WO2001041794A2

WO2001041794A2 - Activated amylase

Info

Publication number: WO2001041794A2
Application number: PCT/FR2000/003445
Authority: WO
Inventors: Jean Chatillon; Paul Chislain Coupan; Murielle Trinquier
Original assignee: Laboratoire Top-Pharm
Priority date: 1999-12-10
Filing date: 2000-12-08
Publication date: 2001-06-14
Also published as: WO2001041794A3; FR2802100A1; FR2802100B1; AU2525101A

Abstract

The invention relates to a novel composition which is characterized in that it contains alpha-amylase and a calcium salt. The enzymatic activity of the amylase is considerably increased and prolonged by several weeks when the amylase and the calcium salt are present in specified relative concentrations. The alpha-amylase is advantageously produced by Bacillus amyloliquefaciens. The calcium salt is especially calcium chloride.

Description

ACTIVATED AMYLASE

The subject of the present invention is a new composition which is remarkable in that it contains an association of amylase of bacterial origin and of a calcium salt having a high amylolytic activity for several weeks.

The enzymes of the family of amylases and in particular the alpha-amylases, are known

10 long-standing and used in various applications. Indeed, thanks to their amylolytic activity these ferments are capable of degrading cellulosic and starchy fractions to reduce them to the state of simple sugars. The mechanism by which alpha-amylase degrades polysaccharide compounds into mono and disaccnaπdes is a hydrolysis reaction where the enzyme plays the role of catalyst. It is this function that has often been * 5 exploited in different ways.

For example, amylases are used for their beneficial action on the digestion of food. US Patent 3,956,483 describes a composition prepared from defatted and dried pancreas, rich in pancreatic juices called "pancreatin". Pancreatin

^2υ contains different enzymes, in particular proteases, lipases and amylases. It is used to aid digestion in patients with chronic pancreatic insufficiency or cystic fibrosis. However, the amylase activity of pancreatin is too weak to be used as it is and requires the addition of an activating additive to achieve acceptable efficacy. Furthermore, this preparation is only intended for a

²⁵ palliative use of intestinal disorders, since the pancreatic extract contains many substances, not only enzymatic anyway, which have a very specific overall effect in the digestion of food, but are undesirable for any other use.

US Patent 4,740,368 describes a breath-purifying product comprising an alpha-amylase, - ^ intended to eliminate food debris trapped in the inter-dental spaces and to limit the development of the bacterial colonies causing unpleasant oral odors. It is an alpha-amylase which can be ingested which is used, for example of fungal origin, or extracted from pancreatic juices from cattle, or the like. The recommended dose is limited due to the gastrointestinal disorders usually associated with the ingestion of amylase.

35

To the disadvantages already noted for the use of amylase, it is necessary to add a certain number of others which prevented their use in particular in the pharmaceutical industry. Unpurified pancreatic extracts, but also purified amylases originating from certain organisms, cause immune reactions in some patients. Cases of allergy to alpha-amylase from the filamentous fungus Aspergillus orizae are described in the literature.

The alpha-amylase finding its applications in demanding fields from the point of view of the criteria of purity, harmlessness, safety, it is essential to control well the physicochemical characteristics of the enzymatic preparations as well as the allergenic properties of the protein enzymatic itself or impurities which may accompany them.

Some of these criteria are met when using alpha-amylase in drugs. Indeed, amylases are also used for their anti-inflammatory and anti-edematous properties, as well as for their antibacterial and antiviral properties which have been detected more recently. Their action is attributed to the fact that they attack substrates rich in carbohydrates, in particular the cell wall and the membrane of many bacteria. Likewise, amylase degrades the carbohydrate constituents present in the cell envelope of viral particles. A composition for therapeutic use has been proposed, intended to treat in particular infections of the oropharyngeal sphere (French patent application No. 99 14102). It is a purified alpha-amylase of bacterial origin which is used in this composition, for which no allergy is reported in the literature.

