EP3193910A1

EP3193910A1 - Mutants of leech derived neutrophil elastase inhibitors and uses thereof

Info

Publication number: EP3193910A1
Application number: EP15703058.6A
Authority: EP
Inventors: Friso Martijn VOERMAN; Gerard Voerman
Original assignee: Reunion Therapeutics BV
Current assignee: VOERMAN, GERARD
Priority date: 2014-08-05
Filing date: 2015-02-03
Publication date: 2017-07-26
Also published as: WO2016020070A1

Abstract

The invention 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. Such an antistasin type serine proteinase (NE) inhibitor is preferably selected from fahsin, guamerin, piguamerin, hirustasin and bdellastasin and mutant homologs.

Description

Title: Mutants of leech derived neutrophil elastase inhibitors and uses thereof. 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.

Various substances extracted from leeches are known to have useful biological activity. These were reviewed by Sawyer, (Sawyer, 1990). Essentially two groups of activity can be recognised. 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).

Further several additions in this field have been published in the patent literature: 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. Such

substances can be derived from all body parts and secretions of the leech, inclusive saliva and gut-, intestinaland skin secretions and mucus.

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:

C (X₄) CS (X₄) C (X₄) CXC (X₄) C L (Xa) C (¾) DXNGC (X₃) CXC in which X may be any amino acid, and L, N, G and C have their normal meaning in the nomenclature of amino acids.

Next to fahsin also other leech derived antistasin type serine proteinase (NE) inhibitors were found to have a similar structure. Examples are guamerin, piguamerin, hirustasin and bdellastasin. The amino acid sequences for these compounds are:

guamerin (Hirudo nipponia): VDENAEDTHG LCGEKTCSPA

QVCLNNECAC TAIRCMIFCP NGFKVDENGC EYPCTCA Piguamerin (Hirudo nipponia): TDCGGKTCSE AQVCKDGKCV CVIGQCRKYC PNGFKKDENG CTFPCTCA

Hirustasin (Hirudo medicinalis): TQGNTCGGET CSAAQVCLKG KCVCNEVHCR IRCKYGLKKD ENGCEYPCSC AKASQ

Bdellastasin (Hirudo medicinalis): FDVNSHTTPC

GPVTCSGAQM CEVDKCVCSD LHCKVKCEHG FKKDDNGCEY ACICADAPQ

These antistasin type serine proteinase (NE) inhibitors can be typically isolated from leech tissue by solvent extraction-techniques;

alternatively they may be isolated from leech secretions (such as leech saliva), although the invention is not limited to specific ways of obtaining the novel protease-inhibitors.

The present inventors now found that 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.

SUMMARY OF THE INVENTION

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. Preferably said antistasin type serine proteinase (NE) inhibitor is selected from fahsin, guamerin, piguamerin, hirustasin and bdellastasin and mutant homologs. In another embodiment 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. In this case the mutant preferably has the sequence DDNCGGKVCS KGQLCHDGHC ECTPIRCIIF CPNGFAVDEN GCELPCSCKH Q. In another embodiment 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. In this case the mutant preferably has the sequence

VDENAEDTHG LCGEKTCSPA QVCLNNECAC TAIRCLIFCP NGFKVDENGC EYPCTCA

In again another embodiment 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.

Further part of the invention is the therapeutical use of the mutants according to the invention. Preferably a mutant according to the invention is 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. Preferably the mutant fahsin-arg is used in the prevention or treatment of pancreatitis.

Lastly, the invention comprises a method for treating

emphysema, arthritis, gingivitis, periodontitis, pancreatitis and/or other inflammatory conditions that are associated with tissue destruction caused by the enzyme human neutrophil elastase, cathepsin G, chymotrypsin and/or Prot3, wherein a mutant protein according to any of claims 1 — 8 is administered to a subject in need thereof. Preferably, said administration is via a controlled or sustained release formulation of said mutant protein.

LEGENDS TO THE FIGURES

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.

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. A: cathepsin G, B: chymotrypsin, C: elastase, D: trypsin, E: plasmin, F:

thrombin

DETAILED DESCRIPTION

Mutants of fahsin, guamerin, piguamerin, hirustasin and bdellastasin have been found to have advantageous properties. The 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). Preferably, 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. In respect of the present invention, this biological function that is maintained is defined as the (human) proteinase inhibitory activity. Thus part of the invention are 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%. Thus, for example, 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

http ://w w w . ncbi . nlm . nih .go /) using the default settings of 11 for wordlength (W), 10 for expectation (E), 5 as reward score for a pair of matching residues (M), -4 as penalty score for mismatches (N) and a cutoff of 100.

