HK1145697A - Protease variants for pharmaceutical use - Google Patents

Description

Protease variants for pharmaceutical use

Relating to sequence listing

The present application contains a sequence listing in computer readable form. The computer readable form is incorporated herein by reference.

Technical Field

The invention relates to a protease which is similar to the protease shown in SEQ ID NO: 1, and which is at least 90% identical to amino acids 1-188 of SEQ ID NO: 1 comprises at least one substitution selected from the following substitutions: A1T; I3V; g12; R14I; T22A; N23D; G34A; r38; T41A; T44K; n47; G48D; E53K; Q54L, D; T68A, R, S; S69T; L73P; V88A; S99P; P124L; e125; M131V; T151I; r165; and T166A. SEQ ID NO: 1 is a wild-type protease from a Nocardiopsis sp.

The invention also relates to the pharmaceutical use of these proteases, optionally in combination with lipases and/or amylases. Other examples of medical indications are: treatment of digestive disorders, Pancreatic Exocrine Insufficiency (PEI), pancreatitis, cystic fibrosis, diabetes type I, and/or diabetes type II.

Background

Several commercial drugs in the form of pancreatin supplements are known for the treatment of exocrine pancreatic insufficiency. The active ingredients of these products are digestive enzymes (mainly amylases, lipases and proteases) which are normally produced in the pancreas and secreted into the upper small intestine (duodenum). The enzymes used in such drugs are derived from bovine or porcine pancreas.

WO 2005/115445 describes SEQ ID NO: 1 and related proteases for use, e.g. in the treatment of PEI.

WO2006/136159 depicts SEQ ID NO: 2and related lipases for use in, e.g., the treatment of PEI.

WO 2006/136161 depicts SEQ ID NO: 3.4 and 5 and related amylases, for example, for the treatment of PEI.

Proteases derived from Nocardiopsis species (SEQ ID NO: 1), their preparation and their various industrial applications are described in WO 88/03947 and WO 01/58276. Related proteases are described in WO2004/111220, WO 2004/111222, WO 2004/111223, WO 2004/111221, WO2005/035747, WO 2004/111219, WO 2005/123911 and JP 2003284571-A (GENESEBP: ADF 43564).

The present invention provides novel proteases with improved properties, e.g., improved apparent protein digestibility in vivo, improved pH-ratio (pH5.6/pH8), and/or reduced toxicity.

Summary of The Invention

The invention relates to a protease which is similar to the protease shown in SEQ ID NO: 1, and which is at least 90% identical to amino acids 1-188 of SEQ ID NO: 1 comprises at least one substitution compared to amino acids 1-188 selected from the following substitutions: A1T; I3V; g12; R14I; T22A; N23D; G34A; r38; T41A; T44K; n47; G48D; E53K; Q54L, D; T68A, R, S; S69T; L73P; V88A; S99P; P124L; e125; M131V; T151I; r165; and T166A.

Furthermore, the invention relates to such proteases (optionally in combination with lipases and/or amylases) for use as medicaments.

Still further, the present invention relates to the use of such protease (optionally in combination with lipase and/or amylase) for the preparation of a medicament for the treatment of digestive disorders, pancreatic exocrine insufficiency, pancreatitis, cystic fibrosis, diabetes type I, and/or diabetes type II.

The invention also relates to such proteases (optionally in combination with lipases and/or amylases) for use in the treatment of digestive disorders, pancreatic exocrine insufficiency, pancreatitis, cystic fibrosis, diabetes type I, and/or diabetes type II.

Furthermore, the present invention relates to a pharmaceutical composition comprising such protease (optionally in combination with lipase and/or amylase) together with at least one pharmaceutically acceptable auxiliary material.

Finally, the present invention relates to a method for the treatment of digestive disorders, pancreatic exocrine insufficiency, pancreatitis, cystic fibrosis, diabetes type I, and/or diabetes type II, by administering a therapeutically effective amount of such protease (optionally in combination with lipase and/or amylase).

Detailed Description

Enzyme

Term "Protease enzyme"is defined herein as a polypeptide having protease activity. Protease refers to an enzyme that hydrolyzes peptide bonds. It includes any enzyme belonging to the EC 3.4 enzyme group (including each of its 13 subclasses, which are hereinafter referred to as "belonging to EC 3.4. -group"). EC numbering refers to the enzyme nomenclature 1992 from NC-IUBMB, academic Press, San Diego, California (including supplements 1-5 published in Eur. J. biochem.1994, 223, 1-5; Eur. J. biochem.1995, 232, 1-6; Eur. J. biochem.1996, 237, 1-5; Eur. J. biochem.1997, 250, 1-6; and Eur. J. biochem.1999, 264, 610-. Naming for regular replenishment and updating; see, for example, the world Wide Web at http:// www.chem.qmul.ac.uk/iumbb/enzyme.

In a specific embodiment, the protease of the invention is selected from the group consisting of proteases derived from strains of the genus nocardiopsis.

The correlation between two amino acid sequences is described by the parameter "identity".

For the purposes of the present invention, the Needle program from EMBOSS package (http:// EMBOSS. org) version 2.8.0 was used to align two amino acid sequences. The Needle program performs the global alignment algorithm described in Needleman, S.B. and Wunsch, C.D. (1970) J.Mol.biol.48, 443-. The substitution matrix used is BLOSUM62, the gap opening penalty is 10, and the gap extension penalty is 0.5.

The degree of identity between an amino acid sequence of the invention ("invention sequence"; e.g., amino acids 1-188 of SEQ ID NO: 6) and a different amino acid sequence ("foreign sequence"; e.g., amino acids 1-188 of SEQ ID NO: 1) is calculated as the number of exact matches in this alignment of the two sequences divided by the shortest of the length of the "invention sequence" or the length of the "foreign sequence". Results are expressed as percent identity.

An exact match occurs when the "invention sequence" and the "foreign sequence" have identical amino acid residues in the same position of overlap (in the alignment example below, this is denoted by "|"). The sequence length is the number of amino acid residues in the sequence (e.g., the length of SEQ ID NO: 6 is 188).

In the purely hypothetical alignment example below, the overlap is the amino acid sequence "HTWGER-NL" of sequence 1; or the amino acid sequence "HGWGEDANL" of sequence 2. In this example, the notch is indicated by "-".

Hypothetical alignment example:

sequence 1: ACMSHTWGER-NL

| ||| ||

Sequence 2: HGWGEDANLAMNPS

Thus, the percentage of sequence 1 identity to sequence 2 was 6/12-0.5, corresponding to 50%.

In a specific embodiment, the amino acid sequence of the polypeptide is determined as follows with or against, for example, seq id NO: 1, i.e., i) aligning two amino acid sequences using the Needle program and the BLOSUM62 substitution matrix, gap opening penalty of 10, and gap extension penalty of 0.5; ii) calculating the number of exact matches in the alignment; iii) dividing the number of exact matches by the shortest length of the two amino acid sequences, and iv) converting the division of iii) into a percentage. Ratios were calculated in a similar manner, or compared to other sequences of the invention such as SEQ ID NO: 2, amino acids 1-269.

In a further embodiment, the protease of the invention is identical to SEQ ID NO: 1 has at least 90%, at least 91%, or at least 92%; a degree of identity of at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%.

In the present invention, a specific numbering of amino acid residue positions is used. The numbering system is derived from the amino acid sequence alignment of the amino acid sequence in SEQ ID NO: 1, or a pharmaceutically acceptable salt thereof.

For sequences corresponding to SEQ ID NO: 1, using the following nomenclature: initial amino acids (in SEQ ID NO: 1), positions (in alignment), substituted amino acids (in another protease). Thus, the use of the peptide in, for example, SEQ ID NO: 6 to SEQ ID NO: the substitution at position 125 of glutamic acid (E) in 1 (by reference to SEQ ID NO: 1) is referred to as "E125D". Multiple mutations are separated by a plus ("+"), e.g., "T44K + S99P" indicating the substitution of threonine (T) with lysine (K), and serine (S) with proline (P), respectively, mutations at positions 44 and 99 (as in, e.g., SEQ ID NO: 8). Furthermore, expressions such as G12D, N, H mean that the glycine (G) at position 12 is exchanged for aspartic acid (D), asparagine (N) or histidine (H).

In SEQ ID NO: the substitution of glycine to any other amino acid in position 12 of 1 is referred to as "G12".

In SEQ ID NO: the substitution of arginine to any other amino acid in position 38 of 1 is referred to as "R38".

In SEQ ID NO: the substitution of asparagine to any other amino acid in position 47 of 1 is referred to as "N47".

In SEQ ID NO: the substitution of glutamic acid to any other amino acid in position 125 of 1 is referred to as "E125".

In SEQ ID NO: 1 to any other amino acid at position 165 is referred to as "R165".

In a further embodiment, the protease of the invention is acid-stable, which means that in the case of the protease corresponding to A₂₈₀The protease activity of the pure protease in the dilution of 1.0 and after incubation for 2 hours at 37 ℃ in a buffer of 100mM succinic acid, 100mM HEPES, 100mM CHES, 100mM CABS, 1mM CaCl was at least 40% (or at least 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or at least 97%) of the reference activity as measured using the assay described in example 2C of WO01/58276 (substrate: Suc-AAPF-pNA, pH 9.0, 25 ℃)₂、150mM KCl、0.01％X-100 and pH 3.5. The term reference activity means that the same protease corresponds to A in pure form₂₈₀Protease activity after 2 hours incubation at 5 ℃ in the following buffer, where the activity is determined as described above, in a dilution of 1.0: 100mM succinic acid, 100mM HEPES, 100mM CHES, 100mM CABS, 1mM CaCl₂、150mMKCl、0.01％X-100 and pH 9.0. Term A₂₈₀By 1.0 is meant such a concentration (dilution) of the pure protease that gives an absorbance of 1.0 at 280nm in a 1cm pathlength cuvette relative to the buffer blank. The term pure protease refers to A₂₈₀/A₂₆₀A ratio higher than or equal to 1.70 (see example 2E of WO 01/58276) and by sweepingCoomassie stained SDS-PAGE gels were traced for at least 95% of the samples in the band whose scanning intensity corresponded to the protease (see example 2A of WO 01/58276).

Preferred proteases of the invention:

and SEQ ID NO: 1, and has at least 90% identity to amino acids 1-188 of SEQ id no: 1 compared to a protease comprising at least one substitution selected from the following substitutions: A1T; I3V; g12; R14I; T22A; N23D; G34A; r38; T41A; T44K; n47; G48D; E53K; Q54L, D; T68A, R, S; S69T; L73P; V88A; S99P; P124L; e125; M131V; T151I; r165; and T166A.

And SEQ ID NO: 1, and has at least 90% identity to amino acids 1-188 of SEQ id no: 1 compared to a protease comprising at least one substitution selected from the following substitutions: G12D, N, H; R38T; N47H, T, S; E125D and R165S, H, G, T.

And SEQ ID NO: 1, and has at least 90% identity to amino acids 1-188 of SEQ id no: 1 comprises at least one substitution compared to amino acids 1-188: G12D, N, H; in particular the protease of G12D.

And SEQ ID NO: 1, and has at least 90% identity to amino acids 1-188 of SEQ id no: 1 comprises at least one substitution compared to amino acids 1-188: N47H, T, S; in particular the protease of N47H.

And SEQ ID NO: 1, and has at least 90% identity to amino acids 1-188 of SEQ id no: 1 comprises at least one substitution compared to amino acids 1-188: R165S, H, G, T; in particular the protease of N165H.

And SEQ ID NO: 1, and has at least 90% identity to amino acids 1-188 of SEQ id no: 1 compared to a protease comprising at least one substitution selected from the following substitutions or combinations of substitutions: G12D; and (N47H + G48D).

And SEQ ID NO: 1, and has at least 90% identity to amino acids 1-188 of SEQ id no: 1 compared to a protease comprising at least one substitution selected from the group consisting of combinations of substitutions: (T41A + T68R + V88A); (G12N + T22A + N23D + N47T + R165H); and (R14I + R38T + T151I).

And SEQ ID NO: 1, and has at least 90% identity to amino acids 1-188 of SEQ id no: 1 compared to a protease comprising at least one substitution selected from the following substitutions or combinations of substitutions: R38T, (T44K + S99P), S69T, (S69T + E125D), E125D, and R165S.

And SEQ ID NO: 1, and has at least 90% identity to amino acids 1-188 of SEQ id no: 1 compared to a protease comprising at least one of the following substitutions or combinations of substitutions: R38T; T44K and S99P; S69T; S69T and E125D; E125D; and R165S.

A protease that hybridizes to SEQ ID NO: 1 comprises at least one of the following substitutions compared to amino acids 1-188: A1T; I3V; g12; R14I; T22A; N23D; G34A; r38; T41A; T44K; n47; G48D; E53K; Q54L, D; T68A, R, S; S69T; L73P; V88A; S99P; P124L; e125; M131V; T151I; r165; and T166A; and furthermore it is selected from the group:

(a) comprises, preferably has a sequence identical to SEQ ID NO: 1 amino acids 1-188 of 1 have an amino acid sequence of at least 90% identity;

(b) a protease encoded by a polynucleotide that hybridizes under very low (preferably low, medium-high, or most preferably very high) stringency conditions with (i) a mature polypeptide coding sequence of seq id no: (i) SEQ ID NO: 1 (nucleotide 900-1463 of SEQ ID NO: 1 of WO2005/035747, incorporated herein by reference), or (ii) the full-length complementary strand of (i); and

(c) SEQ ID NO: 1 further comprises one or more (e.g., several) amino acid substitutions, deletions and/or insertions (preferably substitutions, deletions and/or insertions of a conservative nature).

Very low to very high stringency conditions are defined as an optimal 12 to 24 hour prehybridization and hybridization at 42 ℃ in 5X SSPE, 0.3% SDS, 200 micrograms/ml sheared and denatured salmon sperm DNA, and 25% formamide for very low and low stringency, 35% formamide for medium and medium-high stringency, or 50% formamide for high and very high stringency following standard Southern blotting procedures. The carrier material is finally washed three times each for 15 minutes using 2X SSC, 0.2% SDS, preferably at 45 ℃ (very low stringency), more preferably at 50 ℃ (low stringency), more preferably at 55 ℃ (medium stringency), more preferably at 60 ℃ (medium-high stringency), even more preferably at 65 ℃ (high stringency), and most preferably at 70 ℃ (very high stringency).

Amino acid changes of a conservative nature do not significantly affect the folding and/or activity of the protein, and include small deletions, typically of 1 to about 30 amino acids; small amino-or carboxy-terminal extensions, such as an amino-terminal methionine residue; a small linker peptide of up to about 20-25 residues; or a small extension that facilitates purification by altering the net charge or another function, such as a poly-histidine tract (tract), an antigenic epitope, or a binding domain.

Preferably, the total number of alterations in the variant is 18, 17 or 16. More preferably, the total number of alterations is 15, even more preferably 14, even more preferably 13, even more preferably 12, even more preferably 11, even more preferably 10, even more preferably 9, even more preferably 8, even more preferably 7, even more preferably 6, even more preferably 5, even more preferably 4, even more preferably 3, even more preferably 2, and most preferably 1 amino acid.

A variant produced by shuffling one or more polynucleotides encoding one or more homologous parent proteases, wherein the variant comprises alterations at one or more positions corresponding to one or more positions in the parent protease selected from the group consisting of: 38, 44, 69, 99, 125 and 165, wherein the alterations independently correspond to substitutions of the amino acid occupying that position, and wherein the variant has protease activity.

