NUTRITIONALLY-OPTIMIZED COLLAGEN PEPTIDE

Abstract

The present invention relates to a nutritionally optimized synthetic or recombinant collagen peptide comprising at least one amino acid sequence motif (glycine-X-Y).sub.n, and the collagen peptide according to the invention for use in a method for therapeutic treatment of the human or animal body and products containing the collagen peptide according to the invention.

Claims

1. A synthetic or recombinant collagen peptide comprising at least one amino acid sequence motif (glycine-X-Y).sub.n, wherein X and Y for each amino acid sequence motif (glycine-X-Y) are each independently a naturally occurring amino acid, wherein n is an integer >1, and wherein the collagen peptide contains at least 1.02% sulfur-containing amino acids, at least 0.73% histidine, at least 1.02% isoleucine, at least 2.24% leucine, at least 2.07% lysine, at least 1.1% threonine, at least 0.28% % tryptophan, at least 1.91% tyrosine and/or phenylalanine, and at least 1.3% valine (in each case % by weight based on the total weight of the collagen peptide).

2. The synthetic or recombinant collagen peptide according to claim 1, wherein the collagen peptide comprises 10 to 30% proline (based on the total amount of amino acids of the collagen peptide).

3. The synthetic or recombinant collagen peptide according to claim 1, wherein the collagen peptide comprises 20 to 45% glycine (based on the total amount of amino acids of the collagen peptide).

4. The synthetic or recombinant collagen peptide according to claim 1, wherein X and Y for each amino acid sequence motif (glycine-X-Y) are, independently from one another, an amino acid selected from the group consisting of isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, valine, histidine, cysteine, tyrosine and proline.

5. The synthetic or recombinant collagen peptide according to claim 1, comprising one of the amino acid sequences SEQ ID No. 1 or 2.

6. The synthetic or recombinant collagen peptide according to claim 1, comprising an amino acid sequence according to SEQ ID No. 3 or 4.

7. The synthetic or recombinant collagen peptide according to claim 1, comprising an amino acid sequence according to SEQ ID no. 5 to 28, in particular 7 to 10.

8. The synthetic or recombinant collagen peptide according to claim 1, wherein the collagen peptide has a Protein Digestibility Corrected Amino Acid Score (PDCAAS) of at least 0.4, preferably at least 0.5, preferably at least 0.8, particularly preferably 1.

9. The synthetic or recombinant collagen peptide according to claim 1, wherein the collagen peptide comprises at least 10% leucine (% by weight based on the total weight of the collagen peptide).

10. The synthetic or recombinant collagen peptide according to claim 1, wherein the collagen peptide is hydroxylated.

11. The synthetic or recombinant collagen peptide according to claim 1, wherein the collagen peptide is glycosylated.

12. A method for therapeutic treatment of a human or animal subject, comprising administering to the subject the synthetic or recombinant collagen peptide according to claim 1.

13. A pharmaceutical composition comprising at least one collagen peptide according to claim 1 and at least one pharmaceutically acceptable additive.

14. A food supplement, food or luxury food item comprising at least one collagen peptide according to claim 1 and at least one food-acceptable additive.

15. A cosmetic product comprising at least one collagen peptide according to claim 1 and at least one skin-friendly additive.

Description

[0133] The invention is described below without restricting the general inventive concept on the basis of exemplary sequences, figures, and embodiments.

[0134] In the following:

[0135] FIG. 1A shows the stimulation of the collagen synthesis of human dermal fibroblasts by the collagen peptides according to the invention of SEQ ID No. 7, SEQ ID No. 8, SEQ ID No. 9 and SEQ ID No. 10 in comparison to a mixture of collagen peptides obtained from natural sources with a comparable average molecular weight (Verisol) according to Example 10. The FIGURE shows the factor for the quantitative determination of collagen synthesis compared to an untreated sample. The error bars show the standard error (SEM).

[0136] FIG. 1B shows the stimulation of the synthesis of proteoglycans of human dermal fibroblasts by the collagen peptides according to the invention of SEQ ID No. 7, SEQ ID No. 8, SEQ ID No. 9 and SEQ ID No. 10 in comparison to a mixture of collagen peptides obtained from natural sources with a comparable average molecular weight (Verisol) according to Example 10. The FIGURE shows the factor for the quantitative determination of collagen synthesis compared to an untreated sample. The error bars show the standard error (SEM).

