NOVEL TREATMENTS

Abstract

The present invention provides polypeptides having protease activity for use in the treatment or prevention of microbial infections in a subject with or susceptible to immunodeficiency. For example, the polypeptide may be administered as a mouth spray, nasal spray, lozenge, pastille, chewing gum or liquid to treat or prevent microbial infections in a patient with primary immunodeficiency. In particular, the polypeptides are useful in the treatment or prevention of rhinorrhea and/or fungal infection of the oral cavity and/or gum sores. In one embodiment, the polypeptide is a trypsin enzyme from Atlantic cod, or a fragment or variant thereof.

Claims

1. A method of treating or preventing microbial infections in a subject with or susceptible to an immunodeficiency comprising administering to said subject a polypeptide having protease activity.

2. The method according to claim 1 wherein the protease is selected from the group consisting of serine proteases, threonine proteases, cysteine proteases, aspartate proteases, glutamic acid proteases and metalloproteases.

3. The method according to claim 2 wherein the protease is a serine protease.

4. The method according to claim 3 wherein the protease is a trypsin or chymotrypsin.

5. The method according to claim 1 wherein the immunodeficiency is a secondary or acquired immunodeficiency.

6. The method according to claim 5 wherein the subject is receiving treatment with an immunosuppressant therapy.

7. (canceled)

8. The method according to any claim 1 wherein the immunodeficiency is naturally-occurring and/or is primary.

9. The method according to claim 1 wherein the immunodeficiency is due to a primary immunodeficiency, a cancer chronic infection malnutrition and/or aging.

10.-12. (canceled)

13. The method according to claim 1 wherein said polypeptide treats or prevents rhinorrhea and/or fungal infection of the oral cavity and/or gum sores.

14. The method according to claim 1 wherein the microbial infection is selected from the group consisting of bacterial infections, viral infections, fungal infections and yeast infections.

15. The method according to claim 1 further comprising administering one or more additional anti-microbial treatments.

16. The method according to claim 1 wherein the subject is human.

17. (canceled)

18. (canceled)

19. The method according to claim 1 wherein the polypeptide comprises or consists of an amino acid sequence of SEQ ID NO: 1: TABLE-US-00018 [SEQIDNO:1] IVGGYECTKHSQAHQVSLNSGYHFCGGSLVSKDWVVSAAHCYKSVLRVRL GEHHIRVNEGTEQYISSSSVIRHPNYSSYNINNDIMLIKLTKPATLNQYV HAVALPTECAADATMCTVSGWGNTMSSVADGDKLQCLSLPILSHADCANS YPGMITQSMFCAGYLEGGKDSCQGDSGGPVVCNGVLQGVVSWGYGCAERD HPGVYAKVCVLSGWVRDTMANY or a fragment, variant, derivative or fusion thereof (or a fusion of said fragment, variant or derivative) which retains the trypsin activity of said amino acid sequence.

20.-26. (canceled)

27. The method according to claim 19 wherein the variant is a non-naturally occurring variant.

28. The method according to claim 27 wherein the variant has an amino acid sequence which has at least 50% identity with the amino acid sequence according to SEQ ID NO: 1 or a fragment thereof, for example at least 55%, 60%, 65%, 70%, 75%, 80%, 90%, 95%, 96%, 97%, 98% or at least 99% identity.

29. (canceled)

30. (canceled)

31. The method according to claim 1 wherein the polypeptide having protease activity is selected from the group of polypeptides in Table 3, or comprises or consists of an amino acid sequence of SEQ ID NO: 2 or 3, or a fragment thereof which exhibits an antimicrobial activity.

32. (canceled)

33. (canceled)

34. The method according to claim 19 wherein the polypeptide, or fragment, variant, fusion or derivative thereof, comprises or consists of L-amino acids.

35. The method according to claim 19 wherein the polypeptide, or fragment, variant, fusion or derivative thereof, comprises one or more amino acids that are modified or derivatised.

36. (canceled)

37. The method according to claim 1 wherein the polypeptide is between 10 and 30 amino acids in length, is between 150 and 250 amino acids in length, for example between 200 and 250, 210 and 240, 220 and 230, or 220 and 225 amino acids in length.

38.-41. (canceled)

42. The method according to claim 1 wherein the polypeptide is provided in a mouth spray, nasal spray, lozenge, pastille, chewing gum or liquid.

