NaCl reference solution. In particular, lower air flow position (J2) corresponds with bigger dv(50), with about 60% and 25% of droplets sized below 10 µm and 5 µm, respectively. Better yield is obtained by the J3 setting, which provides higher shear stress and, consequently, lower-sized particles, resulting in more than 80% and more than 40% of droplets being sized below 10 µm and 5 µm, respectively.

Finally Jet nebulization resulted in a more efficient delivery of an aerosol suitable for pulmonary deposition. The biochemical investigation highlighted substantial protein integrity maintenance with the percentage of native plasminogen band > 90%, in accordance with the quality specifications of PLG-OMP. In a coherent way, the specific activity of plasminogen is maintained within the range 4.8–5.6 IU/mg (PLG-OMP pre-nebulization: 5.0 IU/mg.


The study assesses the feasibility of delivering inhalable plasminogen starting from human plasminogen orphan medicine (PLG-OMP). The research has shown that, even if none of the tested conditions massively degraded plasminogen, the ultrasonic and jet nebulization of PLG-OMP were not interchangeable. It is also concluded that PLG-OMP can be nebulized as it is. Additionally, if it would be necessary to dilute PLG-OMP to iPLG-OMP within a clinical setting, it can still be nebulized, although a lower activity of the drug should be expected. Considering the recent approval of clinical trials based on the inhalation of fibrinolytic drugs such as t-PA [42], the present study opens up the opportunity for adjunctive therapy regimes based on the repurposing of PLG-OMP as inhalable medicine for the treatment of ARDS in COVID-19 patients. The acquired preclinical data appear useful for the bench-to-bedside translation of PLG-OMP in the treatment of COVID-19 patients, in view of evaluating safety issues and fine dosage/efficacy correlation.

1. A.M. Piras, Y. Zambito, M. Lugli, B. Ferro , P.Roncucci , F. Mori, A, Salvatore, E. Ascione, M. Bellini and R. Crea Repurposing of Plasminogen: An Orphan Medicinal Product Suitable for SARS-CoV-2 Inhalable Therapeutic. Pharmaceuticals 2020, 13, 425
17 "Targeting Chronic Inflammatory Lung Diseases Using Advanced Drug Delivery Systems":
16. Videira, M.; Llop, J.; Sousa, C.; Kreutzer, B.; Cossío, U.; Forbes, B.; Vieira, I.; Gil, N.; Silva-Lima, B. Pulmonary Administration: Strengthening the Value of Therapeutic Proximity. Front. Med. 2020, 7. [CrossRef] [PubMed]
17. Hertel, S.; Winter, G.; Friess, W. Protein stability in pulmonary drug delivery via nebulization. Adv. Drug Deliv. Rev. 2015, 93, 79–94. [CrossRef] [PubMed]
18. Chang, K.H.; Moon, S.-H.; Oh, J.Y.; Yoon, Y.-S.; Gu, N.; Lim, C.-Y.; Park, B.J.; Nam, K.C. Comparison of Salbutamol Delivery Efficiency for Jet versus Mesh Nebulizer Using Mice. Pharmaceutics 2019, 11, 192. [CrossRef] [PubMed]
19. Adorni, G.; Seifert, G.; Buttini, F.; Colombo, G.; Stecanella, L.A.; Krämer, I.; Rossi, A. Aerosolization Performance of Jet Nebulizers and Biopharmaceutical Aspects. Pharmaceutics 2019, 11, 406. [CrossRef]
42. Nebulised Rt-PA for ARDS Due to COVID-19 (PACA). Identifier: NCT04356833. Available online: (accessed on 10 October 2020).

Need more info? Contact us!