However, it has been found that the amylase activity decreases over time, which raises problems when it is possible to keep preparations based on alpha-amylase long enough to be able to market them while guaranteeing a level of activity. high and therefore a certain therapeutic effect.

This decrease significantly affects the enzyme when it is dissolved. In fact, if the crystallized forms retain at least 80% of their activity after three years, the alpha-amylase solutions lose almost 90% of their activity in a few days.

The fragility of enzymatic substances is well known and is explained by their rapid denaturation as soon as the physico-chemical conditions such as pH, temperature, are modified. Studies have attempted to elucidate the mechanisms of action and inhibition of alpha-amylases in their natural physiological environments, at the cellular or tissue level, but very little work has been devoted to the conditions making industrial application possible. This is what was attempted in the patent US Pat. No. 3,956,483 already cited, where different calcium salts are tested with a view to increasing the activity of a pancreatic extract. The results show that the pancreatic lipase and protease are strongly activated by calcium chloride, unlike the amylolytic activity, which is zero when this salt is used. Tests carried out on other salts lead to the conclusion that only calcium sulphate and calcium acetate produce sufficient activation to allow the exploitation of the pancreatic powder. Finally, calcium chloride is excluded as having no efficacy in stimulating the amylolytic activity of pancreatin.

Thus, until now, a pure alpha-amylase, of constant quality, having a high and controllable level of activity exists in dry form, but it has not been possible to have such an alpha-amylase in solution. , which can be kept while maintaining these characteristics for more than a few hours.

By an original approach, a composition using specific proportions, an alpha-amylase and calcium chloride, was developed. Particularly sought were the conditions which would make it possible to obtain a high amylase activity which persists over time.

This approach consisted in taking a pure enzymatic activity and in measuring its activity according to the concentrations of calcium salts, to seek the maximum enzymatic activity for a dose of employment of the minimum salt.

An activating effect could be demonstrated, manifested by an increase in the enzymatic activity during the dissolution of the alpha-amylase in the presence of calcium chloride. However, activation occurs only within a given range of concentrations which must be determined on pain of not obtaining the desired effect. The determination of the optimum concentrations was carried out according to the method of Walker (Walker JA, Harmon DL, Technical note: A simple, rapid assay for alpha-amylase in bovine pancreatic juice "J. Anim. Sci., (1996), 74 ( 3), 658-662), and conducted in two stages described in detail in Example 1.

It is shown that for concentrations of alpha-amylase between 10 ^"2 and 10- ⁴ g / 1, calcium chloride must be added in an amount of 10" ² to 10 ^-7 mol / 1 to obtain a significant activation, that is to say from 25% to 50%. It is for a solution containing 8.10- ³ g / 1 of alpha-amylase and 10- ⁵ mole / 1 of calcium chloride that the best activation is obtained. Stability tests as a function of time were then carried out on aqueous solutions of the composition according to the invention taken at the optimum concentrations previously defined, to determine the duration of the activation.

Unexpectedly, two new and large-scale effects were discovered.

First of all. 1 initial activation of alpha-amylase has the characteristic of being stable for approximately 4 days, while alpha-amylase alone sees its activity level drop suddenly already a day after it is dissolved

Thus, the composition according to the invention has an initial amylolytic activity of at least 13,000 A / g and preferably at least 14,000 A / g, with an average value of 14,250 A / g on the First 5 hours of storage of the solution, while the activity of an alpha-amylase solution not additive with calcium chloride is between 1,500 A / g and 13,500 A / g, with an average value of 12,500 A / g over the first 5 hours of storage of the solution.

From to to to + 4 days, that is to say between the time of its dissolution and the fourth day of storage, this value is kept constant for the solution of alpha-amylase and calcium chloride. During the same time interval, the activity of alpha-amylase alone drops to less than 4000 A / g, which represents a loss of approximately 70% of its activity.

