The invention relates to LDTI (Leech Derived Tryptase Inhibitor) crystals, methods for their production and slow release formulations comprising these crystals.

Human mast cells contain in their secretory granules high amounts of tryptase, a trypsin-like serine proteinase in a tetrameric, proteoglycan-associated form. Although the physiological function of human tryptase still remains to be elucidated, pathogenic roles of this serine proteinase have been discussed in several diseases, such as allergy, asthma, rheumatoid arthritis, fibrosis, artherosclerosis, scleroderma, anaphylaxis and mastocytosis. Human tryptase is virtually unique among the serine proteinases as it is fully catalytically active both in plasma and in the extracellular space. Among serine proteinase inhibitors derived from non-human species or produced by recombinant DNA technology, the so-called ieech derived tryptase inhibitor (LDTI) is able to modulate tryptase activity (reviewed in Ascenzi et al., Moiec. Aspects Med. (1995) 16, 215-313).

LDTI has been isolated and characterized for the first time in WO-A-9503333 wherein several methods for the isolation and preparation are described. Although, being of great interest WO-A-9503333 failed to provide any evidence in respect to the crystallization of LDTI.

After the isolation of a new protein showing a pharmaceutically valuable activity the crystallization of this protein is always one of the major task. Crystals are an important source of structural data for the exploration of mechanism and structures responsible for the activity of a protein. The structural data can be used for designing antagonists, agonists or for molecular modeling. Another important application of crystallized proteins is their use in pharmaceutical preparations, e.g., for the topical application. Unfortunately, the crystalli¬ zation of proteins is, like their isolation and methods for their folding, a process that has to be developed for each protein on its own. Any prediction about suitable conditions is impossible.

For crystallization usually a large set of experiments, at various temperatures, with varying precipitant, pH, ionic strength, additive, concentration of protein, drop size and

crystallization techniques has to be investigated and it is totally unpredictable which out of the millions of possible combinations is suitable.

For example for the crystallization of LDTI a selection of possible variations is given below:

• precipitants: polyethylene glycol (PEG) 400, 1000, 4000, 6000, 8000, 10000, 20000; polyethylene glycol monomethyl ether (PEG MME) 550, 2000, 5000; sodium chloride, ethylene glycol, dioxane, isopropanol, sodium citrate, sodium acetate, sodium formate, lithium sulfate, glycerol, ammonium sulfate, ammonium phosphate, Na/K phosphate, polyethyleneimine, tert-butanol, Jeffamine M-600, imidazole, ethanol, 2-meth A- pentanediol (MPD), 1 ,6 hexandiol, 2-propanol, magnesium sulfate and magnesium formate;

• pH range: 4.0 to 9.0;

• salts and additives: calcium chloride, sodium citrate, magnesium chloride, ammonium acetate, ammonium sulfate, potassium phosphate, magnesium acetate, zinc acetate, calcium acetate, cetyl-trimethyl ammonium bromide, cobalt chloride, cadmium chloride, K/Na tartrate, ferric chloride, manganese chloride, Na/K phosphate, cesium chloride, zinc sulfate, cadmium sulfate, calcium chloride, nickel chloride and ammonium phosphate;

• protein concentration: from 10 mg/ml to 100 mg/ml.

Moreover, most proteins can be crystallized only in small amounts under extreme conditions and, hence, are unsuitable for any industrial application. Another problem in the industrial application of crystallized proteins is that they are often very sensitive to any change in the chemical or physical surroundings, e.g., due to a low density of the crystals. These crystals are unsuitable for pharmaceutical preparations.

Surprisingly, the crystals obtained according to the current invention show a high density, are very stable and can be produced easily in large amounts. Accordingly, these crystals can be handled without special care and - d in industrial applications.

Detailed description of the invention

The current invention concerns Ieech derived tryptase inhibitor (LDTI) in crystalline form.

LDTI is a protein with 46 amino acids (SEQ ID NO 1). The isolation and characterization of LDTI is described in detail, for example, in WO- A-9503333.

