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WO2016020070A1 - Mutants d'inhibiteurs de l'élastase neutrophile dérivés de sangsue et leurs utilisations - Google Patents

Mutants d'inhibiteurs de l'élastase neutrophile dérivés de sangsue et leurs utilisations Download PDF

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Publication number
WO2016020070A1
WO2016020070A1 PCT/EP2015/052192 EP2015052192W WO2016020070A1 WO 2016020070 A1 WO2016020070 A1 WO 2016020070A1 EP 2015052192 W EP2015052192 W EP 2015052192W WO 2016020070 A1 WO2016020070 A1 WO 2016020070A1
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mutant
fahsin
residue
inhibitor
antistasin
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Gerard Voerman
Friso Martijn VOERMAN
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/81—Protease inhibitors
    • C07K14/815—Protease inhibitors from leeches, e.g. hirudin, eglin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/81—Protease inhibitors
    • C07K14/8107—Endopeptidase (E.C. 3.4.21-99) inhibitors
    • C07K14/811—Serine protease (E.C. 3.4.21) inhibitors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the invention relates to mutants of neutrophil elastase inhibitors, especially neutrophil elastase inhibitors that have been derived from leeches, more specifically mutants of fahsin, guamerin, piguamerin, hirustasin, and bdellastasin and uses thereof for preventing diseases associated with neutrophil elastase, more specifically periodontitis.
  • the first group comprises antithrombotic and fibrinolytic activities
  • the second group comprises enzymes and inhibitors.
  • Well known representatives of the first group include for instance hirudin, a thrombin inhibitor, (Markwardt, 1956; 1988; Petersen, et al, 1984); hementin, a fibrinolytic agent (Budzynski, et al, 1981; Kirschbaum & Budzynski, 1990); antistasin, an inhibitor of
  • coagulation factor Xa (Gasic, et al, 1983), which was reported to have antimetastatic properties as well; and gilanten, another factor X inhibitor (Condra, et al, 1989; Blankenship, et al, 1990).
  • Representatives of the second group are: bdellin, an inhibitor of trypsin and plasmin (Fritz & Krejci, 1976); eglin, an inhibitor of chymotrypsin, elastase and Cathepsin G (Seemuller, 1979), and orgelase, an hyaluronidase (Sawyer, 1986).
  • a fibrinolytic enzyme isolated from Hirudo medicinalis which splits Glutamine-Lysin sequences (EP 0502876).
  • a platelet adhesion inhibitor isolated from Hirudo medicinalis, which inhibits ccllagen-induced platelet aggregation (EP 05522), a thrombin inibitcr from the leech
  • Hirudinaria manillensis (PCT/GB89/D145).
  • an inhibitor of platelet aggregation from leeches from the Hirudinidae family (EP 0348208) and an anticoagulant/moduator reactor isolated from Hirudo medicinalis (EP 0352903), A chymotrypsin- and elastase inhibitor from Hirudinaria manillensis (PCT/NL90/00046).
  • the inventor of the current invention further found a novel family of protease-inhibitors obtainable from Limnatis nilotica (EP 0789764).
  • the reactivity of one member of this family, fahsin has been reported in the literature (De Bruin, E. et al., FEMS Yeast Res. 5:1069-1077, 2005; WO 96/13585). It has been demonstrated in this publication that fahsin is a proteinase that is specific for human neutrophil elastase (hNE) and leaves other important blood-derived serine proteases, such as plasmin, thrombin, tPA, coagulation factors Vila, Xa, XIa and Xlla untouched.
  • hNE human neutrophil elastase
  • substances can be derived from all body parts and secretions of the leech, inclusive saliva and gut-, intestinaland skin secretions and mucus.
  • fahsin The amino acid sequence of fahsin (GenBank DQ097891.1) and the nucleotide sequence coding for said amino acid sequence (GenBank AAY85799.1) has been provided in Fig. 1. As is shown in De Bruin et al., (supra) fahsin is similar to other antistasin-type proteinase inhibitors by comprising a consensus sequence with 10 cysteine residues at specific distances:
  • NE leech derived antistasin type serine proteinase
  • Hirustasin (Hirudo medicinalis): TQGNTCGGET CSAAQVCLKG KCVCNEVHCR IRCKYGLKKD ENGCEYPCSC AKASQ
  • NE inhibitors can be typically isolated from leech tissue by solvent extraction-techniques;
  • leech secretions such as leech saliva
  • the invention is not limited to specific ways of obtaining the novel protease-inhibitors.
  • mutants of these proteinase inhibitors can have specific activity, which is advantageous for the use of such compounds for prevention or therapy of several diseases.