The term "parent" protease refers to a protease that has been modified, e.g., substituted, inserted, deleted and/or truncated, to produce the enzyme variants of the invention. The term also refers to polypeptides that are compared and aligned with the variants. The parent may be a naturally occurring (wild-type) polypeptide, or it may even be a variant thereof prepared by any suitable means. For example, a parent protein may be a variant of a naturally occurring polypeptide that has been modified or altered in amino acid sequence. The parent may also be an allelic variant of a polypeptide encoded by any of two or more alternative forms of a gene occupying the same chromosomal locus.

The term "shuffling" refers to the recombination of nucleotide sequences between two or more homologous nucleotide sequences, resulting in a recombinant nucleotide sequence (i.e., a nucleotide sequence that has undergone a shuffling cycle) having many altered nucleotides compared to the starting nucleotide sequence.

Preferably, the proteases and protease variants according to the respective above examples and other examples are used in the combinations, uses, compositions and methods of the invention as set out herein and in the claims.

Protein digestibility in vivo, pH-ratio (pH5.6/pH8) and/or reduced toxicity.

The proteases and protease variants of the invention exhibit improved properties, such as improved apparent protein digestibility in vivo, improved/altered pH-ratio (pH5.6/pH8) and/or reduced toxicity.

Can be in females with induced PEIApparent protein digestibility in vivo was determined in mini-pigs (Ellegaard). Pigs were fed two meals a day containing 21.3% protein, 51.9% starch, 2.6% fat, which preferably consisted as described in example 4. The pigs are allowed free access to water and preferably, according to a 12: 12h light-dark period captive breeding in cagesIn (1). First, the pigs were fed with 1 liter of water and 0.625g of Cr₂O₃(markers) a single 250g test meal was mixed with varying amounts of the amino acid sequence of SEQ id no: 1 (0mg, 20mg, 50mg and 120mg enzyme protein/meal). For this test, the dosages of the protease of the invention were 20mg, 50mg and 120mg per meal. After the first appearance of the dietary marker in the ileum (green chyme), the ileal chyme was collected on ice for a total of 8 hours and stored at-20 ℃ before analysis. At least one day of flushing (washout) is allowed between separate measurements. The frozen ileal chyme samples were freeze-dried, ground and analyzed for Dry Matter (DM) and crude protein. DM was assessed gravimetrically after freeze drying followed by incubation at 103 ℃ for 8 hours. Crude protein was calculated as nitrogen (N) multiplied by a factor of 6.25. The nitrogen content is determined by combustion, preferably by the Dumas combustion method, more preferably using a "Vario MAX CNS" elemental analyzer. Mixing Cr₂O₃Oxidized to chromate and the chromium content was calculated via extinction at 365 nm. The apparent precoceal protein digestibility was calculated according to the formula shown in example 4, where Cr₂O₃And protein expressed as g/100g dry matter.

For the protease of the invention, preferably the digestibility (e.g. apparent pre-caecal protein digestibility) is improved in at least one of the doses 20, 50 and 120mg enzyme protein/meal compared to the digestibility of a reference protease. Preferably, the improvement is at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, or at least 10%. More preferably, the improvement is at least 11%, at least 12%, at least 13%, at least 14%, at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, or at least 20%. Even more preferably, the improvement is at least 21%, at least 22%, at least 23%, at least 24%, at least 25%, or at least 26%.

In addition, the amount of protease (mg) required to achieve 50% and 60% protein digestibility (% CNA), respectively, can be extrapolated from the individual regression curves (apparent pre-cecal protein digestibility versus enzyme dosage). The improvement factors 50 and 60(IF50 and IF60) were calculated by dividing the amount of reference protease (mg) required to achieve 50% and 60% protein digestibility (% CNA) by the amount of variant protease (mg) required to achieve 50% and 60% protein digestibility (% CNA), respectively. Thus, for the reference protease, both IF50 and IF60 were 1.00.

For the protease of the invention, preferably the IF50 and/or IF60 values are at least 1.05, at least 1.10, at least 1.15, at least 1.20, at least 1.25, at least 1.30, at least 1.35, at least 1.40, at least 1.45 or at least 1.50.

See example 4 for more details.

The pH-ratio is determined using casein as substrate, more specifically using casein derivatives labelled with a suitable red fluorescent dye, which is preferably pH insensitive (e.g. in the range of pH 5.6-8.0). Fluorescence enhancement is proportional to protease activity. The preferred pH8.0 assay buffer is 100mM Tris/base and the preferred pH5.6 assay buffer can be prepared by mixing 25ml of 0.2M succinic acid with 37.5ml of 0.2M NaOH. Incubation is carried out at a suitable temperature (e.g. room temperature, e.g. 22 ℃) for a suitable period of time (e.g. 60 minutes), the preferred fluorescent dye is BODIPYTR-X, in which case the resulting fluorescent peptide can be determined using a standard fluorescein filter (excitation 590nm, emission 635 nm). For the protease of the invention and SEQ ID NO: 1 of the reference protease the ratio of pH5.6 activity to pH8.0 activity was determined and the ratio of the protease of the invention to the ratio of the reference protease was calculated. Preferred proteases of the invention have a ratio of said ratio relative to the ratio of a reference protease of 1 or more, preferably at least 1.05, at least 1.10, at least 1.15, at least 1.20, or at least 1.25. See example 3 for more details.

Toxicity can be determined as in vitro toxicity to human colon adenocarcinoma cell lines such as HT-29 cells (e.g., DSMZ No. ACC 299). Cells are cultured in McCoy's 5A medium (e.g., from Cambrex) supplemented with 10% FBS (e.g., from Sigma, Cat. catalog number F-6178), preferably at a density of 4.104 cells/well/200. mu.l in 96-well culture plates. After cells were acclimated to each well for 24 hours, protease was added in triplicate at 9 different concentrations (w/vol enzyme protein) at 2-fold dilutions in serum-free medium (e.g., DMEM: F12, Invitrogen) supplemented with 0.5g/l Probumin (Millipore), 1% insulin/transferrin/selenium supplement (e.g., from Invitrogen), and 1% penicillin and streptomycin (e.g., from Invitrogen), and incubated for an additional 24 hours. Viability is measured by the metabolic capacity of the cells, which is measured by using Alamar Blue (e.g. from Invitrogen).

The maximum metabolic activity (100%) was determined as the metabolic activity of a control (no protease added). For a given protease to be tested, the toxicity ratio is calculated as the concentration at which 50% of the maximum metabolic activity is obtained for this protease divided by the concentration at which 50% of the maximum metabolic activity is obtained for the reference protease (SEQ ID NO: 1). For the protease of the invention, the toxicity ratio is preferably at least 1.1, preferably at least 1.2, preferably at least 1.3, preferably at least 1.4, preferably at least 1.5, preferably at least 1.6, preferably at least 1.7, preferably at least 1.8, preferably at least 1.9, most preferably at least 2.0. The toxicity ratio is explained in example 6. Good correlation has been found for in vivo and in vitro toxicity results.

In a further particular embodiment, optionally, additional proteases may be used, for example mammalian proteases, for example in the form of a porcine-derived pancreatic extract, or microbial proteases, for example of bacterial or fungal origin, such as Bacillus sp. In particular, the protease may be derived from a strain of Aspergillus, such as Aspergillus oryzae (Aspergillus oryzae) or Aspergillus melleus (Aspergillus melleus), in particular Prozyme6TM (neutral, alkaline protease EC 3.4.21.63), which is a product commercially available from Amano Pharmaceuticals, Japan.

Cloning and introduction of mutations in genes

Standard methods for cloning genes and introducing mutations (random and/or site-directed) can be used to obtain enzymes and enzyme variants, such as the protease variants of the invention. Primers designed to include restriction sites can be used to amplify the gene of interest (e.g., SEQ ID NO: 1 of WO2005/035747, which is the gene encoding SEQ ID NO: 1 herein). For a further description of suitable techniques, reference is made to Sambrook et al (1989), Molecular cloning: a laboratory manual, Cold Spring Harbor lab, Cold Spring Harbor, NY; ausubel, f.m. et al (eds) "Current protocols in Molecular Biology". john wiley and Sons, 1995; harwood, C.R. and Cutting, S.M. (eds.) "Molecular biology methods for Bacillus". John Wiley and Sons, 1990, and WO 96/34946.

After digesting the gene of interest and the above plasmid with the relevant restriction endonuclease for cloning purposes, the gene of interest and the plasmid can be ligated in a ligation method involving a ligase. Following the ligase reaction, the ligation mixture can be used to transform E.coli (E.coli) cells as described in Ausubel, F.M. et al. Transformed E.coli cells can be propagated in liquid media or on solid agar plates, plasmids can be rescued from the transformed cells, and used to transform Bacillus subtilis cells. Suitable competent Bacillus cells, such as MB1510, which is a 168-derivative (e.g.obtainable from BGSC under accession number 1A1168trpC 2) may be transformed, as described in WO 03/095658.

Transformation of Bacillus can be performed using integration cassettes carried by E.coli plasmids used for library construction. The process is described in detail in WO 03/095658. Alternatively, in vitro amplified PCR-SOE products (Melnikov and Youngman, Nucleic Acid Research 27, 1056) may be used.

The plasmid vector may contain the following elements:

i) signal peptide coding regions (e.g., genes obtained from Bacillus NCIB 11837 maltogenic amylase, Bacillus stearothermophilus alpha-amylase, Bacillus licheniformis subtilisin, Bacillus licheniformis alpha-amylase, Bacillus stearothermophilus neutral protease (nprT, nprS, nprM) and Bacillus subtilis prsA), followed by SEQ ID NO: 1 (pro-domain) (residue 405-899 of SEQ ID NO: 1 of WO 2005/035747) and the mature protease variant gene. This sequence may be preceded by and operably connected to:

ii) a DNA sequence comprising a stable segment of mRNA (e.g.derived from the CryIIIa gene as shown in WO 1999/043835);

iii) marker genes (e.g., chloramphenicol resistance genes); and

iv) genomic DNA from Bacillus subtilis as upstream and downstream 5 'and 3' flanking segments, respectively, of the polynucleotide, to achieve genomic integration by homologous recombination between the flanking segments and the Bacillus genome.

The protease of the invention may be reacted withLipase enzymeAre used in combination.

In this context, lipase refers to a carboxylic ester hydrolase, EC3.1.1, which includes activities such as EC3.1.1.3 triacylglycerol lipase, EC 3.1.1.4 phospholipase a1, EC 3.1.1.5 lysophospholipase, EC3.1.1.26 galactolipase, EC 3.1.1.32 phospholipase a1, EC 3.1.1.73 ferulic acid esterase. In a specific embodiment, the lipase is an EC3.1.1.3 triacylglycerol lipase.

In particular embodiments, the lipase is a mammalian lipase, for example in the form of a porcine-derived pancreatic extract, or a microbial lipase, for example derived from a bacterial or fungal strain, such as bacillus, pseudomonas, aspergillus or rhizopus. In particular, the lipase may be derived from a strain of Rhizopus, such as Rhizopus javanicus (Rhizopus javanicus), Rhizopus oryzae (Rhizopus oryzae) or Rhizopus delemar, e.g., the product lipase DAmano 2000 commercially available from Amano Pharmaceuticals, Japan^TM(also known as lipase D2^TM)。

In a further embodiment, the lipase used in the present invention is a recombinantly produced microbial lipase, e.g.derived from a fungus such as Humicola (Humicola) or Rhizomucor (Rhizomucor), a yeast such as Candida (Candida), or a bacterium such as Pseudomonas. In a preferred embodiment, the lipase is derived from a strain of Humicola lanuginosa (Humicola lanuginose) or Rhizomucor miehei (Rhizomucor miehei).

Humicola lanuginosa (Thermomyces lanuginosus) lipase is described in EP 305216, while specific lipase variants are described in, for example, WO 92/05249, WO92/19726, WO 94/25577, WO 95/22615, WO 97/04079, WO 97/07202, WO99/42566, WO 00/32758, WO 00/60063, WO 01/83770, WO 02/055679, WO 02/066622 and WO 2006/136159. Further examples of fungal lipases are cutinases from Humicola insolens, described in EP 785994, and phospholipases from Fusarium oxysporum, described in EP 869167. Examples of yeast lipases are lipases A and B from Candida Antarctica (Candida Antarctica), lipase A being described in EP652945 and lipase B being described, for example, by Uppenberg et al in Structure, 2(1994), 293. An example of a bacterial lipase is a lipase derived from Pseudomonas cepacia (Pseudomonas cepacia), which is described in EP 214761.

In a preferred embodiment, the lipase is reacted with the amino acid sequence of seq id NO: 2 amino acids 1-269 is at least 70% identical. In other preferred embodiments, the peptide has a sequence identical to SEQ ID NO: 2 is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%. Lipases comprising (preferably having) the following amino acid sequence are preferred: (i) SEQ ID NO: 2, (ii) amino acids +1 to +269 of SEQ ID NO: 2, (iii) amino acids-5 to +269 of SEQ ID NO: 2 amino acid-4 to + 269; (iv) SEQ ID NO: 2 amino acid-3 to + 269; (v) SEQ ID NO: 2 amino acid-2 to + 269; (vi) SEQ ID NO: 2, (vii) amino acids-1 to +269 of SEQ ID NO: 2 from amino acid +2 to +269 of (1), and (viii) any mixture of two or more lipases of (i) - (vii). In a particular embodiment, the lipase is selected from the group consisting of lipases of (i), (ii) and any mixtures of (i) and (ii). (i) Preferred mixtures of (i) and (ii) comprise at least 5%, preferably at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or at least 95% of lipase (i), the percentage being determined by N-terminal sequencing using the Edman method, as described in example 5 of WO 2006/136159. Other preferred mixtures are: (a) a composition comprising 35-75%, preferably 40-70%, more preferably 45-65% of lipase (ii); (b) a composition comprising 20-60%, preferably 25-55%, more preferably 30-50%, most preferably 35-47% of lipase (i); (c) a composition comprising up to 30%, preferably up to 25%, more preferably up to 20%, most preferably up to 16% of lipase (vii); and (d) any combination of (a), (b), and/or (c), such as a composition comprising 45-65% lipase (ii), 35-47% lipase (i), and up to 16% lipase (vii).

In a still further preferred embodiment, the lipase (e.g., mammalian pancreatic lipase) is a1, 3-position specific lipase.

The protease of the invention (with or without lipase as described above) may also be used in combination withAmylaseAre used in combination.

In this context, amylases are enzymes that catalyze the internal hydrolysis of starch and other linear and branched oligo-and polysaccharides. The amylose portion of starch is rich in 1, 4-alpha-glucosidic bonds, whereas the amylopectin portion is more branched and contains not only 1, 4-alpha-but also 1, 6-alpha-glucosidic bonds. In a specific embodiment, the amylase is an enzyme belonging to the EC group 3.2.1.1.

In particular embodiments, the amylase is a mammalian amylase, for example in the form of a porcine-derived pancreatic extract, or a microbial amylase, for example derived from a bacterial or fungal strain, such as bacillus, pseudomonas, aspergillus, or rhizopus.

In particular, the amylase may be derived from Aspergillus, such as Aspergillus niger, riceAspergillus or Aspergillus melleus strains, e.g., the Aspergillus oryzae-derived product amylase A1 commercially available from Amano Pharmaceuticals, Japan^TMOr an Aspergillus melleus-derived amylase EC commercially available from Extract-Chemie, Germany^TMAny one of them.