Secondly, at this concentration, the presence of calcium chloride manifests itself over the long term by maintaining the amylolytic activity of the composition at high values, several weeks after the dissolution of the enzyme in the presence of chloride. calcium. This effect is also called "activation" because the activity values measured at a given moment are higher in the presence of calcium chloride than with the enzyme alone. The activation values calculated according to formula (I) above below from the activity measurements carried out in the days and weeks following the dissolution of the alpha-amylase, are of the order of 670% after 7 days and 590% after 27 days

Activation is defined as the ratio of the difference between the activity of amylase alone and amylase in the presence of calcium, to the activity of amylase alone

Activity (-amylase) - Activity (α-amylase +, - CaC)

Activation = x 100 (I)

Activity (α-amylase) The activity is measured using the "AmylaseTM" kit sold by the company Sigma Diagnostics® (USA) according to the standard protocol indicated by the manufacturer. This method has the advantage of being very easy to use and of having good reliability. It is the subject of a detailed description in Example No. 1.

The unit of activity according to the Sigma method, noted "A" is expressed per gram of enzyme. It is linked to classic enzymatic activity units by the relationships: 1 1, 95 A = 1 CEIP unit (international CEIP unit)

= 0.714 Unit of the European Pharmacopoeia or 1 Unit of the European Pharmacopoeia = 16.737 A

The detailed results are the subject of Examples 2 and 3.

Thus, it has been found that the enzymatic activity of alpha-amylase was increased in significant proportions in the presence of calcium ions, but that this effect is only obtained for determined quantities of salt.

The level of enzymatic activation is then extremely high and has the particularly advantageous characteristic of being extended over time for several weeks.

Thus, the composition containing the amylolytic enzyme and a calcium salt has an enzymatic activity at to ^ - days at least equal to 60%, and preferably at least equal to 70% of the enzymatic activity to to. Its an enzymatic activity at to + 27 days is at least equal to 40%, and preferably at least equal to 50% of the enzymatic activity at t ₀ . These values are to be compared with those obtained from a solution of alpha-amylase alone: the latter only has 10% activity at to + 7 days and 7% at t ₀ -τ-27 days.

The time to is the time when the alpha-amylase is dissolved, possibly in the presence of calcium chloride.

The value of the activity at to is defined as the initial value of the activity. It is an average value calculated for each solution on the basis of the activities measured during the plateau phase of the activity curve as a function of the storage time of the alpha-amylase solution. This curve is presented in Example 3. The plateau phase being situated for the measurements carried out between 1 hour and 5 hours, the value of the activity at and> ι of 12,460 A / g for the amylase alone, of 14 248 A / g for the first alpha-amylase + Chloride solution calcium and 14,259 A / g for the second alpha-amylase solution + Calcium chloride.

The enzymatic activation effect brought to light according to the conditions defined by the present invention is particularly enhanced by the judicious choice of a particular alpha-amylase, selected among other reasons for its naturally high stability and activity. Thus, a composition according to the invention has an enzymatic activity at to + 7 days at least equal to 9000 A / g, and preferably at least equal to 9 500 A / g. At to + 27 days, the enzymatic activity is at least equal to 5,500 A / g, and preferably at least equal to 6,000 A / g. After identical delays, the activity of the control solution of alpha-amylase not supplemented with calcium salt is only 1212 A / g and 856 A / g, respectively.

The present invention is therefore remarkable in that it provides a composition having a high amylolytic activity for several weeks, containing an amylase of bacterial origin and calcium chloride. Its characteristics are certified, measurable and reproducible. It provides a powerful and universal technical solution to the problem posed by providing a pure alpha-amylase, of constant quality, with a high and known level of activity, and which can be preserved with maintenance of these characteristics for several weeks or even several months.

The composition according to the invention contains an enzyme with amylolytic activity. It's a

(1,4) -α-D-glucan-glucanohydrolase, commonly called "alpha-amylase", or "alfa-amylase" according to the International Non-proprietary Name recommended by the World Health Organization for pharmaceutical substances. The mechanism by which alpha-amylase degrades polysaccharide compounds is a hydrolysis reaction in which the enzyme acts as a catalyst. This randomly attacks the α- (1,4) bonds of the starch releasing glucose and maltose (maltose is a disaccharide composed of two glucose monomers).