EXAMPLES

Example 1Solid Phase Synthesis

[0137] Collagen peptides according to the invention of the SEQ ID No. 5 to 12 were obtained by solid phase synthesis (Merrifield synthesis) on a polystyrene resin.

Example 2Recombinant Production

[0138] Collagen peptides according to the invention were also obtained by recombinant expression in Pichia pastoris.

Example 3Level of Essential Amino Acids

[0139] For the synthetically or recombinantly produced hydroxylated and non-hydroxylated collagen peptides of SEQ ID No. 5 to 12 from example 1 and SEQ ID No. 19 to 22 (hydroxyproline-containing forms of SEQ ID No. 5, 6, 11 and 12) the percentage (% by weight) of essential amino acids was calculated. The results are shown in Table 3.

TABLE-US-00003 TABLE3 Syntheticallyorrecombinantlyproducedcollagen peptidesaccordingtotheinvention Percentageofessentialamino acids(%byweightbased onthetotalweightofthe SEQIDNo. Aminoacidsequence collagenpeptide) 5 GVPGYTGIKGFLGIPGYPGTQGLPGLNG CYS+MET 3.6 MPGWHGLFGLPGPKGMTGKVGPKGIF HIS 4.0 GAPGKDGVRGLTGPIGPPGPAGAPGDK ILE 4.4 GEAGPSGPFGPTGLCGVPGWHGYPGH LEU 7.9 K LYS 8.7 THR 5.0 TRP 3.7 TYR+PHE 10.5 VAL 3.8 6 GLTGFMGILGYVGPKGPTGNHGCPGPH CYS+MET 3.7 GPIGKFGSKGPLGWTGPIGKPGEVGPM HIS 4.1 GPTGPLGWKGAPGADGPAGAPGTPGP ILE 4.5 QGILGYRGVVGLPGFKGYHGFPGLK LEU 7.9 LYS 8.9 THR 5.1 TRP 3.7 TYR+PHE 10.7 VAL 3.9 7 GL[P]GKMGVFGLTGPIGP[P]GPWGV[P] CYS+MET 4.1 GHKGYLGPT HIS 4.3 ILE 3.5 LEU 10.7 LYS 8.1 THR 6.4 TRP 5.8 TYR+PHE 9.7 VAL 6.3 8 GL[P]GKYGVHGLTGPLGP[P]GPMGI[P] CYS+MET 4.1 GWKGFVGPT HIS 4.3 ILE 3.5 LEU 10.7 LYS 8.1 THR 6.4 TRP 5.8 TYR+PHE 9.7 VAL 6.3 9 GP[P]GPLGMKGL[P]GVWGPFGL[P]GTP CYS+MET 3.8 GPHGITGYKGV HIS 4.0 ILE 3.3 LEU 9.9 LYS 7.3 THR 6.0 TRP 5.2 TYR+PHE 8.8 VAL 5.9 10 GP[P]GPVGTKGL[P]GFYGPLGI[P]GHPG CYS+MET 3.8 PWGMLGTKGV HIS 4.0 ILE 3.3 LEU 9.9 LYS 7.3 THR 6.0 TRP 5.2 TYR+PHE 8.8 VAL 5.9 11 GMTGKVGPKGIFGAPGKDGVRGLTGPI CYS+MET 4.0 GPPGPAGAPGDKGEAGPSGPFGPTGL HIS 2.4 CGVPGWHGYP ILE 3.9 LEU 3.9 LYS 9.0 THR 5.2 TRP 3.2 TYR+PHE 7.9 VAL 5.1 12 GLTGFMGILGYVGPKGPTGNHGCPGPH CYS+MET 4.5 GPIGKFGSKGPLGWTGPIGKPGEVGPM HIS 3.4 GPTGPLGWKGAPGADGPAGAPGTPGP ILE 5.7 QGILGY LEU 7.0 LYS 7.9 THR 6.3 TRP 4.6 TYR+PHE 7.6 VAL 2.4 19 GV[P]GYTGIKGFLGI[P]GY[P]GTQGL[P] CYS+MET 3.6 GLNGM[P]GWHGLFGLPGPKGMTGKVG HIS 4.0 PKGIFGA[P]GKDGVRGLTGPIGP[P]GPA ILE 4.4 GA[P]GDKGEAGPSG[P]FGPTGLCGV[P] LEU 7.9 GWHGY[P]GHK LYS 8.7 THR 5.0 TRP 3.7 TYR+PHE 10.5 VAL 3.8 20 GLTGFMGILGYVGPKGPTGNHGC[P]GP CYS+MET 3.7 HGPIGKFGSKGPLGWTGPIGK[P]GEVG HIS 4.1 PMGPTGPLGWKGA[P]GADGPAGA[P]G ILE 4.5 TPGPQGILGYRGVVGL[P]GFKGYHGF[P] LEU 7.9 GLK LYS 8.9 THR 5.1 TRP 3.7 TYR+PHE 10.7 VAL 3.9 21 GMTGKVGPKGIFGA[P]GKDGVRGLTGP CYS+MET 4.0 IGP[P]GPAGA[P]GDKGEAGPSG[P]FGPT HIS 2.4 GLCGV[P]GWHGY[P] ILE 3.9 LEU 3.9 LYS 9.0 THR 5.2 TRP 3.2 TYR+PHE 7.9 VAL 5.1 22 GLTGFMGILGYVGPKGPTGNHGC[P]GP CYS+MET 4.5 HGPIGKFGSKGPLGWTGPIGK[P]GEVG HIS 3.4 PMGPTGPLGWKGA[P]GADGPAGA[P]G ILE 5.7 TPGPQGILGY LEU 7.0 LYS 7.9 THR 6.3 TRP 4.6
[P] denotes 4-hydroxyproline; essential amino acids are shown in bold.