Other examples of fungal amylases are aspergillus niger amylase (SWISSPROT P56271), which is also described in example 3 of WO 89/01969, and aspergillus oryzae amylase. Examples of variants of Aspergillus oryzae amylase are described in WO 01/34784.

Alpha-amylases derived from Bacillus licheniformis are examples of bacterial alpha-amylases. This amylase is described in e.g. WO99/19467, together with other homologous bacterial alpha-amylases derived from e.g. bacillus amyloliquefaciens (bacillus amyloliquefaciens) and bacillus stearothermophilus, and variants thereof. Additional examples of amylase variants are those described in U.S. patent nos. 4,933,279; EP 722490, EP 904360, and WO 2006/136161.

Preferred amylases are (i) those comprising SEQ ID NO: 5 (e.g., amino acids 1-481, 1-484, or 1-486 thereof), SEQ ID NO: 3 and/or amino acids 1-481 of SEQ ID NO: 4 amino acids 1-483. In a preferred embodiment, the amylase is an amylase having or comprising an amino acid sequence which is at least 70% identical to any one of: (i) SEQ ID NO: 5, (ii) amino acids 1-513 of SEQ ID NO: 3, and/or (iii) amino acids 1-481 of SEQ ID NO: 4, amino acids 1-483. SEQ ID NO: the amylases of 3-5 can be prepared, for example, as described in WO 2006/136161. In other preferred embodiments of any of (i), (ii), or (iii), the degree of identity is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%.

In general, the proteases, lipases and amylases (hereinafter "enzymes") used according to the invention may be automotives, in particular mammalian (e.g. human or porcine enzymes); natural or wild-type enzymes obtained from plants, or from microorganisms, but also any mutants, variants, fragments, etc., which exhibit the desired enzymatic activity, as well as synthetases, such as shuffled enzymes (shuffled enzymes), and consensus enzymes (consensus enzymes).

In a particular embodiment, the enzyme is a hypoallergenic variant designed to elicit a reduced immune response upon exposure to animals (including humans). The term immune response is to be understood as any reaction produced by the immune system of an animal exposed to the enzyme. One type of immune response is an allergic response, resulting in elevated levels of IgE in exposed animals. Hypoallergenic variants can be prepared using techniques known in the art. For example, enzymes may be conjugated to polymeric moieties (moieties) that shield (shield) portions or epitopes of enzymes involved in immune responses. Conjugation to a polymer may involve in vitro chemical coupling of the polymer to an enzyme, for example as described in WO 96/17929, WO 98/30682, WO 98/35026 and/or WO 99/00489. Additionally or alternatively, conjugation may involve in vivo coupling of a polymer to an enzyme. Such conjugation can be achieved by genetically engineering the nucleotide sequence encoding the enzyme, inserting a consensus sequence encoding an additional glycosylation site into the enzyme, and expressing the enzyme in a host capable of glycosylating the enzyme, see, e.g., WO 00/26354. Another way to provide hypoallergenic variants is to genetically modify the nucleotide sequence encoding the enzyme, thereby causing the enzyme to self-oligomerize, achieving that the enzyme monomer can shield epitopes of other enzyme monomers, thereby reducing the antigenicity of the oligomer. Such products and their preparation are described, for example, in WO 96/16177. Epitopes involved in immune responses can be identified by a variety of methods, such as the phage display methods described in WO 00/26230 and WO 01/83559, or the random methods described in EP 561907. Once an epitope has been identified, its amino acid sequence can be altered by known gene manipulation techniques such as site-directed mutagenesis (see, e.g., WO 00/26230, WO00/26354 and/or WO 00/22103) to produce altered immunological properties of the enzyme, and/or conjugation of the polymer can be accomplished sufficiently close to the epitope for the polymer to shield the epitope.

In particular embodiments, the protease, lipase and/or amylase are (i) stable at a pH of 4-8, preferably also at a pH of 3-4, more preferably at a pH of 3.5; (ii) is active at a pH of 4-9, preferably 4-8, more preferably at pH 6.5; (iii) are stable against degradation by pepsin and other digestive proteases (such as pancreatic proteases, i.e. mainly trypsin and chymotrypsin); and/or (iv) is stable and/or active in the presence of bile salts.

The term "in combination with" refers to the combined use of a protease, a lipase and/or an amylase according to the present invention. The combined use may be simultaneous, overlapping or sequential, and the three terms are generally interpreted according to a prescription made by a physician.

The term "simultaneous" refers to the situation where the enzymes are active at the same time, e.g. when they are administered simultaneously as one or more separate pharmaceutical products, or if they are administered in the same pharmaceutical composition.

The term "sequential" refers to the situation where one and/or two enzymes act first, followed by a second and/or third enzyme. The sequential effect can be obtained by: the enzymes in question are administered as separate pharmaceutical formulations at desired time intervals, or as one pharmaceutical composition in which the enzymes in question are formulated differently (partitioned), for example with a view to achieving different release times, providing improved product stability, or optimizing enzyme dosage.

The term "overlap" refers to the situation during which the enzyme activities are neither completely simultaneous nor completely sequential, i.e. there is a period during which both or all of the enzymes are active.

The term "one or one" when used, for example, in the context of the protease, lipase and/or amylase of the invention means at least one or one. In particular embodiments, "one or" an "means" one or more or "one or more" or "at least one or" which in turn means one or one, two or two, three or three, four or four, five or five, etc.

The correlation between two amino acid sequences is described with the parameter "identity", which is described in detail above (in the protease part). The definitions and methods are equally applicable to the lipases and amylases used according to the invention.

Any suitable assay may be used to measure the enzymatic activity of the present invention. In general, the assay pH and assay temperature should be adapted to the enzyme in question. Examples of pH values to be determined are pH2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12. Examples of measured temperatures are 30, 35, 37, 40, 45, 50, 55, 60, 65, 70, 80, 90 or 95 ℃.

Examples of suitable enzyme (primarily protease) assays are included in example 1 herein, and reference may also be made to WO2006/136159 and WO 2006/136161, respectively, for suitable lipase and amylase assays.

Medicine

In this context, the term "medicament" refers to a compound or mixture of compounds that treats, prevents and/or alleviates a disease symptom, preferably a disease symptom. The medication may be prescribed by a physician, or it may be an over-the-counter product.

Pharmaceutical composition

The isolation, purification and concentration of the enzyme of the invention can be carried out by conventional means. For example, they can be recovered from the fermentation broth by conventional methods (including but not limited to centrifugation, filtration, extraction, spray drying, evaporation or precipitation) and further purified by a variety of methods known in the art (including but not limited to chromatography (e.g., ion exchange, affinity, hydrophobic, chromatofocusing, and size exclusion), electrophoretic methods (e.g., preparative isoelectric focusing), differential solubility (e.g., ammonium sulfate precipitation), SDS-PAGE, or extraction) (see, e.g., Protein Purification, code for j.

For example, SEQ ID NO: 2 can be prepared, for example, based on U.S. Pat. No. 5,869,438 by recombinant expression in a suitable host cell of the nucleic acid sequence shown as SEQ ID NO: 1, or a modified DNA sequence corresponding thereto. Such modifications can be made by site-directed mutagenesis, as is well known in the art.

In a specific embodiment, concentrated solid or liquid preparations of each enzyme are prepared separately. These concentrates may also be (at least partially) formulated separately, as explained in more detail below.

In yet another specific embodiment, the enzyme is incorporated in the pharmaceutical composition of the invention in the form of a solid concentrate. The enzyme can be brought to a solid state by a variety of methods as are known in the art. For example, the solid state may be crystalline (in which the enzyme molecules are arranged in a highly ordered form), or a precipitate (in which the enzyme molecules are arranged in a less ordered, or disordered, form).

For example, crystallization can be performed at a pH close to the enzyme pI and at low conductivity (e.g., 10mS/cm or less), as described in EP 691982. In a particular embodiment, the lipase used according to the invention is a crystalline lipase, which can be prepared as described in example 1 of EP 600868B 1. Furthermore, the lipase crystals can be crosslinked as described in WO 2006/044529.

Various precipitation methods are known in the art, including salts (such as ammonium sulfate and/or sodium sulfate); with organic solvents such as ethanol and/or isopropanol; or by precipitation with a polymer, such as PEG (polyethylene glycol). In the alternative, the enzyme may be precipitated from the solution by removing the solvent (typically water) by a variety of methods known in the art, such as lyophilization, evaporation (e.g., under reduced pressure), and/or spray drying.

In yet another specific embodiment, the solid concentrate of the enzyme has an active enzyme protein content of at least 50% (w/w) relative to the total protein content of the solid concentrate. In still further specific embodiments, the active enzyme protein content relative to the total protein content of the solid concentrate is at least 55, 60, 65, 70, 75, 80, 85, 90 or at least 95% (w/w). Protein content can be measured, as is known in the art, for example, by densitometric scanning of coomassie stained SDS-PAGE gels using, for example, a GS-800 calibrated densitometer from BIO-RAD; by using a commercial kit commercially available from Roche, such as protein assay ESL, order number 1767003; or on the basis of the method described in example 8 of WO 01/58276.

Preferably, the enzyme protein constitutes at least 50%, more preferably at least 55, 60, 65, 70, 75, 80, 85, 90, 92, 94, 95, 96 or at least 97% of the protein mass spectrum of the solid enzyme concentrate used according to the invention, as measured by densitometric scanning of a coomassie stained SDS-PAGE gel. Such enzymes may be referred to as "isolated" enzymes or polypeptides.

The pharmaceutical compositions of the present invention comprise the enzyme, preferably in the form of a concentrated enzyme preparation, more preferably a solid concentrate, together with at least one pharmaceutically acceptable auxiliary or supplementary material, such as (i) at least one carrier and/or excipient; or (ii) at least one carrier, excipient, diluent and/or adjuvant. Non-limiting examples of optional additional ingredients, all of which are pharmaceutically acceptable, are disintegrants, lubricants, buffers, wetting agents, preservatives, flavoring agents, solvents, solubilizers, suspending agents, emulsifiers, stabilizers, propellants and vehicles.

In general, the compositions of the invention may be designed for all modes of administration known in the art, including preferably enteral administration (via the digestive tract), depending in particular on the medical indication in question. As such, the compositions may be in solid, semi-solid, liquid or gaseous form, such as tablets, capsules, powders, granules, microspheres, ointments, creams (cream), foams, solutions, suppositories, injections, inhalants, gels, lotions (injections) and aerosols. The medical practitioner will know to choose the most appropriate route of administration and will, of course, avoid a potentially dangerous or otherwise disadvantageous route of administration. The following methods and auxiliary materials are therefore merely illustrative and in no way limiting.

For solid oral preparations, the enzyme may be used alone or in combination with additives suitable for the preparation of pills (pellets), micropellets (micropellets), tablets, microtablets, powders, granules or capsules, for example with conventional carriers such as lactose, mannitol, corn starch or potato starch; with excipients or binders, such as crystals or microcrystals, cellulose derivatives, acacia (acacia), corn starch or gelatin; with disintegrating agents, such as corn starch, potato starch or sodium carboxymethyl cellulose; with lubricants, such as carnauba wax (carnauba wax), white wax, shellac (shellac), anhydrous colloidal silica, polyethylene glycol (PEG, also known under the term Macrogol) 1500 to 20000, in particular PEG 4000, PEG 6000, PEG 8000, polyvinylpyrrolidone (povidone), talc, glycerol monooleate (monolein) or magnesium stearate; and if desired in combination with diluents, adjuvants, buffers, wetting agents, preservatives such as methylparaben (E218), coloring agents such as titanium dioxide (E171), and flavoring agents such as sucrose, saccharin, orange oil, lemon oil, and vanillin. Oral formulations are examples of preferred formulations for the treatment of medical indications for PEI.

Quite generally, the enzymes may also be formulated as liquid oral formulations by dissolving, suspending or emulsifying them in an aqueous solvent such as water, or a non-aqueous solvent such as vegetable oil or other similar oils, synthetic fatty acid glycerides, esters of higher fatty acids, propylene glycol, polyethylene glycols such as PEG 4000, or lower alcohols such as linear or branched C1-C4 alcohols, e.g. 2-propanol; and if desired, with conventional supplementary materials or additives such as solubilizers, adjuvants, diluents, isotonic agents, suspending agents, emulsifiers, stabilizers and preservatives.

The use of liposomes as a delivery vehicle is another approach that may be of general interest. The liposomes fuse with the cells of the target site and deliver the luminal contents intracellularly. Various means for maintaining contact, such as separation, binding agents, and the like, are used to maintain the liposome in contact with the cells for a sufficient time to effect fusion. In one aspect of the invention, the liposomes are designed to be aerosolized for pulmonary administration. Liposomes can be prepared using purified proteins or peptides that mediate membrane fusion, such as Sendai virus or influenza virus. The lipid may be any useful combination of known liposome-forming lipids, including cationic or zwitterionic lipids, such as phosphatidylcholine. The remaining lipids will typically be neutral or acidic lipids, such as cholesterol, phosphatidylserine, phosphatidylglycerol, and the like. For the preparation of liposomes, the liposome prepared by Kato et al (1991) j.biol.chem.266: 3361 the process described.

Unit dosage forms for oral or rectal administration such as syrups, elixirs, powders and suspensions may be provided wherein each dosage unit contains a predetermined amount of the enzyme, for example, a teaspoonful, tablespoonful, capsule, tablet or suppository. Similarly, unit dosage forms for injection or intravenous administration may comprise the enzyme in a composition that is a solution in sterile water, normal saline, or another pharmaceutically acceptable carrier.

As used herein, the term "unit dosage form" refers to physically discrete units suitable as unitary dosages for human and animal subjects, each unit containing a predetermined quantity of enzyme, in an amount sufficient to produce the desired effect.

In a specific embodiment, the pharmaceutical composition of the invention is for enteral (preferably oral) administration.

In further embodiments, the oral composition is (i) a liquid composition comprising an enzyme crystal; (ii) a liquid suspension of a deposit of (highly) purified enzyme; (iii) a gel containing the enzyme in solid or dissolved form; (iv) a liquid suspension of an immobilized enzyme or an enzyme adsorbed to a particle or the like; or (v) a solid composition in the form of an enzyme-containing powder, pellet, granule or microsphere, if desired, in the form of a tablet, capsule or the like (optionally coated with, for example, an acid-stable coating).

In another particular embodiment of the composition, the enzymes are separated, i.e. from each other, by means of, for example, a separating coating.

In a further specific embodiment of the composition, the protease is separated from other enzymatic components of the composition, such as lipase and/or amylase.

Enzyme dosages will vary widely depending on the particular enzyme to be administered, the frequency of administration, the mode of administration, the severity of the symptoms, and the subject's susceptibility to side effects, among other factors. Some specific enzymes can be more potent than others.

Examples of solid oral formulations of the enzymes of the invention include: (i) has the sequence shown in SEQ ID NO: 6. 7, 8, 9, 10 or 11; (ii) and SEQ ID NO: 2, amino acids 1-269 having at least 70% identity; and/or (iii) an amylase having at least 70% identity to an amylase selected from the group consisting of: a) has the sequence shown in SEQ ID NO: 5, b) an amylase having amino acids 1-513 of SEQ ID NO: 3, and c) an amylase having amino acids 1-481 of SEQ ID NO: 4 amino acids 1-483. In a more preferred solid oral formulation of the invention, (ii) the lipase comprises SEQ ID NO: 2, and (iii) the amylase comprises amino acids 1-269 of SEQ ID NO: 5 amino acids 1-486.