  • the current invention now relates to a mutant of an antistasin type serine proteinase (NE) inhibitor wherein said mutant is mutated with respect to the wild-type sequence at the amino acid residue immediately following the sixth cysteine residue.
  • said antistasin type serine proteinase (NE) inhibitor is selected from fahsin, guamerin, piguamerin, hirustasin and bdellastasin and mutant homologs.
  • said antistasin type serine proteinase (NE) inhibitor is fahsin or a mutant homolog of fahsin and the residue after the 6th cysteine residue is an isoleucine residue.
  • the mutant preferably has the sequence DDNCGGKVCS KGQLCHDGHC ECTPIRCIIF CPNGFAVDEN GCELPCSCKH Q.
  • said antistasin type serine proteinase (NE) inhibitor is guamerin, piguamerin, hirustasin or bdellastasin or a mutant homolog thereof and the residue after the 6th cysteine residue is a leucine residue.
  • the mutant preferably has the sequence
  • said antistasin type serine proteinase (NE) inhibitor is fahsin or a mutant homolog of fahsin and the residue after the 6th cysteine residue is an arginine residue.
  • the invention further relates to a mutant as defined above, wherein said mutant is produced through recombinant technology.
  • a further part of the inventions is a nucleotide sequence encoding a mutant according to any of the previous claims.
  • Further part of the invention is an expression vector comprising such a nucleotide sequence operatively connected to a promoter and optionally other regulatory sequences.
  • Also part of the invention is a host cell comprising an expression vector according to claim 10.
  • mutants according to the invention are used in therapy or prevention of emphysema, arthritis, gingivitis, periodontitis, pancreatitis and other inflammatory conditions that are associated with tissue destruction caused by the enzyme human neutrophil elastase, cathepsin G, chymotrypsin and/or Prot3.
  • mutant fahsin-arg is used in the prevention or treatment of pancreatitis.
  • the invention comprises a method for treating
  • a mutant protein according to any of claims 1 — 8 is administered to a subject in need thereof.
  • said administration is via a controlled or sustained release formulation of said mutant protein.
  • Fig.l Nucleotide and amino acid sequence of wild-type fahsin.
  • Fig.2 Alignment of the primary amino acid sequence of five different antistasin-type serine proteinase inhibitors. The similarly spaced cysteine residues in the proteins are indicated in bold. The reactive site (PI) amino acid residue, reflecting the specificity of the inhibitor, is underlined.
  • PI reactive site
  • Fig. 3 Inhibition of human neutrophil elastase by different mutants of guamerin. Indicated on the X-axis is the residue of the mutant at the PI position (met is the wild- type), bianco is only substrate and max is substrate + elastase. The bars represent the time after start of incubation. The y-axis gives the A405-A540 difference measured.
  • Fig. 4 Inhibition of cathepsin G inhibition by mutants of fahsin in different concentrations. Indications of mutants and Y-axis similar as in Fig. 2.
  • Fig. 5 Inhibition of various proteinases by mutants of guamerin.
  • mutants described in the present invention have been changed at the amino acid position after the 6th cysteine residue (the PI site, as indicated in Fig. 2).
  • the remaining residues are kept identical to the wild-type sequence, but it is also possible to modify one or a few further amino acids without losing the biological function.
  • this biological function that is maintained is defined as the (human) proteinase inhibitory activity.
  • mutant proteins that are changed with respect to the wild type protein on the residue following the 6 th cysteine residue and which have a sequence identity with the wild-type protein of at least 70%, preferably at least 75%, more preferably at least 80%, more preferably at least 82%, more preferably at least 84%, more preferably at least 86%, more preferably at least 88%, more preferably at least 90%, more preferably at least 92%, more preferably at least 94%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%.
  • Such mutant proteins will have maintained the above mentioned biological function and are herein defined as 'mutant homologs'.
  • the present invention provides also for nucleotide sequences coding for the abovementioned amino acid sequences and sequences which have a percentage of identity related to such nucleotide sequences of 65% to 95%.
  • the percentage of identity can be at least, 65%, 70%, 75%, 80%, 85%, 90%, or 95%.
  • Sequence identity on nucleotide sequences can be calculated by using the BLASTN computer program (which is publicly available, for instance through the National Center for Biotechnological Information, accessible via the internet on
  • sequence identity can be calculated through the BLASTP computer program (also available through
  • mutants of fahsin which are described below, especially preferred is a mutated guamerin, wherein the methionine residue after the 6th cysteine residue is changed into a leucine residue: VDENAEDTHG LCGEKTCSPA QVCLNNECAC TAIRCLIFCP NGFKVDENGC EYPCTCA.