43.-56. (canceled)

Description

[0207] Preferred, non-limiting examples which embody certain aspects of the invention will now be described.

[0208] FIGS. 1A-1B. Percentage of various infection symptoms per week for a 12-year old male patient diagnosed with CVID and treated weekly with subcutaneous injections of Hizentra (human IgG). Baseline data compiled during 2012 and from January to September of 2013. ColdZyme treatment was maintained for 9 weeks from October to November of 2013.

[0209] FIGS. 2A-2B. Average days per week spent at home from school for a 12-year old male patient diagnosed with CVID and treated weekly with subcutaneous injections of Hizentra (IgG). Baseline data compiled during 2012 and from January to September of 2013. ColdZyme treatment was maintained for 9 weeks from October to November of 2013.

EXAMPLES

Example A

Exemplary Therapeutic Formulation

[0210] An exemplary stock solution of a polypeptide of the invention, trypsin I from Atlantic cod (SEQ ID NO:1), may be formulated as shown in Table A:

TABLE-US-00012 TABLE A Item description Quantity Purified water 50 L Glycerol 50 L Tris buffer stock solution 1 L Trypsin I from Atlantic cod 300 000 U

[0211] The pH is adjusted to 7.5.

[0212] A suitable therapeutic composition of trypsin I from Atlantic cod (SEQ ID NO:1) is also available commercially as ColdZyme (Enzymatica AB, Lund, Sweden).

Example B

Case Study

[0213] Patient: 12-year old male

[0214] Diagnosis: CVID (Common variable immunodeficiency)

[0215] Treatment history: Hizentra, subcutaneous immunoglobulin (Human) Amoxicillin, oral

[0216] Treatment: ColdZyme, 1U per dose, 2 doses per day

[0217] Since 2003, the subject had received weekly subcutaneous injections of human immunoglobulin (Hizentra, 4 g weekly). However, prior to treatment with the polypeptide of the invention in late 2013, the subject suffered recurrent microbial infections of the ears, sinuses, nose, bronchi and lungs. The subject frequently exhibited continuous rhinorrhoea, fungal growth in the oral cavity and gingivitis with wounds in gum. As a consequence, the subject's quality of life had been severally compromised and he usually needed to stay at home from school at least one day every week. The month of November was often particularly challenging month for the subject since the recurrent infections often developed into pneumonia.

[0218] A period of prophylactic treatment with amoxicillin from August 2012 to May 2013 had little effect on the subject's recurrent symptoms.

[0219] The subject commenced twice daily treatment (morning and evening) with ColdZyme mouth spray in October 2013; weekly administration of Hizentra was continued throughout this period.

[0220] Within just three days of commencement of ColdZyme treatment, the subject experienced a marked improvement in symptoms and quality of life (see Table B):

TABLE-US-00013 TABLE B After three days treatment Symptom Before ColdZyme with ColdZyme Rhinorrhoea Continuous No Rhinorrhoea Stuffed nose Not possible to breathe Breathing through nose through nose Oral fungal High Very low infection Wounds in gum High Very low (healed for the first tissue time on several years)

[0221] The effect of treatment with ColdZyme can also be seen clearly from FIGS. 1A, 1B, 2A and 2B.

[0222] FIG. 1(A) shows the percentage of various infection symptoms per week experienced by the subject during three time periods: [0223] (a) During the whole of 2012 (treatment=Hizentra and amoxicillin); [0224] (b) From January to September 2013 (treatment=Hizentra and amoxicillin); and [0225] (c) From October 2013 to November 2013 (treatment=Hizentra and ColdZyme).

[0226] The dramatic reduction in all the observed infection symptoms during the period of treatment with ColdZyme is immediately evident.

[0227] FIG. 1(B) shows the percentage of various infection symptoms per week experienced by the same subject during an extended study period of 58 weeks

[0228] FIG. 2(A) shows the average number of days per week the subject was absent from school due to the severity of infection symptoms during the same three time periods in FIGS. 1A-1B. The dramatic improvement following commencement of treatment with ColdZyme is again immediately evident.

[0229] Such a quick onset of action and near total eradication of infection symptoms in the subject by treatment with the polypeptide of the invention was wholly unexpected, particularly given that the initial treatment period (OctoberNovember) coincided with the time of year during which the subject typically experienced the most severe infections. Understandably, both the subject and his mother were elated at the improvement in quality of life following over ten years of debilitating recurrent infections.