(i) Examples of expected daily clinical doses of the enzymes of (i), (ii) and (iii) are as follows (all in mg enzyme protein per kg body weight (bw)): for the protease of (i): 0.005-500, 0.01-250, 0.05-100 or 0.1-50 mg/kgbw; for the lipase of (ii): 0.01-1000, 0.05-500, 0.1-250 or 0.5-100mg/kg bw; for the amylase of (iii): 0.001-250, 0.005-100, 0.01-50 or 0.05-10mg/kg bw, preferably for the protease of (i): 0.05-100, 0.1-50 or 0.5-25mg/kg bw; for the lipase of (ii): 0.1-250, 0.5-100 or 1-50mg/kg bw; and for the amylase of (iii): 0.01-50, 0.05-10 or 0.1-5mg/kg bw.

For greater stability with respect to oral administration, the amide (peptide) linkage, as well as the amino and carboxyl termini, may be modified. For example, the carboxy terminus may be amidated.

Specific embodiments of the pharmaceutical composition of the invention suitable for the treatment of digestive disorders, PEI, pancreatitis, cystic fibrosis, diabetes type I, and/or diabetes type II may be prepared by incorporating the enzyme of the invention into a pill. The pellets may generally comprise 10-90% (w/w, relative to the dry weight of the resulting pellet) of the physiologically acceptable organic polymer, 10-90% (w/w, relative to the dry weight of the resulting pellet) of cellulose or cellulose derivative, and 80-20% (w/w, relative to the dry weight of the resulting pellet) of the enzyme, the total amount of organic polymer, cellulose or cellulose derivative and enzyme in each case amounting to 100%.

The physiologically acceptable organic polymer may be selected from the group consisting of: polyethylene glycol 1500, polyethylene glycol 2000, polyethylene glycol 3000, polyethylene glycol 4000, polyethylene glycol 6000, polyethylene glycol 8000, polyethylene glycol 10000, polyethylene glycol 20000, hydroxypropyl methylcellulose, polyoxyethylene-polyoxypropylene copolymer and a mixture of the organic polymers. Polyethylene glycol 4000 is preferred as the physiologically acceptable organic polymer.

The cellulose or cellulose derivative may for example be selected from cellulose, cellulose acetate, cellulose fatty acid esters, cellulose nitrate, cellulose ethers, carboxymethyl cellulose, ethyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, methyl ethyl cellulose and methyl hydroxypropyl cellulose. Cellulose, in particular microcrystalline cellulose, is preferred as cellulose or cellulose derivative.

The resulting pellets may be coated with a suitable enteric coating, other non-functional coating or used directly without such a coating. In addition, the resulting pill can be filled in a capsule (e.g., a hard gelatin capsule or a gelatin-free capsule) of suitable size for treating a condition or disease as described in more detail above. In one embodiment of the invention, pellets produced from different enzyme types, in particular from lipases, proteases and/or amylases, can be filled into the capsules. In filling capsules with different enzyme types, dosing (dosing) of a single enzyme type (i.e. lipase, protease or amylase) can be adapted to the specific needs of a certain indication group or a certain patient subgroup by adding a defined amount of any lipase, protease and/or amylase to the capsules, i.e. the amount of lipase: protease: capsules with variations in amylase ratio.

Preferred pharmaceutical compositions of the protease of the invention are described in WO 2005/092370, in particular formulations comprising the preferred excipients mentioned therein. In a particularly preferred embodiment, the pharmaceutical composition comprises a Macrogol glyceride mixture of mono-, di-and triacylglycerides and polyethylene glycol (PEG) mono-and diesters of aliphatic C6-C22 carboxylic acids, and possibly also a small proportion of glycerol and free polyethylene glycol.

Preferably, the polyethylene glycol (PEG) contained in the Macrogol glyceride mixture is a PEG having an average of 6 up to 40 ethylene oxide units per molecule or a molecular weight between 200 and 2000.

A further aspect of the invention provides a pharmaceutical composition of the enzyme of the invention comprising a system consisting of a surfactant, a co-surfactant and a lipophilic phase, the system having an HLB value (hydrophilic-lipophilic balance) greater than or equal to 10 and a melting point greater than or equal to 30 ℃. In a preferred embodiment, the system has an HLB value of from 10 to 16, preferably from 12 to 15, and has a melting point of from 30 to 600 ℃, preferably from 40 to 500 ℃. In particular, the systems characterized by HLB value and melting point are mixtures of mono-, di-and triacylglycerides and polyethylene glycols (PEG) with mono-and diesters of aliphatic carboxylic acids having 8 to 20, preferably 8 to 18, carbon atoms, whereby the polyethylene glycol preferably has about 6 to about 32 ethylene oxide units per molecule, and optionally the systems contain free glycerol and/or free polyethylene glycol. Preferably, the HLB value of this system is regulated by the PEG chain length. The melting point of this system is regulated by the chain length of the fatty acids, the chain length of the PEG and the fatty acid chain saturation and therefore by the starting oil used for the preparation of the Macrogol glyceride mixtures.

"aliphatic C8-C18 carboxylic acid" means a mixture containing, in significant and variable proportions, caprylic (C8), capric (C10), lauric (C12), myristic (C14), palmitic (C16) and stearic (C18) acids, if these acids are saturated, and the corresponding unsaturated C8-C18 carboxylic acids. The proportion of these fatty acids may vary with the starting oil.

Such mixtures of mono-, di-and triacylglycerides and mono-and diesters of polyethylene glycol (PEG) with aliphatic carboxylic acids having from 8 to 18 carbon atoms can be obtained, for example, by reaction between polyethylene glycol having a molecular weight of 200-1500 and a starting oil consisting of a mixture of triglycerides with fatty acids selected from the group consisting of: caprylic acid, capric acid, lauric acid, myristic acid, palmityl aldehyde, stearic acid, oleic acid and linolenic acid (alone or as a mixture). Optionally, the product of such a reaction may also contain a small proportion of glycerol and free polyethylene glycol.

Such mixtures are available, for example, under the trade nameAnd (4) commercialization. An advantageous embodiment of the invention provides thatOf the products known below, in particular "50/13 'and/or'44/14 "represents a mixture suitable for use in a pharmaceutical formulation according to the invention.50/13 is a mixture of mono-and diesters of mono-, di-and triacylglycerides and polyethylene glycol with 40% to 50% and 48% to 58%, respectively, of palmitic (C16) and stearic (C18) acids constituting the major proportion of bound fatty acids. The proportion of octanoic acid (C8) and decanoic acid (C10) is in each case less than 3%, whereas lauric acid (C12) is in each caseAnd myristic acid (C14) in a proportion of less than 5%.44/14 is a mixture of mono-and diesters with mono-, di-and triacylglycerides and polyethylene glycols in proportions of 4 to 25% palmitic acid (C16), 5 to 35% stearic acid (C18), less than 15% caprylic acid (C8), less than 12% capric acid (C10), 30 to 50% lauric acid (C12) and 5 to 25% myristic acid (C14), respectively. Can be prepared, for example, by alcoholysis/esterification reactions using palm kernel oil and polyethylene glycol 150044/14。

A preferred embodiment of the present invention provides a pharmaceutical composition of the enzyme of the invention comprising a system comprising a mixture of mono-, di-and triacylglycerides and polyethylene glycol mono-and diesters of aliphatic C8-C18 carboxylic acids and possibly also a small proportion of glycerol and free polyethylene glycol, the system having a melting point of 40 ℃ to 55 ℃ and an HLB value in the range of 12 to 15. More preferably, the system has a melting point of 44 ℃ to 50 ℃ and an HLB value in the range of 13 to 14. Alternatively, the system has a melting point of around 44 ℃ and an HLB value of 14, or the system has a melting point of around 50 ℃ and an HLB value of 13.

Method of treatment

The proteases used according to the invention, optionally in combination with lipases and/or amylases (enzymes of the invention), can be used for the therapeutic and/or prophylactic treatment of various diseases or disorders in animals. The term "animal" includes all animals, and in particular humans. Examples of animals are non-ruminants, and ruminants such as sheep, goats, and cattle (e.g., beef and dairy cows). In a particular embodiment, the animal is a non-ruminant animal. Non-ruminant animals include monogastric animals, such as horses, pigs (including but not limited to piglets, nursery pigs, and sows); birds such as turkeys, ducks, and chickens (including but not limited to broiler chicks, layers); young calves; pets such as cats and dogs; and fish (including but not limited to salmon, trout, tilapia, catfish, and carp (carp), and crustaceans (including but not limited to shrimp and prawn).

For example, enzymes may be used to treat digestive disorders such as dyspepsia (maldigestion) or dyspepsia (dyspeptia), which are often caused by the production and/or insufficient secretion of digestive enzymes normally secreted from the stomach and pancreas into the gastrointestinal tract.

Furthermore, the enzymes are particularly useful in the treatment of PEI. Can be used in particularTests (JOP. J Pancreas (in-line) 2002; 3 (5): 116-; chronic pancreatitis; tropical pancreatitis; hereditary pancreatitis; Shu-Daidi Syndrome (Shwachman Diamond Syndrome); ductal obstruction of the pancreas or common bile ducts (e.g., due to neoplasms); and/or cystic fibrosis (a genetic disease in which thick mucus blocks the pancreatic ducts). Enzymes may also be useful in treating acute pancreatitis.

The effect of the enzyme on digestive disorders can be measured as generally described in EP 0600868, where example 2 describes an in vitro digestibility test for measuring lipase stability under gastric conditions, and example 3 describes an in vitro digestibility test for lipase activity in the presence of bile salts. Corresponding tests can be established for proteases and amylases. Also, WO 02/060474 discloses suitable tests, such as (1) in vitro tests for measuring lipid digestion in pig test feeds, and (2) in vivo tests with pancreas-incompetent pigs, in which the digestibility of fat, protein and starch is measured.

In a specific embodiment, the in vivo screening assay of example 4 is used to measure the effect of the protease of the invention.

As another example, enzymes are useful in the treatment of type I and/or type II diabetes, particularly for adjunctive treatment in the treatment of diabetes for digestive disorders that commonly accompany such diseases (with a view to reducing late complications).

The effect of the enzyme on diabetes can be determined by one or more of the methods described in WO 00/54799, for example by controlling the glycosylated hemoglobin level, the blood glucose level, the hypoglycemic attack (hypoglycaemic attack), the status of fat soluble vitamins such as vitamins A, D and E, the required daily dose of insulin, the body mass factor and the hyperglycaemic cycle.

The invention described and claimed herein is not to be limited in scope by the specific embodiments herein disclosed, since these embodiments are intended as illustrations of several aspects of the invention. Any equivalent embodiments are intended to be within the scope of the present invention. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description. It is also intended that such modifications fall within the scope of the appended claims. In case of conflict, the present disclosure, including definitions, will control.

Various references are cited herein, the disclosures of which are incorporated by reference in their entirety.

Examples

The chemicals used are commercial products of at least reagent grade.

Example 1: enzyme assay

FIP (F.D. International pharmaceutical Pharmaceutique) and the European pharmacopoeia and the U.S. pharmacopoeia have published assays for lipase, protease and amylase activity in porcine pancreatin (pancreatin). 1 FIP-unit 1 ph. Assays are described, for example, in: international association of pharmacy, scientific section: the International Commission on enzyme standardization for pharmaceutical use (International Commission for the standardization of pharmaceutical enzymes). a) "Pharmaceutical Enzymes", "compiled by R.Ruyssen and A.Lauwers, E.StoryScientia, Ghent, Belgium (1978), b) European pharmacopoeia. See also Deemester et al, Lauwers A, Scharp S (eds): pharmaceutical Enzymes, New York, Marcel Dekker, 1997, page 343-. Suitable enzyme standards may be obtained from: international Commission on Pharmaceutical Enzymes, Standard center (Centre for standards), Harelbekstraat 72, B-9000 Ghent.

The following describes the protease FIP assay as well as other assays suitable for protease, lipase and starch.

Protease FIP assay

Protease activity can be determined using the FIP assay (international association of pharmaceuticals), 1 FIP-unit 1 ph. This assay is described, along with other FIP assays: international association of pharmacy, scientific section: the International Committee for standardization of enzyme for pharmaceutical use. a) "pharmaceutical enzymes" compiled by r.ruyssen and a.lauwers, e.story scientific, Ghent, Belgium (1978), b) european pharmacopoeia. See also Deemester et al, Lauwers A, Scharp S (eds): pharmaceutical Enzymes, New York, Marcel Dekker, 1997, page 343-.

This assay was used to determine protease activity in pancreatin. For determining the FIP activity of microbial proteases, the activation step by addition of enterokinase was omitted.

The principle is as follows: the protease hydrolyzes the substrate casein at pH7.5 and a temperature of 35 ℃. The reaction was stopped by adding trichloroacetic acid and the undegraded casein was filtered off. The amount of peptide remaining in the solution was determined by spectrophotometry at 275 nm.

Definition of Activity: the amount of peptide that was not precipitated by 5.0% (wt/vol, i.e. 5.0g/100ml) trichloroacetic acid solution was determined as protease activity by reference to a pancreatic reference powder (protease reference standard) of known FIP activity. Materials and methods:

casein solution:

1.25g casein (dry weight) (e.g. Calbiochem No. 218680) was suspended in water until a substantially clear solution was obtained. The pH was adjusted to 8.0 and the solution was diluted with water to a final volume of 100 ml. Here and in the following, water means deionized water.

Borate buffer pH 7.5:

2.5g of sodium chloride, 2.85g of disodium tetraborate and 10.5g of boric acid are dissolved in 900ml of water, the pH is adjusted to pH7.5 +/-0.1 and diluted to 1000ml with water.

Filter paper:

folded filter paper having a diameter of 125mm, for example Schleicher & Schuell No. 15731/2. Filter paper test: 5ml of 5.0% trichloroacetic acid were filtered through the filter paper. Using an unfiltered trichloroacetic acid solution as a blank, the absorbance of the filtrate at 275nm should be less than 0.04.

Protease reference standard:

commercially available protease (pancreas) from the International Committee for pharmaceutical enzymes, Standard centre, Harelbekstraat 72, B-9000Ghent, Belgium. The standard has a labelling activity (a) in FIP/ph. The amount corresponding to about 130 protease-FIP/ph. A spatula tip of sea sand was added, moistened with a few drops of ice cold 0.02M calcium chloride (pH 6.0-6.2) and triturated all over with a flat-ended glass rod. Diluted with about 90ml of the same ice-cold calcium chloride solution and the suspension was stirred in an ice bath for 15 to 30 minutes. The pH was adjusted to 6.1 and the volume was adjusted to 100ml with the same calcium chloride solution. 5.0ml of this suspension was diluted to 100ml with borate buffer pH 7.5. For the activity test, 1.0, 2.0 and 3.0ml of this solution were used as a reference (hereinafter referred to as S1, S2 and S3, S stands for standard).

Test suspensions:

the sample suspension was prepared using a sample amount equal to about 260 FIP/ph.eur. -units, as described above for the protease reference standard. The pH was adjusted to 6.1 and water was added to 100 ml. 5.0ml of this solution was mixed with 5ml of calcium chloride solution. 5ml of this dilution was further diluted to 100ml with borate buffer. 2.0ml of this solution was used for the assay (hereinafter the sample is referred to as Un, which represents a sample of unknown activity, number n).