Example 4Nutritional Optimization of Bovine ?1 Chain of Collagen Type I

[0140] Starting from the natural amino acid sequence of the ?1 chain of bovine collagen type I, a conservative amino acid exchange of non-essential amino acids was carried out to provide a nutritionally optimized collagen peptide, i.e. non-essential hydrophobic amino acids were preferably exchanged for hydrophobic essential amino acids. The exchange for polar, aliphatic and aromatic amino acids was carried out in the same way. Care was also taken to ensure that the amino acid motif (Gly-X-Y) remains intact and that the prolines or 4-hydroxyprolines located at the variable positions X and Y in the natural sequence are not exchanged. For the approx. 90 kDa collagen peptide according to the invention obtained in this way, comprising 1014 amino acids (SEQ ID No. 13) and the two halves each comprising 507 amino acids (SEQ ID No. 14 and 15), the percentage of essential amino acids (% by weight based on the total weight of the collagen peptide) were calculated (see Table 4)

TABLE-US-00004 TABLE 4 Nutritionally optimized collagen peptides derived from bovine Type I collagen Percentage of essential amino acids (% by weight based on the total SEQ ID No. weight of the collagen peptide) 13 CYS + MET 4.2 HIS 3.5 ILE 4.6 LEU 8.6 LYS 8.3 THR 4.7 TRP 3.6 TYR + PHE 11.2 VAL 4.0 14 CYS + MET 4.6 HIS 3.9 ILE 5.2 LEU 9.3 LYS 8.2 THR 4.6 TRP 3.0 TYR + PHE 10.9 VAL 3.9 15 CYS + MET 3.6 HIS 3.6 ILE 4.2 LEU 7.7 LYS 8.2 THR 4.6 TRP 3.8 TYR + PHE 11.3 VAL 3.9

Example 5Nutritionally Optimized Collagen Peptide with Increased Leucine Content

[0141] The essential amino acid leucine is of particular importance for muscle building. For the greatest possible effect of the amino acid on muscle-building, a quantity of at least 100 mg per g of total protein is recommended. To provide a nutritionally optimized collagen peptide with an increased leucine content, starting from a nutritionally optimized collagen peptide derived from the ?1 chain of bovine type I collagen according to SEQ ID No. 13, a further amino acid exchange was carried out in order to achieve a leucine content of at least 100 mg/g total protein (SEQ ID NO. 16). SEQ ID NO. 17 and 18 are the two 507 amino acid halves of the collagen peptide according to SEQ ID no. 16.