[0230] FIG. 2(B) shows the average number of days per week the subject was absent from school due to the severity of infection symptoms during the same extended study period in FIG. 1(B).

Example C

Production of Recombinant Serine Protease Polypeptides

[0231] Cloning

[0232] A synthesized gene encoding the serine protease polypeptide of interest was cloned into E. coli expression E3 vector (GenScript) without any tag.

[0233] Nucleic acid encoding wildtype trypsin I from Atlantic cod is shown below in SEQ ID NO: 4 (in pUC57)

TABLE-US-00014 [SEQIDNO:4] 1 GAAGAAGATAAAATCGTTGGCGGCTATGAATGCACGAAACACTCGCAGGCACACCAGGTC 61 TCACTGAACAGCGGTTACCACTTTTGCGGCGGTAGTCTGGTTAGCAAAGATTGGGTTGTT 121 AGTGCGGCCCATTGCTATAAAAGCGTGCTGCGTGTTCGCCTGGGCGAACATCACATTCGT 181 GTGAATGAAGGCACCGAACAGTACATTAGCTCTAGTAGCGTTATCCGCCATCCGAACTAC 241 TCTAGTTACAACATCAACAACGATATCATGCTGATCAAACTGACCAAACCGGCGACGCTG 301 AACCAGTATGTGCACGCCGTTGCACTGCCGACCGAATGCGCAGCGGATGCAACCATGTGT 361 ACCGTGAGCGGCTGGGGTAATACGATGAGCTCTGTTGCGGATGGCGATAAACTGCAGTGC 421 CTGTCTCTGCCGATTCTGAGTCATGCGGATTGTGCCAACTCTTATCCGGGCATGATCACG 481 CAGAGCATGTTTTGCGCCGGTTACCTGGAAGGCGGTAAAGATAGCTGCCAGGGTGATTCT 541 GGCGGTCCGGTGGTTTGTAACGGCGTTCTGCAGGGTGTGGTTAGCTGGGGCTACGGTTGT 601 GCAGAACGTGATCACCCGGGTGTCTATGCTAAAGTCTGTGTGCTGTCGGGCTGGGTCCGT 661 GATACGATGGCGAACTAT

[0234] A number of nucleic acid molecules encoding mutated versions of trypsin I from Atlantic cod were synthesised by conventional techniques, i.e. directed mutagenesis by PCR.

[0235] Refolding and Purification of Trypsin

[0236] Chemically competent E. coli BL21 (DE3) cells were transformed with the E3 vector containing the nucleotide sequence encoding the serine protease polypeptide (trypsin) of interest using standard procedures, i.e. heat shock transformation.

[0237] The zymogen polypeptide (trypsinogen) was overexpressed and formed inclusion bodies in the cytoplasm of the host cells.

[0238] The cells after induction were harvested and lysed by sonication. After centrifugation, inclusion bodies were washed in buffer (50 mM Tris, 10 mM EDTA, 2% Triton X-100, 300 mM NaCl, 2 mM DTT, pH8.0) and dissolved in 50 mM Tris, 8M Urea, pH8.0 and then dialyzed into 1 PBS, 10% Glycerol,pH7.4 at 4 C. overnight.

[0239] The purity of the expressed zymogen polypeptide from refolding exhibited >90% purity and no other purification was deemed necessary.

[0240] The recombinant zymogen polypeptide was then activated by adding wildtype trypsin I purified from Atlantic cod (0.2 U/ml) and incubating at room temperature for 24 hours (see Example D).

[0241] Exemplary Polypeptides

[0242] The following polypeptides were obtained or produced: [0243] (a) Wldtype trypsin I purified from Atlantic cod (WT-Tryp or wildtype); [0244] (b) Recombinantly expressed wildtype trypsin I of Atlantic cod (SEQ ID NO:1, R-Tryp); and [0245] (c) Thirty-eight different mutated versions of trypsin I of Atlantic cod (i.e. mutated sequences of SEQ ID NO:1).

[0246] The sequence mutations of the thirty-eight different mutated versions of cod trypsin I are shown in Table C below.