  • mutant proteins and mutant homologs thereof having a leucine residue after the 6th cysteine residue form part of the invention.
  • mutants that have been made while studying the mutants that are applicable in the present invention comprise a number of fahsin mutants, in which the PI site (i.e. the residue following the 6th cysteine residue) has been changed.
  • PI Arg
  • PI He
  • PI Met
  • PI Val
  • Fahsin-Ile i.e. the residue following the 6th cysteine residue is He
  • Fahsin-Ile is a very specific inhibitor of elastase and does not inhibit chymotrypsin, cathepsin G and proteinase 3.
  • this mutant is very suitable for diseases in which specifically elastase is a causing factor, such as emphysema and psoriasis. Also, this mutant could be very well suited for arthritis, gingivitis, periodontitis and other
  • the invention specifically also covers use of this Fahsin-Ile mutant as a therapeutic compound, especially for the treatment of inflammatory diseases that are related to neutrophil elastase, and in particular for emphysema, periodontitis, arthritis and the like.
  • the above described Leu-mutants of guamerine and other NE inhibitors can be advantageously used for treating inflammation related diseases in which elastase is a causing factor and especially gingivitis and periodontitis.
  • Periodontitis is a multifactorial infectious disease. This disease effects the soft and hard supportive tissues of human teeth. Although the primary cause is bacterial infection, degranulation of proteases from polymorphonuclear leucocytes in the innate immunological reaction on the bacterial infection, degrades these tissues in inflammation (Huynh C, et al: J Clin Pariodontol 1992; 19: 187. Zafiropoulos GGK, et al: J. Periodont. Res. 1991;26: 24.). Leukocytes from gingival crevicular fluid (GCF) degranulate with significant more HNE upon stimulation than same cells from eripheral blood (Benedek-Spat E. et al: Arch. Oral Biol. 1991;36(7):507).
  • GCF gingival crevicular fluid
  • protease inhibitors like ctl-PI, ct2-M, or secretory leukocyte protease inhibitor (SLPI) is largely diminished due to oxidation or chemical degradation in inflammation (Weiss S.J. et al: New Engl J Med 1989; 320: 365. Abbink J.J. et al: Arthr Rheum 1993; 36: 168. Laugisch O. et al: Mol Oral Microbiol. 2012; 27 (1); 45). Genetic factors influencing the immunologic reaction against the infection are also of great importance in the extent and gravity of the disease (Papantonopoulos G, et al: J
  • gingivitis sites as compared to other forms of periodontitis (both chronic and aggressive) contain as much free HNE when related to the presence of lactoferrin, which is related to secondary granule release by PMN's ( Murray M.C. et al: Oral Dis 1995; 1 (3): 106. Gustafsson A. et al: J Periodont Res 1994; 29 (4): 276 ). Free, active HNE was found in all inflamed gingival tissues (gingivitis and
  • Fahsin with the Pl-mutant isoleucine that is the mutation of the residue following the 6th cysteine residue is He
  • Fahsin with the Pl-mutant isoleucine has shown to be more active and particularly more specific than native fahsin.
  • mutant homologs of fahsin-Ile and Pl-Leu mutants of the other NE inhibitors and homolog mutants thereof would be particularly suited for this purpose.
  • a mouthwash containing such a compound is by providing a standard mouthwash solution (preferably without alcohol), with a dosage of the mutant protein of 1 to 10 microgram per ml. solution.
  • a toothpaste preparation containing the usual ingredients, may comprise an addition of mutant protein of 1 to 100 microgram per gram of toothpaste.
  • inhaling an aerosol with said mutant compound e.g. by smoking of an e-cigarette containing said compound, may in itself have a strong preventive action on the generation or maintenance of gingivitis. In all these treatments it would be possible to combine the administration of the mutant NE inhibitor or mutant homolog thereof with existing
  • medication for the prevention or treatment of periodontitis and/or gingivitis such as antibiotics.
  • Antibiotics are especially useful, since a major factor in both gingivitis and periodontitis is the presence of bacterial populations in the affected areas. It is well-known, that the occurrence of gingival crevicular fluid (GCF) in periodontitis with well-established pathological pockets, prohibits any entry of whatever substance into these shallow or deeper pockets (Turkoglu O. et al: BMC Oral Health. 2014; 14: 55. ) Minocycline, doxycycline, and chlorhexidine preparations amongst others have been used as means to reduce bacterial load in shallow or deep pockets in
  • Periodontitis Preparations were used with slow release formulations ( microspheres, local strips, gels) to prevent them from being expelled from these pockets by GCF ( Paolantonio M. et al: J Periodontol 2009; 80(9) : 1479. Chandra R.V. Quintessence Int; 2012 ;43(5) :401.).