TABLE-US-00005 TABLE 5 Collagen peptides according to the invention with an increased leucine content Percentage of essential amino acids (% by weight based on the total SEQ ID No. weight of the collagen peptide) 16 CYS + MET 4.2 HIS 3.0 ILE 4.7 LEU 12.1 LYS 8.3 THR 4.7 TRP 3.4 TYR + PHE 11.2 VAL 4.0 17 CYS + MET 4.6 HIS 3.1 ILE 5.2 LEU 12.1 LYS 8.2 THR 4.6 TRP 3.0 TYR + PHE 11.0 VAL 3.9 18 CYS + MET 3.7 HIS 2.8 ILE 4.2 LEU 12.0 LYS 8.3 THR 4.7 TRP 3.8 TYR + PHE 11.4 VAL 4.0

Example 6Nutritionally Optimized Collagen Peptides of the Bovine ?1-Chain of Collagen Type I with a Defined Minimum PDCAAS Value

[0142] As described in Example 4, based on the natural amino acid sequence of the al chain of bovine collagen type I, a conservative amino acid exchange of non-essential amino acids was carried out, i.e. non-essential hydrophobic amino acids were preferably exchanged for hydrophobic essential amino acids. The exchange for polar, aliphatic and aromatic amino acids was carried out in the same way. Care was also taken to ensure that the amino acid motif (Gly-X-Y) remains intact and that the prolines or 4-hydroxyprolines located at the variable positions X and Y in the natural sequence are not exchanged. The aim of the amino acid exchange was to provide collagen peptides with a PDCAAS value of at least 0.4 or at least 0.6 (each determined according to Table 1). As in Example 4, the percentage of essential amino acids (% by weight based on the total weight of the collagen peptide) was calculated (see Table 6) for the collagen peptides according to the invention with a size of approx. 90 kDa, comprising 1014 amino acids (PDCAAS value at least 0.4: SEQ ID No. 23, PDCAAS value at least 0.6: SEQ ID No. 26) and for the two halves each containing 507 amino acids (PDCAAS value at least 0.4: SEQ ID No. 24 and 25, PDCAAS value at least 0.6: SEQ ID No. 27 and 28).

TABLE-US-00006 TABLE 6 Nutritionally optimized collagen peptides derived from bovine collagen type I with a PDCAAS value of at least 0.4 (SEQ ID No. 23, 24, 25) and PDCAAS value of at least 0.6 (SEQ ID No. 26, 27, 28) Percentage of essential amino acids (% by weight based on the total SEQ ID No. weight of the collagen peptide) 23 CYS + MET 1.2 HIS 0.9 ILE 1.4 LEU 2.6 LYS 5.1 THR 1.8 TRP 0.8 TYR + PHE 2.7 VAL 1.8 24 CYS + MET 1.4 HIS 0.9 ILE 1.2 LEU 2.7 LYS 5.2 THR 1.6 TRP 0.8 TYR + PHE 3.0 VAL 1.5 25 CYS + MET 1.1 HIS 0.9 ILE 1.5 LEU 2.5 LYS 4.9 THR 2.0 TRP 0.8 TYR + PHE 2.4 VAL 2.0 26 CYS + MET 1.5 HIS 1.8 ILE 2.1 LEU 4.3 LYS 5.0 THR 2.1 TRP 0.8 TYR + PHE 4.7 VAL 2.3 27 CYS + MET 1.6 HIS 1.5 ILE 2.5 LEU 4.2 LYS 5.1 THR 2.0 TRP 0.8 TYR + PHE 4.6 VAL 2.2 28 CYS + MET 1.3 HIS 2.1 ILE 1.8 LEU 4.5 LYS 4.8 THR 2.2 TRP 0.8 TYR + PHE 4.7 VAL 2.4

Example 7Bone Health

[0143] To analyze the biological activity of the collagen peptide according to the invention in terms of maintaining bone health and the prophylaxis and treatment of bone diseases, its stimulating effect on the synthesis of extracellular matrix proteins and enzymes that play a role in the structure and mineralization of the matrix is examined via osteoblasts in vitro. This is done by determining the expression of the corresponding mRNA by means of real-time PCR and a semi-quantitative evaluation (based on a control without collagen peptide).