TABLE-US-00015 TABLE C Sequences of exemplary trypsin polypeptides Mutations relative to Polypeptide name SEQ ID NO: 1* EZA-001 (none) EZA-002 D217S, T218N, S69K EZA-003 K133T EZA-004 K133L EZA-005 K133V EZA-006 K133E EZA-007 N80T EZA-008 I81L EZA-009 L165G, P202Y EZA-010 V189I EZA-011 Y194D, M154K EZA-012 Y194H EZA-013 Y194S EZA-014 A206V EZA-015 H10Y EZA-016 H10N EZA-017 H14Y EZA-018 H53D EZA-019 H54N EZA-020 R56K EZA-021 R56E EZA-022 N58T EZA-023 N58L, Y64F EZA-024 T61S EZA-025 T61N EZA-026 K32E, D33Q EZA-027 S69R EZA-028 E6T, H53D, D130S, K133V EZA-029 S158N, V210N EZA-030 M115Q EZA-031 M125K, V128G EZA-032 M154Q EZA-033 L46I, S67A EZA-034 L139I EZA-035 V1181, L139AA162V EZA-036 V103I EZA-037 V30I, V215I, M219I EZA-038 V215I EZA-039 L211Y *the amino acid numbering is according to Protein Data Bank (PDB) entry 2EEK!

[0247] The exemplary trypsin polypeptides were initially expressed as a zymogen polypeptide with the activation peptide MEEDK (SEQ ID NO: 5) fused to the N-terminus.

Example D

Stability of Wildtype and Mutant Forms of Trypsin I of Atlantic Cod, Expressed Recombinantly

[0248] This example summarizes the results from the activation of 39 recombinant trypsin mutants expressed in E. coli. The activity of the recombinant trypsin polypeptides (R-Tryp) was activated by wildtype trypsin I purified from Atlantic cod (WT-Tryp) after a 24 hours incubation.

[0249] Materials & Methods

[0250] Expression of Recombinant Trypsins

[0251] See Example C

[0252] Assessment of Stability

[0253] The experiment designed for the activation and stability analysis of the recombinant samples was performed as follows:

[0254] Day 1: Activation of recombinant trypsin

[0255] Recombinant enzymes (0.2 U/ml) were activated by wild type trypsin (0.2 U/ml) at room temperature during 24 hours in a microtiter plate. The samples were mixed with 20mM Tris-HCl, 1 mM CaCl.sub.2, 50% glycerol, pH 7.6 to a final volume of 200 l.

[0256] Day 2: Activity and stability measurements

[0257] The activated recombinant enzymes were transferred to a new microtiter plate (II) and kept on ice to keep the enzymes stable and stop the activation process.

[0258] (a) Determination of initial activity A0

[0259] The activity of the activated enzyme (A0) was determined in a new microtiter plate (III) by mixing 245 l of Gly-Pro-Arg in assay buffer, with 5 l of recombinant enzyme from microtiter plate (II). The absorbance at 410 nm was followed and the activity was calculated according to the following formula:

[00001]$\begin{array}{cc}U.Math.\text{/}.Math.\mathrm{ml}=\mathrm{.Math.}.Math..Math.\mathrm{mol}.Math.\text{/}.Math.L.s=\frac{{\mathrm{Slope}}_{410.Math..Math.n.Math..Math.m}}{{\mathrm{.Math.}}^{*}.Math.I^{*}}*\mathrm{df}*60*{10}^3& \left(1\right)\end{array}$

[0260] where slope is the slope of the linear regression from the kinetic measurement of the trypsin activity at 30 C. during 200 seconds; df is the dilution factor, 60 is the conversion of seconds to minutes, c is the extension coefficient equal to 8800 M.sup.1 cm.sup.1, I is the length of the light path equal to 0.7109 cm, 10.sup.3 is the conversion mol/l to mol/ml.

[0261] (b) Temperature inactivation

[0262] 100 l of the activated enzyme was transferred from microtiter plate (II) to a new microtiter plate (IV) and diluted to 200 l to a final concentration of 50% glycerol, pH 7.6. Plate (IV) was incubated at 60 C. for 3.5 hours (WT-Tryp loses 90% of the initial activity). The remaining activity was determined as under (a).

[0263] Day 3: Autocatalysis

[0264] 100 l of the activated enzyme was transferred from microtiter plate (II) to a new microtiter plate (V) containing 100 l of 0.1 U/ml trypsin in 25% glycerol and assay buffer, pH 7.6. The plate was incubated at 25 C. for 8 hours (WT-Tryp loses 90% of the initial activity). The activity (A.sub.AX) was determined as described under (a).