  • antibiotics as means for periodontal supportive treatment.
  • Preventive means are especially applicable in gingivitis, and less in periodontitis, where treatment is generally needed.
  • Treatment of periodontal disease is classically performed through root debridement and root planning. This can be done with or without additional supportive antibacterial treatment. The same remark about such supportive action can be placed as with the preventive treatments. Regenerative new attachment after such treatment is generally limited ( less than 1 mm).
  • Results of treatment of periodontitis can be advantageously improved by the application of a mutant NE inhibitor according to the invention, such as fahsin-Ile, preferably in combination with a slow release device.
  • a mutant NE inhibitor according to the invention such as fahsin-Ile, preferably in combination with a slow release device.
  • the latter is useful to withstand the effluent GCF.
  • Treatment of periodontitis thus preferably comprises application of a preparation of a mutant according to the invention, such as fahsin-Ile in a suitable slow-release (sustained release or controlled release) device.
  • a preparation of a mutant according to the invention such as fahsin-Ile in a suitable slow-release (sustained release or controlled release) device.
  • Treatment per element would comprise application of between 6 and 60 microgram mutant to be released over preferably 7 days (see also the calculation further below).
  • Drug delivery system for periodontitis and gingivitis which would be applicable to the presently proposed compounds are known to the skilled person. An overview can, for example, be found in Raheja, I. et al., 2013, Int. J. Pharmacy Pharm. Sci. 5(3):11-16). Among these systems are hollow or matrix-providing fibers or thread-like
  • Slow- release devices generally may consist of systems with are fluid at room- temperature, and become crystalline or solid at body temperature and - moisture.
  • Such system could preferable be, but not limited to, a mixture of mono- and triglycerides, a hydrophilic polymer, and others.
  • Results of professional periodontal treatment are assessed by means of standardized measurements, taken for each affected element present in the mouth of the patient at six places around the element
  • Measurements consist of determining the recession (in mm) and pocket depth (in mm). These two parameters together give rise to the figure of "Loss of Attachment Level” (LAL). Other parameters that may be measured are plaque accumulation (PI) and bleeding on probing (BOP) (all preferably on said six positions per element). Further measurements consist of indicating the presence of root furcation ( class I, if probed with one third of the elements thickness, class II if two thirds can be probed, and class III if the furcation is continuous from one side to the other).
  • Mobility per element is recorded as a classified response when a force of 100 grams of pressure is applied laterally to the element. If the element moves 0— 0.5 mm a class 0 is noted, a movement of 0.5— 1 mm is class 1, a movement of 1 — 2 mm is class 2 and a movement of more than 2 mm is class 3.
  • Standard treatment for three months would provide the result that LAL may have reduced to within 50%, i.e. an improvement of 1 mm for single-rooted elements. Complicated elements (mostly molars) having more than one root, may show less results. The application of fahsin-Ile will show a further reduction ( 1 mm. or more) than is measured in the standard procedure. As a matter of inflammation control, the PI and BOP will be reduced on all positions to a level of maximum of 10% of the original values before treatment.
  • Fahsin-Val and the Fahsin-Met mutant and/or their mutant homologs may be used as elastase-inhibitors in the same way as indicated above for Fahsin-Ile and the Leu-mutants of the other NE inhibitors, although their effect is less specific than the Fahsin-Ile mutant and the wild-type fahsin.
  • Fahsin-Arg i.e with an arginine residue on the PI position.
  • This compound although it only differs in one amino acid from wild-type fahsin does not specifically inhibit elastase, but surprisingly it is an excellent inhibitor of trypsin (and it also inhibits the coagulation factors Xa, XIa and Xlla). Because of these effects, Fahsin-Arg is deemed suitable for inhibition of coagulation and fibrinolysis. Also, Fahsin-Arg may be used in the therapy of pancreatitis.
  • CP Chronic pancreatitis
  • fibrosis and cirrhosis of the pancreas Genetic factors seem to be most important in obtaining CP, but infectious and environmental influences may contribute (Lerch M.M. et al: Dig Dis. 2010; 28(2) :324).
  • Alpha- 1 -antitrypsin is a principal antiprotease which protects the mucosal tissue from the proteolytic effects of trypsin and chymotrypsin by the formation of molar complexes (Kavutharapu S. et al: Saudi J
  • Thrombin was hardly inhibited by any of the mutants. This means that these mutants can specifically be focused on application in diseases or conditions that are characterized by an excess of the enzyme mentioned above.