In a preferred embodiment of the invention the crystalline LDTI is belonging to the orthorhombic space group P2 2ι2 ₁ and in a more preferred form of the invention the crystalline LDTI has the unit cell dimensions of a = 30.60 A, b = 35.47 A, c = 82.33 A, α = β = γ = 90°.

The LDTI is crystallized in one crystal form that is surprisingly stable. This high stability of the inventive LDTI crystals is in contrast to many other protein crystals that are fragile and tend to dissolve easily. The stability of LDTI crystals is likely based on the surprisingly very dense packing.

Another embodiment of the invention concerns a pharmaceutical preparation comprising LDTI in crystalline form as described above. This pharmaceutical preparation may comprise additionally one or more pharmaceutically suitable carrier.

The application of LDTI as a suspension of microcrystals has some advantages over the application in solution because in the crystalline state unspecific absorption to the vessel surfaces are diminished. Additionally, a crystalline formulation of LDTI is more effective than a liquid preparation in minimizing protein denaturation and degradation. In fact, the intermolecular interactions in a crystal lattice are often similar to the intramolecular forces that specify the folded conformation. Crystallization conditions that favor crystal lattice formation therefore tend to stabilize the folded structure (Darby and Creighton, (1993) Protein Structure, In Focus, Oxford University Press, Walton Street, Oxford).

Another embodiment of the invention concerns the use of the pharmaceutical preparation described above in a method of treatment, preferably in a slow release formulation and more preferably for topical application.

The inventive pharmaceutical preparations are suitable, for example, in allergy, asthma, rheumatoid arthritis, fibrosis, artheroscierosis, scleroderma, anaphylaxis and mastocytosis and, especially in Psoriasis vulgaris.

The dosage depends essentially on the specific method of administration and on the purpose of the treatment or prophylaxis The size of the individual doses and the administration program can best be determined based on an individual assessment of the relevant case The methods required to determine the relevant factors are familiar to the expert.

A preparation for topical application can be in the form of an aqueous solution, lotion or jelly, an oily solution or suspension or a fatty or, particularly, emulsion ointment. A preparation in the form of an aqueous solution is obtained, e g. by suspending the substance according to the invention in an aqueous buffer solution of pH 4.0 to 6.5 and, if desired, adding one or more further substance thereto The concentration of the active ingredient is approximately 0.08 to approximately 1.5 mg, preferably 0.25 to 1.0 mg, in approximately 10 ml of a solution or 10 g of a jelly

An oily form of application for topical administration is obtained, e.g. by suspending the substance according to the invention or a therapeutically useful salt thereof in an oil, optionally with the addition of swelling agents, such as aluminum stearate, and/or surface- active agents (surfactants) the HLB value (hydrophilic- pophilic balance) of which is less than 10, such as fatty acid monoesters of polhydπc alcohols, e.g. glycerol monostearate, sorbitan monolaurate, sorbitan monostearate or sorbitan monooleate. A greasy ointment is obtained, e.g. by suspending the substance according to the invention or the salts in a spreadable greasy base, optionally with the addition of a surfactant having an HLB value of less than 10 An emulsion ointment is obtained by trituration of an aqueous solution of the substance according to the invention or the salts in a soft spreadable greasy base with the addition of a surfactant, the HLB value of whicn is less than 10 All of these forms of topical application can also contain a preserving agent The concentration of the active ingredient is approximately 0 08 to approximately 1.5 mg, preferably 0.25 to 1.9 mg in approximately 10 g of the matrix

Another embodiment of the invention concerns a method for the preparation of LDTI in crystalline form as described above

The crystalline form can be achieved using, for example, the 'nangmg drop' method or, preferably, batch crystallization wherein the vessel containing the LDTI to be crystallized and a vessel containing the crystallization buffer in excess are closed up together in a container The latter method is, e.g., suitable for large scale production of crystallized LDTI