[0265] Results

[0266] Activity, thermostability and autocatalysis of thirty-nine exemplary serine protease polypeptides is reported in Table D (recombinant wildtype cod trypsin, EZA-001, and thirty-eight mutants thereof). There is a considerable difference in activity among the mutants. Several mutants expressed improved temperature stability in comparison to wildtype trypsin that only had 5% remaining activity and several mutants showed substantially improved autocatalytic stabilities in comparison to wildtype trypsin.

TABLE-US-00016 TABLE D Activity of 39 exemplary serine protease polypeptides Thermostability: Autocatalytic stability: Remaining activity after Remaining activity after Relative Relative Initial activity inactivation at 60 C., Ax inactivation at 25 C., Ac thermostability autocatalytic Sample ID A0 (U/mg) (U/ml) (U/ml) (Ax/A0) stability (Ac/A0) EZA001 0.52 0.10 0.07 0.20 0.13 EZA002 0.48 0.05 0.01 0.11 0.02 EZA003 0.52 0.09 0.05 0.18 0.09 EZA004 0.48 0.09 0.04 0.19 0.07 EZA005 0.36 0.07 0.02 0.19 0.06 EZA006 0.39 0.10 0.03 0.26 0.07 EZA007 0.30 0.10 0.01 0.33 0.04 EZA008 0.36 0.11 0.09 0.31 0.26 EZA009 0.35 0.06 0.03 0.16 0.09 EZA010 0.44 0.12 0.12 0.28 0.28 EZA011 0.36 0.10 0.11 0.28 0.31 EZA012 0.35 0.13 0.11 0.37 0.31 EZA013 0.36 0.07 0.05 0.20 0.13 EZA014 0.41 0.10 0.07 0.25 0.17 EZA015 0.39 0.09 0.11 0.24 0.27 EZA016 0.36 0.22 0.21 0.60 0.56 EZA017 0.35 0.14 0.15 0.41 0.42 EZA018 0.39 0.05 0.02 0.13 0.04 EZA019 0.37 0.10 0.10 0.27 0.27 EZA020 0.39 0.07 0.03 0.19 0.08 EZA021 0.38 0.11 0.09 0.30 0.23 EZA022 0.49 0.09 0.17 0.18 0.34 EZA023 0.39 0.18 0.16 0.47 0.41 EZA024 0.43 0.09 0.07 0.21 0.17 EZA025 0.39 0.17 0.14 0.43 0.37 EZA026 0.33 0.15 0.15 0.44 0.46 EZA027 0.34 0.10 0.06 0.30 0.17 EZA028 0.35 0.16 0.18 0.45 0.51 EZA029 0.35 0.16 0.16 0.46 0.45 EZA030 0.33 0.16 0.11 0.50 0.33 EZA031 0.40 0.14 0.15 0.35 0.36 EZA032 0.44 0.07 0.02 0.16 0.03 EZA033 0.42 0.12 0.10 0.27 0.24 EZA034 0.41 0.13 0.11 0.31 0.27 EZA035 0.42 0.12 0.16 0.29 0.38 EZA036 0.38 0.11 0.13 0.30 0.34 EZA037 0.36 0.10 0.16 0.27 0.44 EZA038 0.41 0.08 0.05 0.20 0.12 EZA039 0.38 0.10 0.04 0.26 0.11 Wildtype 0.16 0.01 0.01 0.05 0.08

Example E

Activity Measurement of Recombinant Mutated Forms of Cod Trypsin I

[0267] Materials & Methods

[0268] Expression of Recombinant Polypeptides

[0269] Polypeptides corresponding to the wildtype amino acid sequence of trypsin I from Atlantic cod and thirty-eight mutated versions thereof were produced using the methods described in Example C.

[0270] Activation

[0271] Activation of recombinant enzymes (approximately 0.01 mg/ml) was achieved by adding wild type trypsin (0.2 U/ml) at room temperature and incubate for 24 hours. The mixture was made in 20 mM Tris-HCl, 1 mM CaCl.sub.2, 50% glycerol, pH 8.0 to a final volume of 200 l.

[0272] Activity Assay to Determine Kinetic Constants

[0273] The substrate (Gly-Pro-Arg) was used at concentrations 0.005-0.15 mM in assay buffer containing 1% DMSO. 245 L of substrate solutions were pipetted into a 96-well plate. The reaction was started by adding 5 L of the sample mixture (above) and monitored at 410 nm in a SpectraMax plate reader. Kinetic measurement was performed every minute of a continuous 15-min run.