  • the enzyme is neutrophil elastase
  • the application of the inhibitor(s) can advantageously be of use in the treatment or prevention of inflammation related phenomena, such as COPD, emphysema, psoriasis, arthritis and particularly gingivitis and periodontitis, while the Arg mutant can best be applied in pancreatitis and the Leu mutant for treatment of edema and photoaging.
  • HNE human neutrophil elastase
  • GCF GCF ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇
  • the released HNE inhibitor will enter the bloodstream.
  • a dose of 60 ⁇ g fahsin per week it is assumed that per 15 min. 0.1 will enter the blood stream.
  • Such a low dose in insufficient to provide any effects, which means that unaffected elements or contralateral sides of an element that are not affected may serve as a control (placebo) site.
  • the person skilled in the art may easily determine the effective dosis and route of administration.
  • the route of administration will preferably be intravenous, but also enteral administration, such as oral administration may be used.
  • enteral administration such as oral administration
  • the (mutant) proteins of the present invention are extremely stable and will not be harmed by heat, acid or base treatments that would affect other proteins.
  • the above discussed slow-release formulations may be used for prevention or treatment of other conditions or diseases.
  • FA-3 5'- CCAATTAGATGTTTGATTTTCTGTCCAAACGGTTTCGCTGTTGACGAGA ACGGTTGTGAG-3'
  • P. pastoris GS115 (his4, see Cregg, J. et al., Mol. Cell. Biol.
  • plasmid pPIC9Fahsin was linearized with Sail (Invitrogen). After growth for 3 days on selective plates at 30°C, several colonies were selected for PCR confirmation using the vector primers 5AOX1 and 3AOX1 (Invitrogen).
  • the rFahsin was separated using anion-exchange chromatography on a SP Sepharose FF column and eluted using a 1 M NaCl in citrate buffer (20 mM, pH 4.0) on Akta explorer (GE Healthcare). With a chromogenic assay the activity of rFahsin containing chromatography fractions on NE was determined and active fractions were pooled and subsequently dialysed against 20 mM Tris- HC1, pH 8.0 to remove the NaCl. In a last anion exchange chromatography step on Q-Sepharose Fast Flow or Q-Sepharose High Performance
  • Fahsin mutants with different amino acids at the PI position were made through site- directed mutagenesis in the strain that was used for producing the recombinant fahsin (Example 1, P. pastoris GS115). They were tested on several protein assays for testing the activity on other (serine) proteinases. As an example the effects of these mutants on cathepsin G is shown in Fig. 4.
  • guamerin and guamerin mutants expressing yeast strains were made in the same way as for fahsin.
  • the guamerin Lys mutant was shown to strongly inhibit elastase (Fig. 3 and Fig. 5C).
  • Fig. 5 a summary is given of the inhibiting effects of guamerin and its mutants on 6 different proteinases.

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Abstract

La présente invention concerne un mutant d'un inhibiteur d'une sérine protéinase de type antistasine, dans lequel ledit mutant est muté par rapport à la séquence de type sauvage au niveau du résidu d'acide aminé immédiatement à la suite du résidu de la sixième cystéine. Un tel inhibiteur de protéinase sérine de type antistasine est de préférence choisi parmi la fahsine, la guamérine, la piguamérine, l'hirustasine, la bdellastasine et leurs homologues mutants.
PCT/EP2015/052192 2014-08-05 2015-02-03 Mutants d'inhibiteurs de l'élastase neutrophile dérivés de sangsue et leurs utilisations Ceased WO2016020070A1 (fr)

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Cited By (5)

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CN105646702A (zh) * 2016-04-13 2016-06-08 中国科学院昆明动物研究所 菲牛蛭Kazal型胰蛋白酶抑制剂Bdellin-HM及其编码基因和应用
WO2021053058A1 (fr) 2019-09-17 2021-03-25 Mereo Biopharma 4 Limited Alvélestat destiné à être utilisé dans le traitement du rejet de greffe, du syndrome de bronchiolite oblitérante et de la maladie du greffon contre l'hôte
WO2021209740A1 (fr) 2020-04-16 2021-10-21 Mereo Biopharma 4 Limited Procédés impliquant l'alvélestat, un inhibiteur de l'élastase des neutrophiles, pour le traitement d'une infection à coronavirus
CN115025127A (zh) * 2022-08-10 2022-09-09 云南三江本草药业有限公司 菲牛蛭在制备治疗慢性阻塞性肺病的药物中的应用及中药制剂
WO2023067103A1 (fr) 2021-10-20 2023-04-27 Mereo Biopharma 4 Limited Inhibiteurs de l'élastase neutrophile utilisés dans le traitement de la fibrose

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