[0144] For this purpose, human osteoblasts are first isolated from knee joints by incubating bone material under vigorous agitation at 37? C. for 1 h in Hanks' solution, supplemented with 7 mg/ml hyaluronidase type I and III-S and 5 mg/ml pronase. The digestion is then continued at 37? C. for 3-5 h in Hanks' solution supplemented with 16 mg/ml collagenase type CLS IV. The primary osteoblasts obtained are cultivated after enzymatic digestion in Ham's F12 medium, supplemented with 10% fetal calf serum, 20 U/ml penicillin-streptomycin, 50 ?g/ml partricin, 0.05 mg/ml ascorbic acid and 0.15 mg/ml glutamine. Alternatively, primary osteoblasts (Article No. C-12760; 2019) may also be obtained from PromoCell GmbH, Heidelberg, Germany, for investigating the biological effectiveness. The cells are then cultivated in Ham's F12 medium, supplemented with 10% fetal calf serum, 20 U/ml penicillin-streptomycin, 50 ?g/ml partricin and 0.15 mg/ml glutamine.

[0145] To investigate the biological activity, monolayer cell cultures of the isolated human osteoblasts are incubated for a period of 24 hours in a medium that is supplemented with 0.5 mg/ml of the respective collagen peptide. A control is incubated in each case in a medium without peptide. The respective mRNA expression is then determined.

Example 8Skin Health

[0146] The stimulation of the synthesis of collagen (type I) and the proteoglycans biglycan and versican is investigated in vitro on human dermal fibroblasts (skin cells). For this purpose, the cells are incubated for 24 hours with 0.5 mg/ml of a low molecular weight or the collagen peptide according to the invention, and the expression of collagen RNA, biglycan RNA and versican RNA is then determined by real-time PCR and semi-quantitatively (based on a control without peptide).

Example 9Cartilage Health

[0147] For the cell cultures, porcine or human chondrocytes are isolated from cartilage tissue in a known manner and sown on culture plates at a density of approximately 350,000 cells/cm 2. Ham's F12 medium with 10% fetal calf serum, 10 ?g/ml gentamicin and 5 ?g/ml amphotericin B is used as the culture medium. As an alternative to 10 ?g/ml gentamicin, 10 ?g/ml penicillin-streptomycin may also be used. The cultivation took place at 37? C. in an oxygen-reduced atmosphere (5% O.sub.2, 5% CO.sub.2 and 90% N2).

[0148] Determination of Collagen Biosynthesis:

[0149] The quantification of the collagen synthesized by the chondrocytes (essentially type II) is carried out by radioactive labeling with .sup.14C-proline, which is incorporated into the collagen.

[0150] .sup.14C-proline is first added to the culture medium and the chondrocytes are cultivated under these conditions until the time of the determination. In order to be able to distinguish the incorporated from non-incorporated .sup.14C-proline during the detection, the isotope-containing culture medium is then replaced by pure culture medium for a period of 3 days. The culture medium is then discarded and the adherent cell layer is mixed with distilled water in order to destroy the cell membranes through osmotic stress and to release cytosolic, unbounded .sup.14C-proline. The cell debris with the synthesized extracellular matrix is pelleted by centrifugation. The pellet is re-suspended in fresh distilled water and a xylene scintillation cocktail is added. The amount of synthesized collagen may then be quantified by detecting the .sup.14C-Proline with a beta counter.

[0151] Alternatively, the quantification may be carried out using the Sircol Collagen Assay Kit (Article No. 054S5000, 2019, tebu-bio, Offenbach, Germany, or Biocolor Ltd., UK) according to manufacturer instructions (see Example 10).

[0152] Determination of Proteoglycan Biosynthesis:

[0153] The proteoglycans synthesized by the chondrocytes are quantified by means of Alcian blue staining and photometric determination of the glycosaminoglycans (GAG), which are components of the proteoglycans.

[0154] In order to determine the GAG content in the cell culture, the culture medium is first discarded and the adherent cell layer is rinsed with PBS buffer (pH 7). The cells are then fixed in a 10% formaldehyde solution in PBS at 4? C. for 2 hours. After removing the formaldehyde, the Alcian blue staining reagent (5% Alcian blue in 3% acetic acid) is applied to the cell lawn and incubated at 4? C. overnight. Unbound Alcian blue is discarded and washed out by carefully rinsing three to four times with PBS. By adding acidic guanidine solution (8 mol/I), the GAG complexes are released from the cell layer. The amount of glycosaminoglycans may then be quantified photometrically at a wavelength of 620 nm.