[0274] Results

[0275] The results are shown in Table E below.

TABLE-US-00017 TABLE E Sample Parameter Value Relative to WT-Trp WT-Trp Vmax (Kcat) 0.05372 100 (purified) Km 0.00125 100 Vmax/Km 43.07 100 EZA-001 Vmax 0.05309 99 Km 0.00087 70 Vmax/Km 61.10 142 EZA-002 Vmax 0.05292 99 Km 0.00110 88 Vmax/Km 48.09 112 EZA-003 Vmax 0.05162 96 Km 0.00050 40 Vmax/Km 104.07 242 EZA-004 Vmax 0.04380 82 Km 0.00123 99 Vmax/Km 35.47 82 EZA-005 Vmax 0.05162 96 Km 0.00094 75 Vmax/Km 54.95 128 EZA-006 Vmax 0.05289 98 Km 0.00095 76 Vmax/Km 55.81 130 EZA-007 Vmax 0.05313 99 Km 0.00114 91 Vmax/Km 46.72 108 EZA-008 Vmax 0.05084 95 Km 0.00083 67 Vmax/Km 60.90 141 EZA-009 Vmax 0.05287 98 Km 0.00087 70 Vmax/Km 60.98 142 EZA-010 Vmax 0.05046 94 Km 0.00085 68 Vmax/Km 59.44 138 EZA-011 Vmax 0.05045 94 Km 0.00077 62 Vmax/Km 65.55 152 EZA-012 Vmax 0.04208 78 Km 0.00101 81 Vmax/Km 41.74302 97 EZA-013 Vmax 0.05006 93 Km 0.00068 55 Vmax/Km 73.65 171 EZA-014 Vmax 0.05177 96 Km 0.00083 66 Vmax/Km 62.64 145 EZA-015 Vmax 0.05060 94 Km 0.00085 68 Vmax/Km 59.36 138 EZA-016 Vmax 0.05378 100 Km 0.00103 83 Vmax/Km 51.99 121 EZA-017 Vmax 0.05457 102 Km 0.00104 83 Vmax/Km 52.53124 122 EZA-018 Vmax 0.05962 111 Km 0.00198 159 Vmax/Km 30.04 70 EZA-019 Vmax 0.05408 101 Km 0.00115 92 Vmax/Km 47.21 110 EZA-020 Vmax 0.04421 82 Km 0.00095 76 Vmax/Km 46.77 109 EZA-021 Vmax 0.05309 99 Km 0.00128 103 Vmax/Km 41.41 96 EZA-022 Vmax 0.05436 101 Km 0.00119 95 Vmax/Km 45.85 106 EZA-023 Vmax 0.05470 102 Km 0.00137 110 Vmax/Km 40.06 93 EZA-024 Vmax 0.05120 95 Km 0.00098 78 Vmax/Km 52.36 122 EZA-025 Vmax 0.05145 96 Km 0.00090 72 Vmax/Km 57.43 133 EZA-026 Vmax 0.05042 94 Km 0.00084 68 Vmax/Km 59.70 139 EZA-027 Vmax 0.05195 97 Km 0.00094 76 Vmax/Km 55.01 128 EZA-028 Vmax 0.04167 78 Km 0.00076 61 Vmax/Km 54.60 127 EZA-029 Vmax 0.05058 94 Km 0.00091 73 Vmax/Km 55.40 129 EZA-030 Vmax 0.05109 95 Km 0.00080 65 Vmax/Km 63.47 147 EZA-031 Vmax 0.05174 96 Km 0.00103 83 Vmax/Km 50.07 116 EZA-032 Vmax 0.06226 116 Km 0.00246 197 Vmax/Km 25.31 59 EZA-033 Vmax 0.05942 111 Km 0.00166 133 Vmax/Km 35.80 83 EZA-034 Vmax 0.05672 106 Km 0.00144 116 Vmax/Km 39.29 91 EZA-035 Vmax 0.05807 108 Km 0.00162 130 Vmax/Km 35.79 83 EZA-036 Vmax 0.04887 91 Km 0.00210 168 Vmax/Km 23.28 54 EZA-037 Vmax 0.05754 107 Km 0.00172 138 Vmax/Km 33.54 78 EZA-038 Vmax 0.05786 108 Km 0.00157 126 Vmax/Km 36.74 85