For amino acids, the sequence identity can be calculated through the BLASTP computer program (also available through

http://www.ncbi.nlm.nih.gov/).

Next to 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.

It has been shown (results not shown) that such a mutated guamerin is insensitive to both chemical and biological oxidation and it has further been shown that this mutated protein also appeared to be a strong inhibitor of NE like the wild-type fahsin molecule. It is believed that for all mentioned family members listed in Fig. 2 changing this specific residue (PI, see Fig. 2), which in the other molecules mentioned above is an arginine residue (piguamerin and hirustasin) or a lysine residue (bdellastasin) into a leucine residue also provides mutant proteins that have an improved reactivity towards NE and also are more stable than the wild-type proteins.

As such, the mutant proteins and mutant homologs thereof having a leucine residue after the 6th cysteine residue form part of the invention.

Other 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. Several fahsin mutants were made with PI = Arg, PI = He, PI = Met and PI is Val. These mutants were made via site- directed mutagenesis. It appeared that Fahsin-Ile (i.e. the residue following the 6th cysteine residue is He) is a very specific inhibitor of elastase and does not inhibit chymotrypsin, cathepsin G and proteinase 3. This means that 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

inflammatory conditions that are associated with tissue destruction caused by the enzyme human neutrophil elastase (HNE). Hence, 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. Also 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). The

physiological presence of 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

Periodontol 2013; 84 (7): 974. Jong T.M. de, et al: J Clin Periodontol 2014; 41 (6): 531).

There is also large evidence that environmental and lifestyle factors play an important role in the establishment of the disease (Han Y.W. et al: Adv Dent Res 2014; 26 (1): 46. Schafer A.S. et al: Circ Cardiovasc Genet 2014; Dec 2, in press). Also is there evidence that periodontitis plays an important role in the establishment and gravity of various general conditions, a. o. like diabetes, CVD, emphysema ( Loos B.G. et al., Ned Tijdschrift Tandheelk 2014; 121 (9): 428. Bochenek G et al: Hum Mol Genet 2013; 22 (22) : 4516).

It is well-known that a severe form of early-onset aggressive periodontitis (prepuberal juvenile periodontitis) can lead to complete destruction of all supporting tissues of teeth before the age of 20 years.

Prevention of this form of periodontitis depends particularly on the early detection, mainly in routine inspection by dentists (Piergallini G. et al:

Pediatr Med Chir. 2014; 36 (4): 95. Spoerri A. et al: Eur Arch Paediatr Dent. 2014; 15 (6): 443).

It was further found that 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

periodontitis). It was proven, that the physiologal HNE inhibitor al- PI was oxidized and ineffective in inhibiting elastolysis. (Figueredo CM, Gustafsson A.: J Clin Periodontol. 1998 ;25(7):531; Giannopoulou C. et al: Arch Oral Biol. 1994;39(9):741).

Prevention and treatment of periodontitis, but also of gingivitis is therefore of evident importance. Fahsin with the Pl-mutant isoleucine (that is the mutation of the residue following the 6th cysteine residue is He) has shown to be more active and particularly more specific than native fahsin. Also 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.

Prevention and/or treatment of periodontitis could be done by adding said fahsin and/or the other mentioned mutants in mouth care products. Preparation of 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. Furthermore, 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.).

However, one must be restrictive in proposing such antibiotic compounds as a treatment of periodontitis. Especially with locally applied antimicrobial treatment the danger of building resistant microbial species at the site of the infection exists. Therefore, care should be taken when applying 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). More important gains in attachment level can be obtained in surgical treatment with application of growth-factor like compounds (EMD, PDGF, beta-TCP), (Reynolds M.A. J Periodontol. 2014 Oct 15:1). This requires, however, extensive and costly treatments.

Results of treatment of periodontitis, as described here-above (root- debridement and planning) in closed situations (i.e. no surgery), 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. 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.

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

structures, micro- and/or nanoparticles, gels, films, and the 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

(mesiobuccally, buccally, distobuccally, distolingually, lingually, and mesiolingually). 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. These standard figures of improvement are only

obtainable if the patient has abstained from smoking, and has sincerely followed the oral hygiene instructions. Also the 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.

A further very useful other fahsin mutant is 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.

Chronic pancreatitis (CP) is the progressive and irreversible destruction of the pancreas (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).