Preferably, the buffer for the crystallization comprises PEG (polyethylene glycol), wherein PEG 4000, 6000 or PEG monomethylether 2000 are preferred A preferred concentration of PEG is in a range of 20 to 40 % (v/v)

A preferred range for LDTI concentration is from 15 to 60 mg/ml, more preferred from 20 to 40 mg/ml

The preferred pH of the buffer is from 4 7 to 6.0

Advantageously, the buffer comprises additionally sodium cacodylate or sodium acetate, wherein the preferred concentration is in a range from 50 to 100 mM

The crystallized LDTI as described above can be used also to identify the three-dimensional structure of the whole protein or at least of the areas responsible for activity Conventional methods for the identification of the three-dimensional structure are, for example, X-ray studies or NMR studies The data received with these or comparable methods may be used directly or indirectly for the identification of compounds showing the same activity or influencing the activity of this serine proteinase inhibitor, like low molecular weight compounds The identified compounds may be less susceptible to proteolysis and/or show a better specificity or activity than LDTI A commonly used method in this respect is, for example, computer aided drug design or molecular modeling

Accordingly, a further embodiment of the invention concerns a method for drug design, characterized in that crystallized LDTI as described above is used for determination of the structure; essentially for the identification or the design of compounds that inhibit serine proteases and especially for the identification or the design of compounds that inhibit tryptase Also embraced by the scope of the invention are compounds identified with this method and a data carrier comprising the structure data of LDTI

EXAMPLES

Example 1 : Isolation of LDTI

Recombinant LDTI is prepared as described in WO-A-9503333.

The supernatant of the fermentation process (3 I) is filtrated, concentrated to 1 I using a ultrafiltration unit and filtered again through a 0.2 μm filter. The pH is adjusted to 8.0 with sodium hydroxide and 10 g SP-Sephadex C25 equilibrated with 20 mM Na-phosphate pH 8.0 are added. After stirring at room temperature for 2 h, the resin is allowed to settle, then the supernatant is decanted and the residual slurry poured into a 2.6x40 cm column. After wash with 10 bed volumes of 20 mM Na-phosphate pH 8, the column is washed with additional 3 bed volumes of 25 mM NaCl in the same buffer. The protein is eluted with 40 mM NaCl in 20 mM Na-phosphate pH 8. The LDTI containing fraction is adjusted to pH 3 and concentrated to ca. 10 ml in a stirred ultrafiltration cell. Finally LDTI is purified with an HPLC column (HPLC Column: Vydac C18 (1 x25 cm); Gradient: 12 to 22 % acetonitrile in 0.2 % (v/v) aqueous trifluoro-acetic acid over 40 minutes).

Example 2: Protein preparation

Lyophilized recombinant LDTI (SEQ ID NO 1 ) in a salt form (trifluoroacetate salt) accounting to a final product of 90 %, w/w, is dissolved in 20 mM Tris-HCl, pH 8.0 to a final protein content of 20-40 mg/ml, as determined by UV-spectroscopy. A theoretical absorption coefficient of ε ₂₈ o=1810 M ^"1 cm ^"1 was calculated using molar extinction coefficients at 280 nm of 1480 M ¹ cm ^"1 for tyrosine (Mach et al., (1992) Anal. Biochem. 200, 74-80) and of 1 10 M cm ^"1 for cystine. The freshly prepared LDTI solution is centrifuged at 10,000 x g for 15 minutes and then filtered with 0.2 μm filters. Aliquots of the protein solution are immediately used for crystallization experiments or stored at -70°C.

Example 3: Crystallization

The "hanging drop" method (McPherson, (1982) Preparation and Analysis of Protein Crystals, John Wiley and Sons, Inc., New York) is used. The reservoir solution consists of 25-36 % PEG 6000 (v/v) and 0.2 M ammonium sulfate in 0.1 M sodium cacodylate buffer at pH 5.95. A 2 μl aliquot of a 40 mg/ml LDTI solution in 20 mM Tris-HCl at pH 8.0 is mixed with 2 μl of reservoir solution on a siliconized cover slip. The cover slip is inverted and

placed over the reservoir vessel which is filled with 600 μl of reservoir solution. Reservoir vessel and coverslip are sealed with pure vaseline. After 4 days at room temperature the first crystals (size 0.4 x 0.08 x 0.02 mm ³ ) are found. The crystals reach their maximum size after approximately 10 days. Similar size and type crystals are obtained in 33 out of 36 identical crystallization experiments.