Assay method (activity test):

the assays were performed on three reference suspensions (S1, S2, S3) and on a sample suspension (Un) (triplicate of homogeneous). One blank per sample was sufficient (designated S1b, S2b, S3b, and Unb, respectively). A blind sample (B) was prepared in the absence of sample/standard as a compensation liquid for the spectrophotometer (A bland (B) amplified with out sample/standard complex for the spectrophotometer). Borate buffer was added to the tubes as follows: blind sample (B)3.0 ml; 1.0ml of sample (Un); standards (S1, S2, and S3) were 2.0, 1.0, and 0ml, respectively. Protease reference standards were added to S1, S2, and S3 as follows: 1.0, 2.0 and 3.0ml respectively. The test suspension was added to the sample tube (Un) as follows: 2.0 ml. To all blinded samples (S1B, S2B, S3B, Un and B) 5ml of trichloroacetic acid was added, followed by immediate mixing. All tubes were stoppered with glass stoppers and placed in a water bath at constant temperature (35+/-0.5 ℃) with the substrate solution. Upon reaching temperature equilibrium, at time 0, 2.0ml of casein solution was added to tubes S1, S2, S3 and Un, followed by immediate mixing. Just after 30 minutes, 5.0ml of trichloroacetic acid was added to each tube S1, S2, S3 and Un, followed by immediate mixing. The tube was withdrawn from the water bath and allowed to stand at room temperature for 20 minutes to complete precipitation of the protein. The contents of each tube were filtered through the same filter paper twice and the absorbance of the filtrate at 275nm was measured using the filtrate from tube B as the compensation liquid. The activity of the sample (Un) in FIP units was calculated relative to the known marker activity (a) of the standards (S1, S2, S3). The absorbance values subtracted for each blind sample (e.g., S1 absorption minus S1b absorption) should be in the interval 0.15-0.60.

Protease Protazyme AK assay

Substrate: 2.0ml 1 Protazyme AK tablet (Megazyme T-PRAK1000) suspended in 0.01% Triton X-100. A homogeneous suspension was prepared by stirring.

Temperature: 37 deg.C

Determination of buffer: 100mM HEPES/NaOH, 0.01% Triton X-100, pH 7.0

500ul (microliters) of Protazyme AK substrate suspension and 500ul of assay buffer were mixed in an Eppendorf tube and placed on ice. 20ul protease samples (diluted in 0.01% Triton X-100) were added. The assay was started by transferring the Eppendorf tube to an Eppendorf thermostated mixer (which was set to 37 ℃). The tubes were incubated for 15 minutes on an Eppendorf thermostated mixer at their highest shaking rate (1400 rpm). The incubation was stopped by transferring the tube back to an ice bath. After a few minutes, the tubes were centrifuged in a cold centrifuge (15000rpm, 3 minutes). 200ul of supernatant was transferred to a microtiter plate. OD650 was read as a measure of protease activity. Buffer blinding (instead of enzyme) was included in the assay.

Protease Suc-A APF-pNA assay

Substrate: Suc-AAPF-pNA (S-7388)。

Determination of buffer: 100mM succinic acid, 100mM HEPES, 100mM CHES, 100mM MCAS, 1mM CaCl₂、150mM KCl、0.01％X-100, adjusted to pH 9.0 with HCl or NaOH.

Measuring temperature: at 25 ℃.

Mu.l of the diluted protease sample was mixed with 1.5ml assay buffer by adding 1.5ml pNA substrate (50mg dissolved in 1.0ml DMSO and 0.01%X-100 further diluted 45X) to start the active reaction and after mixing, monitor a spectrophotometrically₄₀₅The increase is a measure of protease activity. The protease samples were diluted before activity measurements were made to ensure that all activity measurements fell within the linear portion of the dose response curve of the assay.

Lipase enzyme

Substrate: p-Nitro-phenyl (pNP) pentanoate

And (3) measuring the pH: 7.7

Measuring temperature: 40 deg.C

Reaction time: 25 minutes

The digestion product with yellow color has a characteristic absorbance at 405 nm. The amount thereof was measured by spectrophotometry. Lipase activity can be measured relative to enzyme standards of known activity. The activity can be expressed in Lipolase Units (LU). 1 LU (Lipolase units) is the amount of enzyme that releases 1mmol of titratable butyric acid per minute under standard conditions as above. 1KLU 1000 LU.

Amylase

Substrate: phadebas tablets (Pharmacia Diagnostics; cross-linked, insoluble, bluing starch polymers, mixed with bovine serum albumin and buffer substances, and tableted).

Measuring temperature: 37 deg.C

And (3) measuring the pH: 4.3

Reaction time: 20 minutes

After suspension in water, the starch is hydrolyzed by alpha-amylase to give a soluble blue fragment. The absorbance of the resulting blue solution measured at 620nm is a function of the alpha-amylase activity. 1 fungal alpha-amylase unit (1 FAU) is the amount of enzyme that breaks down 5.26g starch per hour under standard assay conditions (Merck, soluble starch Erg.B.6, batch 9947275).

Example 2: preparation of proteases

Preparation of SEQ ID NO: 6. 7, 8, 9, 10 and 11, in brief: random and/or site-directed mutagenesis is introduced into the gene, the Bacillus subtilis host cell is transformed with the mutated gene, the transformed host cell is fermented (e.g., as described in example 1 of WO 2004/111220), and the protease is purified from the fermentation broth. The reference protease (SEQ ID NO: 1) was recombinantly produced in Bacillus subtilis in a similar manner.

For purification of larger amounts of protease, the culture broth was centrifuged (13.000rpm. for 20 minutes) to give a clear supernatant and the supernatant was filtered through 0.45 μm filter paper to remove remaining bacillus host cells. The pH of the filtrate was adjusted to pH 9.0 with 3M Tris and the protease solution was loaded onto a MEP Hypercel column (PALL Life Sciences) equilibrated in 50mM Tris/HCl, pH 9.0. After washing the column with several column volumes of equilibration buffer, 50mM CH was used₃COOH/NaOH, pH 4.0. Fractions collected during elution were tested for protease activity (using the end-point ProtazymeAK assay of example 1). The active fractions were pooled, the pH adjusted to pH 4.5, and the pool diluted with deionized water to give a mixture with 20mM CH₃COOH/NaOH、50mM H₃BO₃、1mM CaCl₂pH 4.5(SP equilibration buffer) at the same conductivity. The adjusted pool was loaded onto an SP Sepharose HP column equilibrated in SP equilibration buffer. After washing the column with several column volumes of SP equilibration buffer, the column was eluted with a linear NaCl gradient (0- - > 0.5M) in the same buffer in 5 column volumes. Fractions collected during elution were tested for protease activity (using the Protazyme AK assay).The active fractions from the column were pooled as purified protease product.

For the preparation of smaller amounts of protease (micro-purification), the culture broth was sterile filtered through 0.45 μm filter paper. To each well of a filter plate (Whatman, Unifilter 800. mu.l, 25-30 μm MBPP) was added approximately 100. mu.l of MEP-HyperCel chromatography media slurry. The chromatography medium was washed twice with 200. mu.l 25mM Tris, 25mM sodium borate, 2mM CaCl2, pH 8.5 as follows: incubate for 5 minutes at room temperature with vigorous shaking (Heidolph, Titramax101, 1000rpm) to stir the chromatographic medium, followed by removal of the liquid by vacuum (Whatman, UniVac 3). Then 100. mu.l binding buffer (0.5M Tris, 25mM sodium borate, 10mM CaCl)₂pH 8.5) and 400. mu.l of culture supernatant were transferred to each well of the filter plate. Typically 4 wells are micro-purified for each protease. To bind the protease to the chromatography medium, the filter plate was incubated for 30 minutes with vigorous shaking. After removal of unbound material by vacuum, the binding step was repeated: add 100. mu.l binding buffer and 400. mu.l culture supernatant, incubate for 30 minutes with shaking, and remove unbound material by vacuum. The MEP-HyperCel medium was then incubated with 0.1M Tris, 25mM sodium borate, 2mM CaCl₂Washing once at pH 8.5 with 25mM Tris, 25mM sodium borate, 2mM CaCl₂Washed once at pH 8.5 and with 10mM Tris, 25mM sodium borate, 2mM CaCl₂And once washing at pH 8.5. In each washing step, 200. mu.l of buffer was added and the plate was incubated for 10 minutes at room temperature with vigorous shaking and the buffer was removed by vacuum. To release the protease from the chromatography medium, 100. mu.l of elution buffer (50mM sodium acetate, 2mM CaCl) was added₂Ph4.3) and incubate the filter plates for 10 minutes at room temperature with vigorous shaking. The elution buffer containing the protease was transferred to a 96-well plate by vacuum. The elution procedure was repeated by adding 100. mu.l of elution buffer, shaking l0 minutes at room temperature, and collecting in the same 96-well plate. Pooled micro-purified protease was stored at-18 ℃.

Enzyme protein concentrations were determined by active site titration as described below, or were calculated based on the a280 value and the amino acid sequence (amino acid composition), using the principles outlined in s.c. gill and p.h. von Hippel, Analytical Biochemistry 182, 319-326, (1989). The enzyme protein concentration may also be determined by amino acid analysis, for example as described in example 3 of WO 2004/111221.

Tightly binding barley chymotrypsin inhibitor 2A (CI-2A; see Ludvigsen, s., Shen, h.y., Kjaer, m., Madsen, j.c., Poulsen, f.m.: reference element of three-dimensional solution structure of barrel serum protease inhibitor 2and composition with the structure in crystals.j.mol.biol., volume 222, pages 621-635, 1991) was used to determine enzyme concentration by active site titration as follows. In a microtiter plate, 20 μ l aliquots of the micro-purified protease (diluted appropriately with 0.1M Tris, 0.0225% Brij 35 (polyoxyethylene (23) lauryl ether), pH 8.6) were mixed with 20 μ l CI-2A (diluted to 2, 1.5, 1, 0.5, 0.25, 0.125, 0.0625 and 0 μ M, usually with 0.1M Tris, 0.0225% Brij 35, pH 8.6). After 1 hour incubation with shaking, the remaining activity was measured by: mu.l of substrate solution (typically 0.4mg/ml Suc-Ala-Ala-pNA in 0.1M Tris, 0.0225% Brij 35, pH 8.6 prepared from 200mg/ml stock solution in DMSO) was added and the absorbance at 405nm was measured every 10 seconds for 3 minutes (Spectramax, Molecular Devices). Active protease concentration was calculated from linear regression of residual activity versus inhibitor concentration for wells with (significant) residual activity.

Table 1 is a list of selected variants of the invention. These proteases were tested as described in the examples below.

Table 1: list of variants

Example 3: protease with improved pH-ratio

Protease variant VAR294(SEQ ID NO: 6) protease activity was tested at pH5.6 and pH8.0 and compared to the corresponding activity of the reference protease (SEQ ID NO: 1) as described below.

Protease assay

Using a sample from Molecular Probes (Invitrogen, Cat. ID. E6639)Protease assay kit. As substrate, it uses a red fluorescence which is insensitive to pHThe TR-X dye heavily labels casein derivatives, resulting in almost total quenching of the fluorescence of the conjugate. Protease-catalyzed hydrolysis releases peptides that are highly labeled with the fluorescent BODIPY TR-X dye. The concomitant increase in fluorescence is proportional to protease activity.

Reagent:

a tube of the EnzChek protease assay kit (200. mu.g) for red fluorescence was placed in 200. mu.l of 0.1M NaHCO₃(pH 8) to give a stock solution of 1 mg/mL.

The assay buffer pH8 was prepared by adjusting 100mM Tris/base to pH8.0 with HCl. 6.25. mu.g/ml of labeled substrate was added.

The assay buffer pH5.6 was prepared by mixing 25ml of 0.2M succinic acid with 37.5ml of 0.2M NaOH (reference: Gomori, meth. enzymol.1, 141 (1955)). To compensate for the protease added, 1.143ml of 1M HCl was added per 100ml of assay buffer. Thereby, the pH of the buffer was lowered to about 5.0. 5. mu.g/ml of labeled substrate was added.

And (3) sample analysis:

mu.l of 0.5. mu.M protease solution (reference protease, protease variant, both in duplicate) was mixed with 40. mu.l of each buffer in 384-well plates. Incubation at room temperature for 60 minutes; shaking vigorously at 750 rpm/min. The fluorescence was read in a fluorescence microplate reader at t-0 and t-60 minutes. The BODIPYTR-X labeled peptide has an excitation/emission maximum of 589/617 nm. BODIPY TR-X dye-labeled peptides were detected using standard fluorescein filters (filters) (excitation 590nm, emission 635 nm).

And (3) calculating:

the reading at ph5.6 at t 60 minutes must be 4 times higher than when t 0. The ratio between the reading at pH5.6 and the reading at pH8 was calculated. The resulting number must be 1.4 times the respective number of reference enzymes. The ratio of variants was compared to the average ratio of 8 reference wells per 96-well plate. For the calculation, t-0 is subtracted from t-60 at both pH values.

Results

The variants listed in table 2 below have different activity ratios at pH5.6/pH 8.0 compared to the reference protease with a pH5.6/pH8 ratio of 1.00.

TABLE 2

Example 4: in vivo screening test for protease efficacy

3-4 females with induced exocrine pancreatic insufficiency (PEI)Purified protease variants VAR294, VAR295, VAR375, VAR213 and VAR307(SEQ ID NOS: 6, 7, 8, 9 and 10) were studied in a protease screening test in a group of mini-pigs (Ellegaard). Pancreatic Exocrine Insufficiency (PEI) was induced in mini-pigs by ligation of the pancreatic duct, and they were also equipped with a ileo-cecal reentry cannula (both in isofluoran)Under anesthesia and at a weight of about 25 kg), as otherwise described in Tabeling et al (1999): "students on nutrient diagnostic agents (pre-capture and total) in recombinant product-derived pigs and the effects of enzyme catalysis", J.anim.Physiol.A.anim.Nutr.82: 251-263) and Gregory et al (1999): "Growth and differentiation in cultural products branched pigs, Effect of enzymology" in "Biology of the Pancodes in Growing Animals" (coded by SGPierzyynowski and R.Zabielski), Elsevier Science BV, Amsterdam, p.381-393). The study was allowed to recover from surgery for a period of at least 4 weeks before the study was started. Prior to the start of the study, the PEI status of each pig was confirmed via a fecal chymotrypsin test (commercially available from ImmundanagistikAG, Wiesenstrasse 4, D-64625 Bensheim, Germany under the product catalog number K6990).

Assay method

During the study, the ratio of 12: the pigs were housed in modified metabolic cages for 12h light-dark cycles, allowed free access to water, and were fed two meals per day.

Test meal

The test meal contained 21.3% protein, 51.9% starch, 2.6% fat and had the following composition (g/100g dry matter): fish meal 3.5, poultry meal 10.2, wheat flour 29.5, shell rice (shelled rice)14, potato starch 11, corn starch 14, casein 5.9, cellulose powder 4.3, vitamins, minerals and trace elements 7.6 (according to the nutritional needs of pigs/piglets, see e.g. table a of WO 01/58276).

Performance of

To evaluate the protease efficacy, pigs were fed 1 liter of water, 0.625g Cr₂O₃(chromium oxide marker) and varying amounts of SEQ ID NO: 1 (0mg, 20mg, 50mg and 120mg enzyme proteins, equal to 0, 500, 1250 and 3000 FIP U proteinsEnzyme/meal) of a single 250g test meal.