Trypsin, the most important digestive enzyme plays a central role in the regulation of all other digestive enzymes. Chymotrypsin, an

endopeptidase hydrolyzes peptides at amino acids with aromatic side chains. 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

Gastroenterol. 2012; 18(6): 364. In CP both trypsin and chymotrypsin are elevated compared to healthy controls. The physiologic protease inhibitor alpha- 1 -antitrypsin is lowered in CP patients. There is an altered balance between the proteases and the protease inhibitor in CP patients. A therapy, containing application of fahsin- Arg is very useful in the treatment of chronic pancreatitis patients. It should be used in a well-powered study (Thrower E. et al: Curr Opin Gastroenterol. 2008 Sep;24(5):580). Fahsin-Arg is also a stronger cathepsin G inhibitor than the other fahsin mutants. This means that it can also be used as a cathepsin g inhibitor, and thus that it would be useful to treat or prevent inflammation, especially where inflammation leads to edema, to treat or prevent

photoaging. Also, this function enhances the antithrombotic effects of Fahsin-Arg.

For guamerin, next to the above discussed Leu mutant, also other mutants have been made with He, Arg,, Lys or Val at the PI position (see Fig. 1). As discussed above, the Leu mutant was the most effective in inhibition of human neutrophil elastase, while also the wild- type (with Met at the PI position) showed some effects. The other three mutants were less effective. However, the Arg mutant appeared to be the best inhibitor of chymotrypsin, with the Leu mutant coming second. These two were also the best inhibitors of cathepsin G. The Lys mutant, however, proved to be a specific inhibitor of trypsin and plasmin, where for the other mutants only guamerin-Arg could show some effects. 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. Where 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.

For an effective treatment of a disease or condition where human neutrophil elastase (HNE) is an associated factor, it depends on the amount of HNE that becomes available what would be an effective dos of the mutant of the invention. With respect to periodontitis, it has been established that the total concentration of HNE in the GCF is about 0 - 350 μ /ml in stable pockets and about 300 - 900 μ /ml in active pockets. The total concentration of HNE consists of bound and unbound (=active) HNE and the respective concentrations of unbound HNE are 0 - 100 and 0 - 350 μ /ml. The amount of GCF will, of course, depend on the severity of the disease, but is has been measured (Cimasoni G., Monographs in Oral Sciences, eds. Meyers, H.M. et al., Vol. 12, Karger, Basel, 1974, rev. 1983) that the total GCF production is about 0.5 - about 2.4 ml per day, but this may be increased due to an increased production in cases of periodontitis. Production of HNE in the GCF is provided by the polymorphonuclear leukocytes (PMN). The concentration of PMNs per element per 15 minutes has been measured to be 25,000 (Cimasoni G.: Monogr Oral Sci. 1983;12:III-VII, 1-152; Andersen E ., Cimasoni G: J Clin Periodontol. 1993;20(9):651). Degranulation of these 25,000 PMNs produces about 0.1 μg HNE; this means that per week 70 μg HNE is produced per element, assuming that all PMNs degranulate. This will not be the case and it is assumed that 50% degranulates which means a weekly HNE production of about This will not be the case and it is assumed that 50% degranulates which means a weekly HNE production of about 5 μg. This can be inhibited by 6 μg of fahsin mutant. To be on the safe side (and since toxicity of fahsin does not play a role at these low concentrations) it is deemed advisable to provides a dose per week that is ten times as high: about 60 μg.

Of course the released HNE inhibitor will enter the bloodstream. With a dose of 60 μg 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.

Having established that a weekly dose of about 60 μg of the mutant antistasin type serine proteinase HNE inhibitor is needed, it follows that for a slow release formulation that needs to be inserted in the pocket of the affected element this amount should be provided in a volume of 0.04 ml (40 μΐ). It is submitted that it would be advantageous to provide a drug sustained release formulation that will provide a continuous drug release for several weeks to minimize the handling of placing of new drug supplies in the dental pockets (and possibly removal of old, empty delivery vehicles). This means that a concentration of the drug in amounts of several hundreds of micrograms should be available in the volume of 40 μΐ. It has appeared that the mutant proteins of the present invention can easily be solved in such concentrations in standard vehicles, such as water.

For treatments other than gingivitis/periodontitis, the person skilled in the art may easily determine the effective dosis and route of administration. For example, for pancreatitis, the route of administration will preferably be intravenous, but also enteral administration, such as oral administration may be used. It is submitted that 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. Also, the above discussed slow-release formulations may be used for prevention or treatment of other conditions or diseases.