Crystals can be obtained also under the following variations of the conditions. The pH can vary between 4.70 and 6.0. PEG 6000, 4000 and PEG monomethyl ether 2000 give the same crystal form. The protein concentration can vary between 20-40 mg/ml. The buffer can be varied from 50 to 100 mM sodium cacodylate and sodium acetate.

Example 4: Characterization

The crystals are rod-shaped and mechanically stable. They belong to the orthorhombic space group P2ι2 _! 2 _! with unit cell dimensions of a = 30.60 A, b = 35.47 A, c = 82.33 A, α = β = γ = 90° and diffract to a maximum resolution of 2.5 A. The asymmetric unit contains two LDTI molecules (Vm=2.3 A ³ /Da, solvent content= 43%). They can be manipulated by hand without cracking. In the reservoir buffer they are stable for at least one year. These crystals dissolve in 0.1 M sodium cacodylate buffer at pH 5.95 or in water.

Example 5: Batch crystallization

In order to allow the scaling up of the crystallization process from 80 μg to, e.g., 1 g or more of LDTI, the experimental set-up is switched from the "hanging drop" method to the "batch crystallization" method. Batch crystallization experiments are performed as follows:

Corning 50 mm/tissue culture dishes are filled with 3 ml of 25-32% PEG 6000 (v/v) and 0.2 M ammonium sulfate in 0.1 M sodium cacodylate buffer at pH 5.95. A 10 μl aliquot of this solution is mixed with 10 μl of the 40 mg/ml LDTI solution in 20 mM Tris-HCl at pH 8.0, inside an open round plastic miniwell (10 mm ø, 5 mm height). This last container is put to float in the culture dish. The whole system is sealed with vaseline and parafilm and let to equilibrate at room temperature. After 3 days, when the equilibration between the solution into the dish and the solution into the plastic miniwell, across the gaseous phase, has taken place, the first crystals appear. The batch crystallization experiment can be scaled up easily.

SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT:

(A) NAME: CIBA-GEIGY AG

(B) STREET: Klybeσkstr. 141

(C) CITY: Basel

(E) COUNTRY: Switzerland

(F) POSTAL CODE (ZIP): 4002

(G) TELEPHONE: +41 61 696 11 11 (H) TELEFAX: + 41 61 696 79 76 (I) TELEX: 962 991

(A) NAME: UCP Gen-Pharma

(B) STREET: Kraftstrasse 6

(C) CITY: Zuerich

(E) COUNTRY: Switzerland

(F) POSTAL CODE (ZIP): 8044

(ii) TITLE OF INVENTION: Novel Crystals

(iii) NUMBER OF SEQUENCES: 1

(iv) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk

(B) COMPUTER: IBM PC σonpatible

(C) OPERATING SYSTEM: PC-DOS/MS-DOS

(D) SOFTWARE: Patentin Release #1.0, Version #1.30 (EPO)

(2) INFORMATION FOR SEQ ID NO: 1:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 46 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(ix) FEATURE:

(A) NAME/KEY: Protein

(B) LOCATION:!..46

(D) OTHER INFORMATION:/label= LDTI

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:

Lys Lys Val Cys Ala Cys Pro Lys lie Leu Lys Pro Val Cys Gly Ser 1 5 10 15

Asp Gly Arg Thr Tyr Ala Asn Ser Cys lie Ala Arg Cys Asn Gly Val 20 25 30

Ser lie Lys Ser Glu Gly Ser Cys Pro Thr Gly lie Leu Asn

35 40 45