For the test itself, the protease variants of the invention were administered at the mg enzyme protein (20mg, 50mg and 120 mg/meal) dose to compare in vivo efficacy with the reference protease.

After the first appearance of dietary markers in the ileum (green chyme), ileal chyme was collected on ice for a total of 8 hours and stored at-20 ℃ prior to analysis. At least one day of rinsing was allowed between separate assays.

Analysis of

The frozen ileal chyme samples were freeze-dried, ground and analyzed for Dry Matter (DM) and crude protein. DM was assessed gravimetrically after freeze drying followed by incubation at 103 ℃ for 8 hours.

The crude protein was calculated by multiplying nitrogen (N) by a factor of 6.25, i.e.crude protein (g/kg) ═ N (g/kg) x6.25, as specified in Animal Nutrition, 4 th edition, Chapter 13 (P.McDonald, ed. R.A.Edwards and J.F.D.Greenhalgh eds., Longman Scientific and Technical, 1988, ISBN 0-582-. Nitrogen content was determined by the Dumas Combustion method (PG Wiles, IK Gray, RC kissing, J AOAC int.1998 May-Jun; 81 (3): 620-32) using a "Vario MAX CNS" elemental analyzer (Elementaranalysesme GmbH).

Cr₂O₃Oxidized to chromate and the chromium content is calculated via extinction at 365nm (spectrophotometer) as described by Petry and Rapp in Zeitung fur Tierphysiologiogie (1970), Vol.27, pp.181-189 (Petry and Rapp 1970; Z.Tierphysiol.27; 181-189).

Calculation of the apparent pre-cecal protein digestibility was performed by the marker method according to the following formula:

wherein Cr is₂O₃And protein expressed as g/100g dry matter.

In addition, the amount of protease (mg) required to achieve 50% and 60% protein digestibility (% CNA), respectively, was extrapolated from the individual regression curve (excel). In order to more effectively compare the 50% and 60% protein digestibility (% CNA) of the reference protease, a so-called improvement factor "IF" was calculated. IF50 and IF60 values for each protease variant were determined by dividing the amount of reference protease (mg) required to achieve 50% and 60% protein digestibility (% CNA) by the amount of variant protease (mg) required to achieve 50% and 60% protein digestibility (% CNA), respectively.

Results and conclusions

The ileal protein digestion results are shown in table 3 below. The protease dose is indicated in milligrams of enzyme protein per meal (mg/meal).

All variants mentioned in table 4 were also tested. They all have an IF of 0.8 or more.

Example 5: pharmaceutical protease compositions

Pill preparation

A liquid concentrate of protease variant VAR295(SEQ ID NO: 7) was prepared as described in example 2. The liquid concentrate was subjected to microbial filtration, spray-dried, and the protease protein content of the dried powder was measured. Preferably, the protease protein content is higher than 50% (according to regulatory requirements). 500g of dried protease powder was mixed with 200g of microcrystalline cellulose and 300g of polyethylene glycol 4000 (Macrogol)^TM4000) Pre-mixed together in a commercially available mixer. A sufficient amount of the commonly used wetting agent is added and the resulting wet mass (wet mass) is mixed well at room temperature. Then in commerceExtruders are available which extrude homogenized material, equipped with a punching die having a certain hole diameter (for example about 0.8mm) to form cylindrical pellets. The resulting extrudate was rounded into spherical pellets with a commercially available spheronizer (spheronizer) by adding the necessary amount of a commonly used wetting agent. The pellets were dried in a commercially available vacuum dryer at a product temperature of about 40 ℃. The dried pellets are then separated by using a mechanical sieving machine with a suitable size screen (e.g. with 0.7 and 1.4mm screens) to obtain the desired sieving fraction. The collected sieve fractions (e.g.. gtoreq.0.7 mm and. ltoreq.1.4 mm) are collected and the fractions containing the desired standardized active substance content are filled into capsules of suitable size.

The resulting pellets were tested for proteolytic activity by applying a modified FIP method against trypsin from pancreatic powder with the activation step omitted as described in example 1.

The resulting pellets were then tested for Disintegration according to the european pharmacopoeia 2.9.1 (section "Disintegration articles and capsules") (test solution: water-500 mL, 37 ℃).

Example 6: toxicity in vitro

In vitro screening for protease toxicity was performed using a cellular assay with human colon adenocarcinoma cell lines. The assay measures the metabolic capacity of the cell, and thus viability.

In vitro toxicity assay with HT-29 and Caco-2 cells

HT-29 cells (ACC 299 from German Collection of microorganisms and Cell Cultures, DSMZ) were cultured in McCoy's 5A medium (Cambrex) supplemented with 10% FBS (Sigma, Cat. catalog number F-6178). For this experiment, culture plates were incubated in 96 wells at 4. multidot.10⁴Cells were cultured at a density of 200. mu.l per well. After conditioning the cells for 24 hours per well, they were supplemented with 0.5g/l Probumin (Millipore), 1% insulin/transferrin/selenium supplement (I)nvitrogen) and 1% penicillin and streptomycin (Invitrogen) serum-free medium (DMEM: f12, Invitrogen) was added test components (protease) at 9 different concentrations (weight/volume enzyme protein) in triplicate at 2-fold dilutions and incubated for a further 24 hours. Viability was measured by the metabolic capacity of the cells, which was achieved by measurement using Alamar blue (Invitrogen). Caco-2 cells were cultured in DMEM (Invitrogen11960-044) supplemented with 10% fetal bovine serum, 2mM glutamine and 1% non-essential amino acids, and humidified 5% CO₂Culturing in the air. For this experiment, the assay was performed in a 96-well plate at 3.10⁴Cells were cultured at a density of 200. mu.l per well. After 24 hours of acclimation to each well, test components were added to serum-free medium to avoid binding of the protease by protease inhibitors present in the serum, and incubated for an additional 24 hours, after which viability was measured. Cell viability was measured by measuring the ability of cells to metabolize Alamar blue. All experiments for testing protease variants were done under serum-free conditions.

The maximum metabolic activity was observed in wells without any protease added (and set as 100%). The concentration at which 50% of the maximum metabolic activity was obtained for the tested protease was divided by the concentration at which 50% of the maximum metabolic activity was obtained for the reference protease and the resulting "toxicity ratio" was apparent from table 4 below. The higher the concentration, the less toxic; and the higher the ratio to the reference protease, the more toxicity is reduced. Thus, the toxicity of the protease VAR203 is reduced compared to the reference protease. Variants have been tested on both Caco-2 and HT-29 cells.

TABLE 4

Example 7: digestion Performance in vitro

Table of measurements in an in vitro digestion model comprising a1 hour pH3 (gastric) step and a2 hour pH6 (intestinal) step4(SEQ ID NO: 11) and other variants and compared to the performance of the reference protease (SEQ ID NO: 1). The protease was purified as described in example 2, and by A₂₈₀The enzyme protein content in mg/ml was measured.

The diet (which was the same as the test meal of example 4) was dissolved in 0.1M HCl to give a working slurry of 0.2g diet/ml. The pH was adjusted to pH 2.5 with HCl. Mu.l of the diet slurry was added to each well in MTP (microtiter plate) and mixed with 20. mu.l pepsin (Merck VL 317492437, Cat. No. 1.0792.0001, 700mg/l, final concentration 93. mu.g/ml) and 30. mu.l diluted enzyme (diluted to 10. mu.M (0.2 mg/ml)). The final pH in all wells was 2.8 to 3.0. Duplicate 4 concentrations (0.2, 0.1, 0.05 and 0.025mg/ml) were prepared for each enzyme (diluted in 20mM acetate, 0.01% Triton X-100, pH 5). This was incubated at 37 ℃ for 1 hour at 750rpm (Eppendorf thermostatic mixer) and defined as the gastric step of the in vitro digestion model.

To begin the intestinal step, the pH was increased to 6.0 to 6.05 by adding 25 μ l of buffer (0.8M MES, 0.8M imidazole, 0.8M acetate, 40%/60% mix, pH 5/9) to each well. In addition, 25. mu.l of bile salt (80 mM bile salt dissolved in deionized water, Millipore milliQ, bile salt mixture from Solvay pharmaceuticals, batch 176.01-PA-7374) was added to a final concentration of 10mM, followed by incubation at 37 ℃ for 2 hours at 750 rpm. Terminating the bowel step by: the protease was separated from the diet slurry by centrifugation at 2700rpm for 10 minutes at 4 ℃.

Protease activity was determined by quantifying free amino groups in the supernatant using the OPA method (o-phthalaldehyde). The number of free amino acids in the wells with enzyme minus the wells with "pepsin only" reflects the protease performance. The supernatant was diluted 10X in enzyme dilution buffer (20mM acetate pH5, 0.01% Triton X-100) and 20 μ l of the diluted supernatant was mixed with 200 μ l OPA reagent (3.81 g disodium tetraborate decahydrate, 1mL 10% SDS, 88mg DTT, and 80mg OPA dissolved in 2mL 96% ethanol was added followed by deionized water to a total volume of 100 mL). A serine dilution series (0.5 mg/ml stock solution diluted 2-fold) was included as a standard for quantification of free amino groups. The absorbance at 340nm was measured.

Calculation of the apparent Improvement Factor (IF) of the tested variants relative to the reference protease was performed by fitting the absolute data of the hydrolyzed amino groups (obtained by OPA assay) corrected for the absence of enzyme to a three-parameter logistic equation:

wherein NH₂Is the amount of free amino groups (mM), NH₂(Max) is the maximum amount of free amino groups that the protease can release from the diet, concentration is the protease concentration (mg enzyme per meal (250g)), the slope is the slope of the parallel curve (see below), and I (50) is the variable for calculating IF. Inverted V means exponent (exp). Experimental NH-reduction using very high doses of reference protease₂(max) was determined to 20 mM.

To fit the obtained data to this equation, two assumptions were made. First, it was assumed that all the curves obtained for hydrolyzed amino groups versus mg enzyme dosed were parallel (constant slope). Second, substrate availability is a limiting factor for activity, and thus a plateau in the amount of hydrolyzed amino groups (NH) is obtained at significantly higher enzyme concentrations₂(max)). The Improvement Factor (IF) is defined as:

IF ═ I (50) (reference)/I (50) (variant)

Wherein I (50) (reference) is for obtaining half NH₂(maximum) required concentration of reference enzyme and I (50) (variant) means to obtain half NH₂(maximum) required variant concentration.

The protease variant VAR203 has a factor of improvement of 2.6 and the reference protease (by definition) has a factor of improvement of 1.0. This means that 2.6times less (2.6 timekeeper around) of VAR203 protease is required to obtain a similar effect as the reference protease. All variants listed in table 4 have an IF above 0.7.

Example 8

Toxicology assessment Using a rat model of gastric catheterization

Test system

Experience has shown that following oral administration by protease gavage to rats, there is an increased risk of reflux (regorgitation) of the test article, followed by risk of unintended exposure to the lungs. Thus, a special experimental approach was used to distinguish the in vivo toxicity of the protease variant to the gastrointestinal tract from the wild type (SEQ ID No: 1). Gastric catheterized rats supplied by Charles River Laboratories Germany GmbH were used in these experiments. The test article is administered directly into the stomach via a gastric catheter for 14 days per day, which eliminates the risk of mis-dosing (mis-dosing) into the trachea and reduces the risk of reflux of the test article from the stomach. The volume administered was 10mL/kg for all proteases and food was withdrawn about 4 hours prior to administration and re-provided 4 hours after dose addition. Animals were housed individually and catheter blockage was prevented; after each application and once a week it was rinsed with tap water in the afternoon.

Measuring

Rats were observed individually before and after dose addition for behavioral changes, response to treatment or any signs of disease. The temperature of all animals was measured three times during the study with an anal probe before (pre-dose) and 1 hour after dose addition. Body weight and food consumption were measured at weekly intervals, while water consumption was measured by daily visual inspection of the water bottle.

At termination, all animals were subjected to detailed autopsy (autopsy) and histopathology was performed on potential target organs, including stomach, trachea and lung (studies).

Results

The mortality observed in this study was mainly related to reflux of the test article into the respiratory tract, but also to technical problems including leakage of the test article from the site of application (stomach). Mortality was most significant in the group treated with wild-type protease at the 700mg/kg dose level.

In histopathological examination, forestomach inflammation associated with squamous epithelial cell proliferation of the epithelium is considered a target toxicity. Based on the local effects induced by these test articles on the gastric mucosa, the wild-type protease was more toxic than the variants as a group.

In summary, combined mortality and histopathological data show that wild type is more toxic than variants.

Table 5: mortality of wild type and variants in 14-day toxicity studies

Protease enzyme	Dosage (mg/kg/day)	Dead/treated rats	% mortality
				Control (negative)		0/11	0
Wild type	700	4/6	67
				G12D	810	1/8	13
G12N，T22A，N23D，N47T，R165H	1000	0/6	0
				R14I，R38T，T151I	1000	0/6	0
N47H，G48D	1000	0/6	0

Table 6: histopathological stimulation findings in gastric catheterized male rats (given as the number of affected animals)

	Control (negative)	Wild type	G12D	G12N，T22A，N23D，N47T，R165H	R14I，R38T，T151I	N47H，G48D
							Dosage (mg/kg/day)		500	500/810	1000	1000	1000
Number of	11	17	8/8	6	6	6
							Squamous epithelial cell hyperplasia	0	11	0/0	3	6	5
Infiltration of monocytes	2	3	0/0	1	1	0
							Inflammation of the anterior stomach	0	8	0/0	1	1	2

Sequence listing

<110> Novozymes corporation (Novozymes A/S)

Sorvier Pharmaceuticals GmbH (Solvay Pharmaceuticals GmbH)

<120> protease variants for pharmaceutical use

<130>11144.204-WO

<160>11

<170>PatentIn version 3.4

<210>1

<211>188

<212>PRT

<213> Nocardiopsis sp

<220>

<221> mat _ peptide (mature peptide)

<222>(1)..(188)

<400>1

Ala Asp Ile Ile Gly Gly Leu Ala Tyr Thr Met Gly Gly Arg Cys Ser

1 5 10 15

Val Gly Phe Ala Ala Thr Asn Ala Ala Gly Gln Pro Gly Phe Val Thr

20 25 30

Ala Gly His Cys Gly Arg Val Gly Thr Gln Val Thr Ile Gly Asn Gly

35 40 45

Arg Gly Val Phe Glu Gln Ser Val Phe Pro Gly Asn Asp Ala Ala Phe

50 55 60

Val Arg Gly Thr Ser Asn Phe Thr Leu Thr Asn Leu Val Ser Arg Tyr

65 70 75 80

Asn Thr Gly Gly Tyr Ala Thr Val Ala Gly His Asn Gln Ala Pro Ile

85 90 95

Gly Ser Ser Val Cys Arg Ser Gly Ser Thr Thr Gly Trp His Cys Gly

100 105 110

Thr Ile Gln Ala Arg Gly Gln Ser Val Ser Tyr Pro Glu Gly Thr Val

115 120 125

Thr Asn Met Thr Arg Thr Thr Val Cys Ala Glu Pro Gly Asp Ser Gly

130 135 140

Gly Ser Tyr Ile Ser Gly Thr Gln Ala Gln Gly Val Thr Ser Gly Gly

145 150 155 160

Ser Gly Asn Cys Arg Thr Gly Gly Thr Thr Phe Tyr Gln Glu Val Thr

165 170 175

Pro Met Val Asn Ser Trp Gly Val Arg Leu Arg Thr

180 185

<210>2

<211>274

<212>PRT

<213> Artificial

<220>

<223> variants of Humicola lanuginosa lipase (T231R + N233R)