Preferably, said amylolytic enzyme is an alpha-amylase of bacterial origin.

In a particular embodiment, the alpha-amylase secreted naturally by a bacterial microorganism, preferably by Bacillus amyloliquefaciens, is used. This bacterium, recognized as a full-fledged species in 1987, was previously considered as an atypical representative of the species Bacillus subtilis (Priest et al., Int. J. Syst. Bacteriol. 37:69 1987). The alpha-amylase produced naturally by B amyloliquefaciens is a 58 kDa protein, of which the first ten amino acids, determined by sequencing are:

Val - Asn - Gly - Thr - Leu - Met - Gin - Tyr - Phe - Glu

The alpha-amylase used in the composition according to the invention can be produced by any microbiological culture process using a microorganism competent for the production and excretion of said alpha-amylase, whether it is genetically transformed genetic or not. Preferably, the bacterial alpha-amylase is obtained by fermentation in a reactor of B amyloliquefaciens. The processes for the production of enzymes by microbiological fermentation and for purification are well known to those skilled in the art and are implemented by warehouses specialized in the corresponding field.

In particular, the alpha-amylase used in the claimed composition is produced by a process which puts it at the abπ of contamination by BSE (Bovine Spongiform Encephalopathy). Indeed, no raw material used throughout the production cycle (fermentation, extraction, finishing) is of bovine or ovine origin. On ^the other hand, all the raw materials used in fermentation (carbon source and protein nitrogen, salt minerals) are steπlisés at 125 ° C for 30 minutes.

The alpha-amylase thus produced is presented as a fine cream to beige powder having no foreign matter, soluble in water and complying with the quality and safety standards required for products for pharmaceutical use (absence of metals, sterility). It is marketed in France and in many countries, by the Top Pharm Company (Revel, France).

In the composition according to the invention, the calcium salt is preferably calcium chloride. Indeed, surprisingly, the chloride develops properties which have not been able to be demonstrated until now. Its action with respect to alpha-amylase manifests itself at determined concentrations relative to the alpha-amylase content and which can vanish around an optimum.

According to the present invention, the concentration of the calcium chloride composition is in a ratio of 10-5 to 10 moles for a gram of amylase and preferably from 0.125 to 2.5 moles for a gram of amylase

When the composition is dissolved, the concentrations of enzyme and of calcium chloride for which consequent activation is obtained have been studied According to the invention the amylase is employed at a concentration of 10- ² g / 1 to 10- ⁴ g / 1, with an optimum concentration of 8.10-3 g / 1. In this case, the calcium chloride concentration is between 10- ² mol / 1 and 10 ^-7 mol / 1, and pr f rence between 10-3 mol / 1 and 10- ⁵ mol / 1. Mani re Advantageously the composition according to the invention is employed in solution with an amylase concentration of 8.10-3 g / 1 _e t a calcium chloride concentration of 10- ⁵ mol / 1.

Thanks to the composition according to the invention, it is now possible to offer therapeutic products based on alpha-amylase in liquid form, having a shelf life sufficient to eliminate the risks of denaturation of the drug during storage or during storage. treatment period. More varied formulations can thus be considered, such as drinkable syrups.

Furthermore, the composition according to the invention can be used in admixture with other compounds having a chemical or biological activity complementary to or combined with that of alpha-amylase. Thus it is for example advantageous to prepare a medicament incorporating on the one hand the composition according to the invention for its anti-inflammatory and anti-infectious action, and on the other hand a combination of-menthol and beneficial eucalyptol for its analgesic and antiseptic properties.