[0155] Alternatively, the quantification may be carried out using the Blyscan Glycosaminoglycan Assay Kit (Article No. 05463000, 2019, tebu-bio, Offenbach, Germany, or Biocolor Ltd., UK) according to manufacturer instructions (see Example 10).

Example 10Influence of Synthetic Collagen Peptides According to the Invention on the Biosynthesis of Matrix Proteins

[0156] Cell Culture:

[0157] The human cells used were obtained from tebu-bio GmbH, Offenbach, Germany. The chondrocytes (Cat. No. 402-05a) or dermal fibroblasts (Cat. No. 106-05a) were first sown in 12-well culture plates and cultured at 37? C., 5% CO.sub.2 in Ham's F12 medium, to which 10% fetal calf serum, 20 U/ml penicillin-streptomycin and 50 ?g/ml ascorbic acid were added. On every other day, the culture medium was replaced by new culture medium until the cells had reached 80% confluence. To investigate the influence of the collagen peptides according to the invention of SEQ ID No. 7, 8, 9 and 10 on the biosynthesis of matrix proteins, the cell culture medium was then replaced by a special stimulation medium to which 0.5 mg/ml of the specific collagen peptides according to the invention were added.

[0158] Collagen Assay:

[0159] To investigate the collagen metabolism, the amount of newly synthesized collagen was determined after stimulating the cells with 0.5 mg/ml BCP for three weeks. The synthesized collagen was isolated using the Sircol assay (Article No. 054S5000, 2019, tebu-bio, Offenbach, Germany, or Biocolor Ltd., UK) according to the manufacturer's instructions. In brief, the culture medium was initially discarded and the adherent cell layers were digested with 0.1 mg pepsin solution in 0.5 M acetic acid at 4? C. overnight. The cell suspensions were neutralized by adding 100 ?l of an acid-neutralizing reagent. The synthesized collagen was then separated off by adding 200 ?l of an isolation and concentration solution with vigorous shaking at 4? C. overnight. After centrifugation (12,000 rpm, 10 min) and discarding the supernatant, the isolated collagen was resuspended in 1 ml of Sircol dye solution. After 30 minutes of shaking and another centrifugation, the collagen pellet was covered with 750 ?l of cold acidic solid washing reagent. After another centrifugation, the supernatant was again discarded and the enriched collagen was taken up in 250 ?l of alkaline solution. In each case 200 ?l of the sample solutions were used for photometric quantification of the synthesized collagen. The absorbance was measured at a wavelength of 492 nm. The amount of collagen synthesized in each case was determined on the basis of standardized collagen solutions.

[0160] An increased collagen synthesis by dermal fibroblasts compared to untreated controls could be found, which dermal fibroblasts were incubated with the hydroxylated collagen peptides according to the invention of SEQ ID No. 7 (27% increase), SEQ ID No. 8 (22% increase), SEQ ID No. 9 (21% increase), SEQ ID No. 10 (21% increase) (FIG. 1A).

[0161] Proteoglycan Assay:

[0162] The Blyscan glycosaminoglycan assay (Article No. 05463000, 2019, tebu-bio, Offenbach, Germany, or Biocolor Ltd., UK) was used to determine proteoglycans. The biosynthesis of proteoglycans was determined after stimulating dermal fibroblasts for two weeks. According to the manufacturer's instructions, after discarding the culture medium, the cell layers were covered with 1 ml of papain extraction solution and incubated for three hours at 65? C. with vigorous shaking. The cell suspensions were then centrifuged (10,000 g, 10 min) and the supernatants were collected. After adding 1 ml of Blyscan dye solution and shaking (30 min), the supernatants were centrifuged again (12,000 rpm, 10 min) and then discarded. The isolated proteoglycan pellets were resuspended in 500 ?l of dissociation solution. A photometric determination of the synthesized proteoglycan in 200 ?l sample solution was carried out at a wavelength of 656 nm in comparison to untreated control experiments.

[0163] As seen in FIG. 1B, the synthesis of proteoglycans by dermal fibroblasts was significantly increased in comparison to untreated controls when the fibroblasts were incubated with collagen peptides according to the invention of SEQ ID No. 7 (26% increase), SEQ ID No. 8 (22% increase), SEQ ID No. 9 (21% increase), SEQ ID No. 10 (22% increase).