<220>

<221>PROPEP

<222>(1)..(5)

<220>

<221> mat _ peptide (mature peptide)

<222>(6)..(274)

<400>2

Ser Pro Ile Arg Arg Glu Val Ser Gln Asp Leu Phe Asn Gln Phe Asn

5 -1 1 5 10

Leu Phe Ala Gln Tyr Ser Ala Ala Ala Tyr Cys Gly Lys Asn Asn Asp

15 20 25

Ala Pro Ala Gly Thr Asn Ile Thr Cys Thr Gly Asn Ala Cys Pro Glu

30 35 40

Val Glu Lys Ala Asp Ala Thr Phe Leu Tyr Ser Phe Glu Asp Ser Gly

45 50 55

Val Gly Asp Val Thr Gly Phe Leu Ala Leu Asp Asn Thr Asn Lys Leu

60 65 70 75

Ile Val Leu Ser Phe Arg Gly Ser Arg Ser Ile Glu Asn Trp Ile Gly

80 85 90

Asn Leu Asn Phe Asp Leu Lys Glu Ile Asn Asp Ile Cys Ser Gly Cys

95 100 105

Arg Gly His Asp Gly Phe Thr Ser Ser Trp Arg Ser Val Ala Asp Thr

110 115 120

Leu Arg Gln Lys Val Glu Asp Ala Val Arg Glu His Pro Asp Tyr Arg

125 130 135

Val Val Phe Thr Gly His Ser Leu Gly Gly Ala Leu Ala Thr Val Ala

140 145 150 155

Gly Ala Asp Leu Arg Gly Asn Gly Tyr Asp Ile Asp Val Phe Ser Tyr

160 165 170

Gly Ala Pro Arg Val Gly Asn Arg Ala Phe Ala Glu Phe Leu Thr Val

175 180 185

Gln Thr Gly Gly Thr Leu Tyr Arg Ile Thr His Thr Asn Asp Ile Val

190 195 200

Pro Arg Leu Pro Pro Arg Glu Phe Gly Tyr Ser His Ser Ser Pro Glu

205 210 215

Tyr Trp Ile Lys Ser Gly Thr Leu Val Pro Val Arg Arg Arg Asp Ile

220 225 230 235

Val Lys Ile Glu Gly Ile Asp Ala Thr Gly Gly Asn Asn Gln Pro Asn

240 245 250

Ile Pro Asp Ile Pro Ala His Leu Trp Tyr Phe Gly Leu Ile Gly Thr

255 260 265

Cys Leu

<210>3

<211>481

<212>PRT

<213> Artificial

<220>

<223> variants of Bacillus licheniformis (Bacillus licheniformis) amylase

<220>

<221> mat _ peptide (mature peptide)

<222>(1)..(481)

<400>3

Val Asn Gly Thr Leu Met Gln Tyr Phe Glu Trp Tyr Thr Pro Asn Asp

1 5 10 15

Gly Gln His Trp Lys Arg Leu Gln Asn Asp Ala Glu His Leu Ser Asp

20 25 30

Ile Gly Ile Thr Ala Val Trp Ile Pro Pro Ala Tyr Lys Gly Thr Ser

35 40 45

Gln Ala Asp Val Gly Tyr Gly Ala Tyr Asp Leu Tyr Asp Leu Gly Glu

50 55 60

Phe His Gln Lys Gly Thr Val Arg Thr Lys Tyr Gly Thr Lys Gly Glu

65 70 75 80

Leu Gln Ser Ala Ile Lys Ser Leu His Ser Arg Asp Ile Asn Val Tyr

85 90 95

Gly Asp Val Val Ile Asn His Lys Gly Gly Ala Asp Ala Thr Glu Asp

100 105 110

Val Thr Ala Val Glu Val Asp Pro Ala Asp Arg Asn Arg Val Ile Ser

115 120 125

Gly Glu His Leu Ile Lys Ala Trp Thr His Phe His Phe Pro Gly Arg

130 135 140

Gly Ser Thr Tyr Ser Asp Phe Lys Trp Tyr Trp Tyr His Phe Asp Gly

145 150 155 160

Thr Asp Trp Asp Glu Ser Arg Lys Leu Asn Arg Ile Tyr Lys Phe Gln

165 170 175

Gly Lys Thr Trp Asp Trp Glu Val Ser Asn Glu Phe Gly Asn Tyr Asp

180 185 190

Tyr Leu Met Tyr Ala Asp Ile Asp Tyr Asp His Pro Asp Val Val Ala

195 200 205

Glu Ile Lys Arg Trp Gly Thr Trp Tyr Ala Asn Glu Leu Gln Leu Asp

210 215 220

Gly Phe Arg Leu Asp Ala Val Lys His Ile Lys Phe Ser Phe Leu Arg

225 230 235 240

Asp Trp Val Asn His Val Arg Glu Lys Thr Gly Lys Glu Met Phe Thr

245 250 255

Val Ala Glu Tyr Trp Ser Asn Asp Leu Gly Ala Leu Glu Asn Tyr Leu

260 265 270

Asn Lys Thr Asn Phe Asn His Ser Val Phe Asp Val Pro Leu His Tyr

275 280 285

Gln Phe His Ala Ala Ser Thr Gln Gly Gly Gly Tyr Asp Met Arg Lys

290 295 300

Leu Leu Asn Gly Thr Val Val Ser Lys His Pro Leu Lys Ser Val Thr

305 310 315 320

Phe Val Asp Asn His Asp Thr Gln Pro Gly Gln Ser Leu Glu Ser Thr

325 330 335

Val Gln Thr Trp Phe Lys Pro Leu Ala Tyr Ala Phe Ile Leu Thr Arg

340 345 350

Glu Ser Gly Tyr Pro Gln Val Phe Tyr Gly Asp Met Tyr Gly Thr Lys

355 360 365

Gly Asp Ser Gln Arg Glu Ile Pro Ala Leu Lys His Lys Ile Glu Pro

370 375 380

Ile Leu Lys Ala Arg Lys Gln Tyr Ala Tyr Gly Ala Gln His Asp Tyr

385 390 395 400

Phe Asp His His Asp Ile Val Gly Trp Thr Arg Glu Gly Asp Ser Ser

405 410 415

Val Ala Asn Ser Gly Leu Ala Ala Leu Ile Thr Asp Gly Pro Gly Gly

420 425 430

Ala Lys Arg Met Tyr Val Gly Arg Gln Asn Ala Gly Glu Thr Trp His

435 440 445

Asp Ile Thr Gly Asn Arg Ser Glu Pro Val Val Ile Asn Ser Glu Gly

450 455 460

Trp Gly Glu Phe His Val Asn Gly Gly Ser Val Ser Ile Tyr Val Gln

465 470 475 480

Arg

<210>4

<211>483

<212>PRT

<213> Artificial

<220>

<223> variants of Bacillus sp amylase

<220>

<221> mat _ peptide (mature peptide)

<222>(1)..(483)

<400>4

His His Asn Gly Thr Asn Gly Thr Met Met Gln Tyr Phe Glu Trp Tyr

1 5 10 15

Leu Pro Asn Asp Gly Asn His Trp Asn Arg Leu Arg Ser Asp Ala Ser

20 25 30

Asn Leu Lys Asp Lys Gly Ile Ser Ala Val Trp Ile Pro Pro Ala Trp

35 40 45

Lys Gly Ala Ser Gln Asn Asp Val Gly Tyr Gly Ala Tyr Asp Leu Tyr

50 55 60

Asp Leu Gly Glu Phe Asn Gln Lys Gly Thr Ile Arg Thr Lys Tyr Gly

65 70 75 80

Thr Arg Asn Gln Leu Gln Ala Ala Val Asn Ala Leu Lys Ser Asn Gly

85 90 95

Ile Gln Val Tyr Gly Asp Val Val Met Asn His Lys Gly Gly Ala Asp

100 105 110

Ala Thr Glu Met Val Lys Ala Val Glu Val Asn Pro Asn Asn Arg Asn

115 120 125

Gln Glu Val Ser Gly Glu Tyr Thr Ile Glu Ala Trp Thr Lys Phe Asp

130 135 140

Phe Pro Gly Arg Gly Asn Thr His Ser Asn Phe Lys Trp Arg Trp Tyr

145 150 155 160

His Phe Asp Gly Val Asp Trp Asp Gln Ser Arg Lys Leu Asn Asn Arg

165 170 175

Ile Tyr Lys Phe Arg Gly Lys Gly Trp Asp Trp Glu Val Asp Thr Glu

180 185 190

Phe Gly Asn Tyr Asp Tyr Leu Met Tyr Ala Asp Ile Asp Met Asp His

195 200 205

Pro Glu Val Val Asn Glu Leu Arg Asn Trp Gly Val Trp Tyr Thr Asn

210 215 220

Thr Leu Gly Leu Asp Gly Phe Arg Ile Asp Ala Val Lys His Ile Lys

225 230 235 240

Tyr Ser Phe Thr Arg Asp Trp Ile Asn His Val Arg Ser Ala Thr Gly

245 250 255

Lys Asn Met Phe Ala Val Ala Glu Phe Trp Lys Asn Asp Leu Gly Ala

260 265 270

Ile Glu Asn Tyr Leu Asn Lys Thr Asn Trp Asn His Ser Val Phe Asp

275 280 285

Val Pro Leu His Tyr Asn Leu Tyr Asn Ala Ser Lys Ser Gly Gly Asn

290 295 300

Tyr Asp Met Arg Gln Ile Phe Asn Gly Thr Val Val Gln Lys His Pro

305 310 315 320

Met His Ala Val Thr Phe Val Asp Asn His Asp Ser Gln Pro Glu Glu

325 330 335

Ala Leu Glu Ser Phe Val Glu Glu Trp Phe Lys Pro Leu Ala Tyr Ala

340 345 350

Leu Thr Leu Thr Arg Glu Gln Gly Tyr Pro Ser Val Phe Tyr Gly Asp

355 360 365

Tyr Tyr Gly Ile Pro Thr His Gly Val Pro Ala Met Lys Ser Lys Ile

370 375 380

Asp Pro Ile Leu Glu Ala Arg Gln Lys Tyr Ala Tyr Gly Arg Gln Asn

385 390 395 400

Asp Tyr Leu Asp His His Asn Ile Ile Gly Trp Thr Arg Glu Gly Asn

405 410 415

Thr Ala His Pro Asn Ser Gly Leu Ala Thr Ile Met Ser Asp Gly Ala

420 425 430

Gly Gly Asn Lys Trp Met Phe Val Gly Arg Asn Lys Ala Gly Gln Val

435 440 445

Trp Thr Asp Ile Thr Gly Asn Lys Ala Gly Thr Val Thr Ile Asn Ala

450 455 460

Asp Gly Trp Gly Asn Phe Ser Val Asn Gly Gly Ser Val Ser Ile Trp

465 470 475 480

Val Asn Lys

<210>5

<211>513

<212>PRT

<213> Artificial

<220>

<223> variants of Bacillus stearothermophilus amylase

<220>

<221> mat _ peptide (mature peptide)

<222>(1)..(486)

<400>5

Ala Ala Pro Phe Asn Gly Thr Met Met Gln Tyr Phe Glu Trp Tyr Leu

1 5 10 15

Pro Asp Asp Gly Thr Leu Trp Thr Lys Val Ala Asn Glu Ala Asn Asn

20 25 30

Leu Ser Ser Leu Gly Ile Thr Ala Leu Trp Leu Pro Pro Ala Tyr Lys

35 40 45

Gly Thr Ser Arg Ser Asp Val Gly Tyr Gly Val Tyr Asp Leu Tyr Asp

50 55 60

Leu Gly Glu Phe Asn Gln Lys Gly Thr Val Arg Thr Lys Tyr Gly Thr

65 70 75 80

Lys Ala Gln Tyr Leu Gln Ala Ile Gln Ala Ala His Ala Ala Gly Met

85 90 95

Gln Val Tyr Ala Asp Val Val Phe Asp His Lys Gly Gly Ala Asp Gly

100 105 110

Thr Glu Trp Val Asp Ala Val Glu Val Asn Pro Ser Asp Arg Asn Gln

115 120 125

Glu Ile Ser Gly Thr Tyr Gln Ile Gln Ala Trp Thr Lys Phe Asp Phe

130 135 140

Pro Gly Arg Gly Asn Thr Tyr Ser Ser Phe Lys Trp Arg Trp Tyr His

145 150 155 160

Phe Asp Gly Val Asp Trp Asp Glu Ser Arg Lys Leu Ser Arg Ile Tyr

165 170 175

Lys Phe Arg Gly Lys Ala Trp Asp Trp Glu Val Asp Thr Glu Phe Gly

180 185 190

Asn Tyr Asp Tyr Leu Met Tyr Ala Asp Leu Asp Met Asp His Pro Glu

195 200 205

Val Val Thr Glu Leu Lys Asn Trp Gly Lys Trp Tyr Val Asn Thr Thr

210 215 220

Asn Ile Asp Gly Phe Arg Leu Asp Ala Val Lys His Ile Lys Phe Ser

225 230 235 240

Phe Phe Pro Asp Trp Leu Ser Tyr Val Arg Ser Gln Thr Gly Lys Pro

245 250 255

Leu Phe Thr Val Gly Glu Tyr Trp Ser Tyr Asp Ile Asn Lys Leu His

260 265 270

Asn Tyr Ile Thr Lys Thr Asp Gly Thr Met Ser Leu Phe Asp Ala Pro

275 280 285

Leu His Asn Lys Phe Tyr Thr Ala Ser Lys Ser Gly Gly Ala Phe Asp

290 295 300

Met Arg Thr Leu Met Thr Asn Thr Leu Met Lys Asp Gln Pro Thr Leu

305 310 315 320

Ala Val Thr Phe Val Asp Asn His Asp Thr Glu Pro Gly Gln Ala Leu

325 330 335

Gln Ser Trp Val Asp Pro Trp Phe Lys Pro Leu Ala Tyr Ala Phe Ile

340 345 350

Leu Thr Arg Gln Glu Gly Tyr Pro Cys Val Phe Tyr Gly Asp Tyr Tyr

355 360 365

Gly Ile Pro Gln Tyr Asn Ile Pro Ser Leu Lys Ser Lys Ile Asp Pro

370 375 380

Leu Leu Ile Ala Arg Arg Asp Tyr Ala Tyr Gly Thr Gln His Asp Tyr

385 390 395 400

Leu Asp His Ser Asp Ile Ile Gly Trp Thr Arg Glu Gly Gly Thr Glu

405 410 415

Lys Pro Gly Ser Gly Leu Ala Ala Leu Ile Thr Asp Gly Pro Gly Gly

420 425 430

Ser Lys Trp Met Tyr Val Gly Lys Gln His Ala Gly Lys Val Phe Tyr

435 440 445

Asp Leu Thr Gly Asn Arg Ser Asp Thr Val Thr Ile Asn Ser Asp Gly

450 455 460

Trp Gly Glu Phe Lys Val Asn Gly Gly Ser Val Ser Val Trp Val Pro

465 470 475 480

Arg Lys Thr Thr Val Ser Thr Ile Ala Arg Pro Ile Thr Thr Arg Pro

485 490 495

Trp Thr Gly Glu Phe Val Arg Trp Thr Glu Pro Arg Leu Val Ala Trp

500 505 510

Pro

<210>6

<211>188

<212>PRT

<213> Artificial

<220>

<223> variant E125D of Nocardiopsis sp protease

<220>

<221> mat _ peptide (mature peptide)