EXAMPLE 1

Production and characterization of recombinant fahsin Production and purification of fahsin was performed as described in De Bruin, E. et al., FEMS Yeast Res. 5:1069-1077, 2005. In short, a synthetic fahsin gene was constructed by overlap extension PCR of four long oligonucleotides, codon usage optimized for the host Pichia pastoris:

FA-1: 5'-

GGGGTATCTCTCGAGAAAAGAGACGACAACTGTGGTGGTAAGGTTTGT TCTAAGGGTCAA-3' FA-2: 5'-

AATCAAACATCTAATTGAGTACACTCACAGTGACCGGTCGTGACACAAT TGACCCTTAGAACAAAC-3'

FA-3: 5'- CCAATTAGATGTTTGATTTTCTGTCCAAACGGTTTCGCTGTTGACGAGA ACGGTTGTGAG-3'

FA-4: 5'-

GCTGGCGGCCGCTCATTGGTGCTTACAAGAACATGGCAACTCACAACC GTTCTCGTC-3'

After cloning of the PCR product using the pGEMT-easy cloning kit (Promega, Madison, WI, USA) and subsequent DNA sequencing, the proper gene was cloned into the Pichia vector pPIC9, using the Xhol and Noil restriction endonucleases (Invitrogen, Carlsbad, CA, USA).

P. pastoris GS115 (his4, see Cregg, J. et al., Mol. Cell. Biol.

5:3376-3385, 1985) was transformed by electroporation. Prior to

transformation, 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).

After selection of rFahsin producing P. pastoris transformants, fermentations were conducted in a 5 liter BioFlo 3000 fermentor (New Brunswick Scientific, Edison, NJ, USA) in minimal basal- salt medium supplemented with 0.2% (v/v) PTMi-trace salts (Invitrogen). Methanol fed- batch fermentations (Potter, K. et al., Protein Expr. Purif. 19:393-402, 2000) were performed and rFahsin was purified from the fermentation broth using overnight dialysis against 20 mM Tris-buffer, pH 8,0. 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

substantially pure (>90%) as determined by HPLC (C8 reverse phase) was obtained.

EXAMPLE 2

Fahsin mutants

Fahsin mutants with different amino acids at the PI position (i.e. the Leu residue after the 6th cysteine residue) 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.

EXAMPLE 3

Guamerin mutants

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). In Fig. 5 a summary is given of the inhibiting effects of guamerin and its mutants on 6 different proteinases.

For these assays 25 μΐ of mutant sample in PBS/0.2% Tween 20 or a dilution thereof in the same solvent was preincubated with 25 μΐ of proteinase for 60 minutes at 37°C. Then suitable substrate was added (50 μΐ) and the mixture was allowed to react for 1, hr (elastase, trypsine), 2 hrs (cathepsin G, chymotrypsin, thrombin) or 4 hrs (plasmin) at 37°C.

After incubation the absorbance was measured at 405 and 540 Angstrom and the difference of these values was plotted in Figs. 5A-F.

Claims

1. 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.

2. A mutant according to claim 1, wherein said antistasin type serine proteinase (NE) inhibitor is selected from fahsin, guamerin, piguamerin, hirustasin and bdellastasin and mutant homologs.

A mutant according to claim 1 or 2, wherein 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.

4. A mutant according to claim 3 having the sequence

DDNCGGKVCS KGQLCHDGHC ECTPIRCIIF CPNGFAVDEN GCELPCSCKH Q.

5. A mutant according to claim 1 or 2, wherein said antistasin type serine proteinase (NE) inhibitor is guamerin, piguamerin, hirustasin or bdellastasin or a mutant homolog and the residue after the 6th cysteine residue is a leucine residue.

6. A mutant according to claim 5 having the sequence

VDENAEDTHG LCGEKTCSPA QVCLNNECAC TAIRCLIFCP NGFKVDENGC EYPCTCA.

7. A mutant according to claim 1 or 2, wherein 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.

8. A mutant according to any of the previous claims, wherein said

mutant is produced through recombinant technology.

9. A nucleotide sequence encoding a mutant according to any of the previous claims.

10. An expression vector comprising a nucleotide sequence according to claim 9 operatively connected with a promoter and optionally other regulatory sequences.

11. A host cell comprising an expression vector according to claim 10.

12. A mutant according to any of claims 1 - 8 for use in therapy.

13. A mutant according to any of claims 1 - 8 for use 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.

14. A mutant according to claims 5 or 6 for use in prevention or

treatment of pancreatitis.

15. A method for treating emphysema, arthritis, gingivitis, periodontitis, pancreatitis and/or other inflammatory conditions that are associated with tissue destruction caused by the enzyme human neutrophil elastase, cathepsin G, chymotrypsin and/or Prot3, wherein a mutant protein according to any of claims 1— 8 is administered to a subject in need thereof.

16. A method according to claim 15, wherein said administration is via a controlled or sustained release formulation of said mutant protein.