<222>(1)..(188)

<400>6

Ala Asp Ile Ile Gly Gly Leu Ala Tyr Thr Met Gly Gly Arg Cys Ser

1 5 10 15

Val Gly Phe Ala Ala Thr Asn Ala Ala Gly Gln Pro Gly Phe Val Thr

20 25 30

Ala Gly His Cys Gly Arg Val Gly Thr Gln Val Thr Ile Gly Asn Gly

35 40 45

Arg Gly Val Phe Glu Gln Ser Val Phe Pro Gly Asn Asp Ala Ala Phe

50 55 60

Val Arg Gly Thr Ser Asn Phe Thr Leu Thr Asn Leu Val Ser Arg Tyr

65 70 75 80

Asn Thr Gly Gly Tyr Ala Thr Val Ala Gly His Asn Gln Ala Pro Ile

85 90 95

Gly Ser Ser Val Cys Arg Ser Gly Ser Thr Thr Gly Trp His Cys Gly

100 105 110

Thr Ile Gln Ala Arg Gly Gln Ser Val Ser Tyr Pro Asp Gly Thr Val

115 120 125

Thr Asn Met Thr Arg Thr Thr Val Cys Ala Glu Pro Gly Asp Ser Gly

130 135 140

Gly Ser Tyr Ile Ser Gly Thr Gln Ala Gln Gly Val Thr Ser Gly Gly

145 150 155 160

Ser Gly Asn Cys Arg Thr Gly Gly Thr Thr Phe Tyr Gln Glu Val Thr

165 170 175

Pro Met Val Asn Ser Trp Gly Val Arg Leu Arg Thr

180 185

<210>7

<211>188

<212>PRT

<213> Artificial

<220>

<223> variants of the Nocardiopsis sp protease R38T

<220>

<221> mat _ peptide (mature peptide)

<222>(1)..(188)

<400>7

Ala Asp Ile Ile Gly Gly Leu Ala Tyr Thr Met Gly Gly Arg Cys Ser

1 5 10 15

Val Gly Phe Ala Ala Thr Asn Ala Ala Gly Gln Pro Gly Phe Val Thr

20 25 30

Ala Gly His Cys Gly Thr Val Gly Thr Gln Val Thr Ile Gly Asn Gly

35 40 45

Arg Gly Val Phe Glu Gln Ser Val Phe Pro Gly Asn Asp Ala Ala Phe

50 55 60

Val Arg Gly Thr Ser Asn Phe Thr Leu Thr Asn Leu Val Ser Arg Tyr

65 70 75 80

Asn Thr Gly Gly Tyr Ala Thr Val Ala Gly His Asn Gln Ala Pro Ile

85 90 95

Gly Ser Ser Val Cys Arg Ser Gly Ser Thr Thr Gly Trp His Cys Gly

100 105 110

Thr Ile Gln Ala Arg Gly Gln Ser Val Ser Tyr Pro Glu Gly Thr Val

115 120 125

Thr Asn Met Thr Arg Thr Thr Val Cys Ala Glu Pro Gly Asp Ser Gly

130 135 140

Gly Ser Tyr Ile Ser Gly Thr Gln Ala Gln Gly Val Thr Ser Gly Gly

145 150 155 160

Ser Gly Asn Cys Arg Thr Gly Gly Thr Thr Phe Tyr Gln Glu Val Thr

165 170 175

Pro Met Val Asn Ser Trp Gly Val Arg Leu Arg Thr

180 185

<210>8

<211>188

<212>PRT

<213> Artificial

<220>

<223> variants of the protease of Nocardiopsis sp (T44K + S99P)

<220>

<221> mat _ peptide (mature peptide)

<222>(1)..(188)

<400>8

Ala Asp Ile Ile Gly Gly Leu Ala Tyr Thr Met Gly Gly Arg Cys Ser

1 5 10 15

Val Gly Phe Ala Ala Thr Asn Ala Ala Gly Gln Pro Gly Phe Val Thr

20 25 30

Ala Gly His Cys Gly Arg Val Gly Thr Gln Val Lys Ile Gly Asn Gly

35 40 45

Arg Gly Val Phe Glu Gln Ser Val Phe Pro Gly Asn Asp Ala Ala Phe

50 55 60

Val Arg Gly Thr Ser Asn Phe Thr Leu Thr Asn Leu Val Ser Arg Tyr

65 70 75 80

Asn Thr Gly Gly Tyr Ala Thr Val Ala Gly His Asn Gln Ala Pro Ile

85 90 95

Gly Ser Pro Val Cys Arg Ser Gly Ser Thr Thr Gly Trp His Cys Gly

100 105 110

Thr Ile Gln Ala Arg Gly Gln Ser Val Ser Tyr Pro Glu Gly Thr Val

115 120 125

Thr Asn Met Thr Arg Thr Thr Val Cys Ala Glu Pro Gly Asp Ser Gly

130 135 140

Gly Ser Tyr Ile Ser Gly Thr Gln Ala Gln Gly Val Thr Ser Gly Gly

145 150 155 160

Ser Gly Asn Cys Arg Thr Gly Gly Thr Thr Phe Tyr Gln Glu Val Thr

165 170 175

Pro Met Val Asn Ser Trp Gly Val Arg Leu Arg Thr

180 185

<210>9

<211>188

<212>PRT

<213> Artificial

<220>

<223> variant S69T of Nocardiopsis sp protease

<220>

<221> mat _ peptide (mature peptide)

<222>(1)..(188)

<400>9

Ala Asp Ile Ile Gly Gly Leu Ala Tyr Thr Met Gly Gly Arg Cys Ser

1 5 10 15

Val Gly Phe Ala Ala Thr Asn Ala Ala Gly Gln Pro Gly Phe Val Thr

20 25 30

Ala Gly His Cys Gly Arg Val Gly Thr Gln Val Thr Ile Gly Asn Gly

35 40 45

Arg Gly Val Phe Glu Gln Ser Val Phe Pro Gly Asn Asp Ala Ala Phe

50 55 60

Val Arg Gly Thr Thr Asn Phe Thr Leu Thr Asn Leu Val Ser Arg Tyr

65 70 75 80

Asn Thr Gly Gly Tyr Ala Thr Val Ala Gly His Asn Gln Ala Pro Ile

85 90 95

Gly Ser Ser Val Cys Arg Ser Gly Ser Thr Thr Gly Trp His Cys Gly

100 105 110

Thr Ile Gln Ala Arg Gly Gln Ser Val Ser Tyr Pro Glu Gly Thr Val

115 120 125

Thr Asn Met Thr Arg Thr Thr Val Cys Ala Glu Pro Gly Asp Ser Gly

130 135 140

Gly Ser Tyr Ile Ser Gly Thr Gln Ala Gln Gly Val Thr Ser Gly Gly

145 150 155 160

Ser Gly Asn Cys Arg Thr Gly Gly Thr Thr Phe Tyr Gln Glu Val Thr

165 170 175

Pro Met Val Asn Ser Trp Gly Val Arg Leu Arg Thr

180 185

<210>10

<211>188

<212>PRT

<213> Artificial

<220>

<223> variants of Nocardiopsis sp protease R165S

<220>

<221> mat _ peptide (mature peptide)

<222>(1)..(188)

<400>10

Ala Asp Ile Ile Gly Gly Leu Ala Tyr Thr Met Gly Gly Arg Cys Ser

1 5 10 15

Val Gly Phe Ala Ala Thr Asn Ala Ala Gly Gln Pro Gly Phe Val Thr

20 25 30

Ala Gly His Cys Gly Arg Val Gly Thr Gln Val Thr Ile Gly Asn Gly

35 40 45

Arg Gly Val Phe Glu Gln Ser Val Phe Pro Gly Asn Asp Ala Ala Phe

50 55 60

Val Arg Gly Thr Ser Asn Phe Thr Leu Thr Asn Leu Val Ser Arg Tyr

65 70 75 80

Asn Thr Gly Gly Tyr Ala Thr Val Ala Gly His Asn Gln Ala Pro Ile

85 90 95

Gly Ser Ser Val Cys Arg Ser Gly Ser Thr Thr Gly Trp His Cys Gly

100 105 110

Thr Ile Gln Ala Arg Gly Gln Ser Val Ser Tyr Pro Glu Gly Thr Val

115 120 125

Thr Asn Met Thr Arg Thr Thr Val Cys Ala Glu Pro Gly Asp Ser Gly

130 135 140

Gly Ser Tyr Ile Ser Gly Thr Gln Ala Gln Gly Val Thr Ser Gly Gly

145 150 155 160

Ser Gly Asn Cys Ser Thr Gly Gly Thr Thr Phe Tyr Gln Glu Val Thr

165 170 175

Pro Met Val Asn Ser Trp Gly Val Arg Leu Arg Thr

180 185

<210>11

<211>188

<212>PRT

<213> Artificial

<220>

<223> variants of the protease of Nocardiopsis sp (S69T + E125D)

<220>

<221> mat _ peptide (mature peptide)

<222>(1)..(188)

<400>11

Ala Asp Ile Ile Gly Gly Leu Ala Tyr Thr Met Gly Gly Arg Cys Ser

1 5 10 15

Val Gly Phe Ala Ala Thr Asn Ala Ala Gly Gln Pro Gly Phe Val Thr

20 25 30

Ala Gly His Cys Gly Arg Val Gly Thr Gln Val Thr Ile Gly Asn Gly

35 40 45

Arg Gly Val Phe Glu Gln Ser Val Phe Pro Gly Asn Asp Ala Ala Phe

50 55 60

Val Arg Gly Thr Thr Asn Phe Thr Leu Thr Asn Leu Val Ser Arg Tyr

65 70 75 80

Asn Thr Gly Gly Tyr Ala Thr Val Ala Gly His Asn Gln Ala Pro Ile

85 90 95

Gly Ser Ser Val Cys Arg Ser Gly Ser Thr Thr Gly Trp His Cys Gly

100 105 110

Thr Ile Gln Ala Arg Gly Gln Ser Val Ser Tyr Pro Asp Gly Thr Val

115 120 125

Thr Asn Met Thr Arg Thr Thr Val Cys Ala Glu Pro Gly Asp Ser Gly

130 135 140

Gly Ser Tyr Ile Ser Gly Thr Gln Ala Gln Gly Val Thr Ser Gly Gly

145 150 155 160

Ser Gly Asn Cys Arg Thr Gly Gly Thr Thr Phe Tyr Gln Glu Val Thr

165 170 175

Pro Met Val Asn Ser Trp Gly Val Arg Leu Arg Thr

180 185

Claims (19)

1. A protease that hybridizes to SEQ ID NO: 1, and which is at least 90% identical to amino acids 1-188 of SEQ ID NO: 1 comprises at least one substitution selected from the following substitutions: A1T; I3V; g12; R14I; T22A; N23D; G34A; r38; T41A; T44K; n47; G48D; E53K; Q54L, D; T68A, R, S; S69T; L73P; V88A; S99P; P124L; e125; M131V; T151I; r165; and T166A.

2. The protease according to claim 1, comprising at least one of the following substitutions: G12D, N, H; R38T; N47H, T, S; E125D and R165S, H, G, T.

3. The protease according to claim 2 comprising at least one of the following substitutions or combinations of substitutions: G12D; and (N47H + G48D).

4. The protease according to claim 1 comprising at least one of the following substitutions or combinations of substitutions: R38T, (T44K + S99P), S69T, (S69T + E125D), E125D, and R165S.

5. A protease according to claim 1 for use as a medicament.

6. The protease according to claim 1 in combination with a lipase or an amylase for use as a medicament.

7. The protease enzyme according to claim 6 in combination with a lipase or an amylase, wherein

(i) The lipase is similar to a lipase having the sequence shown in SEQ ID NO: 2, amino acids 1-269 is at least 70% identical; and/or

(ii) The amylase has at least 70% identity to an amylase selected from the group consisting of:

a) has the sequence shown in SEQ ID NO: 3 amino acids 1-481,

b) has the sequence shown in SEQ ID NO: 4 amino acids 1-483, and

c) has the sequence shown in SEQ ID NO: 5 amino acids 1-513.

8. The protease according to claim 1 for use in the treatment of digestive disorders, pancreatic exocrine insufficiency, pancreatitis, cystic fibrosis, diabetes type I and/or diabetes type II.

9. The protease according to claim 8 in combination with a lipase or an amylase.

10. The protease enzyme according to claim 9 in combination with a lipase or an amylase, wherein

(i) The lipase is similar to a lipase having the sequence shown in SEQ ID NO: 2, amino acids 1-269 is at least 70% identical; and/or

(ii) The amylase has at least 70% identity to an amylase selected from the group consisting of:

a) has the sequence shown in SEQ ID NO: 3 amino acids 1-481,

b) has the sequence shown in SEQ ID NO: 4 amino acids 1-483, and

c) has the sequence shown in SEQ ID NO: 5 amino acids 1-513.

11. A pharmaceutical composition comprising a protease as defined in claim 1 together with at least one pharmaceutically acceptable auxiliary material.

12. The composition according to claim 11, further comprising a lipase or an amylase.

13. A composition according to claim 12, wherein

(i) The lipase is similar to a lipase having the sequence shown in SEQ ID NO: 2, amino acids 1-269 is at least 70% identical; and/or

(ii) The amylase has at least 70% identity to an amylase selected from the group consisting of:

a) has the sequence shown in SEQ ID NO: 3 amino acids 1-481,

b) has the sequence shown in SEQ ID NO: 4 amino acids 1-483, and

c) has the sequence shown in SEQ ID NO: 5 amino acids 1-513.

14. A method for the treatment of digestive disorders, pancreatic exocrine insufficiency, pancreatitis, cystic fibrosis, diabetes type I, and/or diabetes type II, by administering a therapeutically effective amount of a protease as defined in any one of claims 1-2.

15. The method according to claim 14, further comprising administering a therapeutically effective amount of a lipase or an amylase.

16. The method of claim 15, wherein

(i) The lipase is similar to a lipase having the sequence shown in SEQ ID NO: 2, amino acids 1-269 is at least 70% identical; and/or

(ii) The amylase has at least 70% identity to an amylase selected from the group consisting of:

a) has the sequence shown in SEQ ID NO: 3 amino acids 1-481,

b) has the sequence shown in SEQ ID NO: 4 amino acids 1-483, and

c) has the sequence shown in SEQ ID NO: 5 amino acids 1-513.

17. Use of a protease as defined in claim 1 for the preparation of a medicament for the treatment of digestive disorders, pancreatic exocrine insufficiency, pancreatitis, cystic fibrosis, diabetes type I, and/or diabetes type II.

18. Use according to claim 17, further comprising the use of a lipase or an amylase.

19. Use according to claim 18, wherein

(i) The lipase is similar to a lipase having the sequence shown in SEQ ID NO: 2, amino acids 1-269 is at least 70% identical; and/or

(ii) The amylase has at least 70% identity to an amylase selected from the group consisting of:

a) has the sequence shown in SEQ ID NO: 3 amino acids 1-481,

b) has the sequence shown in SEQ ID NO: 4 amino acids 1-483, and

c) has the sequence shown in SEQ ID NO: 5 amino acids 1-513.