EXAMPLE 1

Demonstration of the activating effect of calcium chloride on alpha-amylase and search for effective doses

Amylase activation has been studied according to the Walker method described in Walker J.A., Harmon D.L., "Technical note: A simple, rapid assay for alpha-amylase in bovine pancreatic juice" J. Anim. Sci., (1996), 74 (3), 658-662). In this method, the enzyme is made to act on its natural substrate, namely potato amylopectin, the enzymatic activity being measured by the variations in optical density of the aqueous solution containing the various constituents.

Two series of tests were carried out, first by varying the concentrations of amylase and of calcium chloride, then by fixing the concentration of amylase and by seeking the optimum value for the calcium ion. The activation percentages are determined by comparison with control tests carried out in the absence of the calcium ion.

A) - First series:

The optical density measurements were carried out in different solutions comprising the substrate by varying the concentrations of amylase and of calcium chloride. The results are given in Table A below.

TABLE A

Amylase H20 10-2 M CaCI2 10-3 M CaCI2 density activation density optical optical activation

4.10-3 g / L 1, 1 52 0.844 25% 0.81 8 30%

6.10-3 g / L 0.966_ 0.663 30% 0.541 44%

8.10-3 g / L 0.81 9 0.472 42% 0.414 49%

The results demonstrate a significant activation of the enzymatic activity in the presence of the Ca ^{4 "" 1 "} ion, this activity being maximum for an amylase concentration of 8 × 10 −3 g / liter.

B) - Second series:

To determine the optimal activation conditions. measurements of the variation of the optical density were carried out in different solutions by only varying the concentration of calcium chloride, the concentration of amylase remaining constant at 8 × 10 −3 g / liter. The results of these measurements are given in Table B below.

TABLE B

10-3 M CaCI2 10-4 M CaCI2 10-5 M CaCI2 10-6 M CaCI2 10-7 M CaCI2 density activa- density activa- density activa- density activa- density activa- tion optical optic optic optic opt. optical tion

0.41 48% 0.414 48% 0.417 48% 0.43 46% 0.6 25%

The results show that the activation is constant in the presence of chloride. calcium up to a concentration of 10- ⁵ M. At lower concentrations, the potentiating effect decreases to reach 25% at the concentration of I ⁷ M.

From these tests it can be concluded that the activity of calcium chloride is optimal at a concentration of 10-5 M for an amylase concentration of 8.10-3 g / liter.

At these concentrations, the potentiation of the enzymatic effect of amylase is close to 50%.

EXAMPLE 2

Method for assaying amylolytic activity with the SIGMA reagent

This method was chosen for monitoring enzyme activity over time, because it offers the advantage of being very easy to use with good reliability.

PRINCIPLE:

The Amylase ™ reagent, sold by SIGMA Diagnostics® (St. Louis, USA), uses a p-nitrophenylglycoside as substrate and more particularly 4.6-ethilidene p-nitrophenol a-D-maltoheptaoside (Ey-G -PNP).

α-amylase

5And-G _7- PNP> 2And-G ₅ + 2G ₂ -PNP + 2And-G ₄ + 2G ₃ -PNP + Et-G ₃ + G ₄ -PNP

α-glucosidase 2 G2-PNP + G3-PNP> 4 PNP ^ 10 Glucose

/ /> tracking at 405 nm

The alpha-amylase hydrolyzes Et-G -PNP to G ₂ , G ₃ and to fragments of G ₄ -PNP. Alpha glucosidase hydrolyzes G ₂ PNP and G ₃ PNP to form p-nitrophenol (PNP) and glucose. Five moles of substrate are hydrolyzed to form 4 moles of p-nitrophenol, giving a stoichiometric coefficient of 1.25. P-nitrophenol absorbs light at 405 nm, so the increase in absorbance at this wavelength is directly proportional to the alpha-amylase activity of the sample. PROTOCOL:

• The Amylase ™ reagent is prepared by adding the amount of water indicated on the bottle.

• The wavelength of the spectrometer is set to 405 nm and the temperature to 30 or 37 ° C.

• In a spectrophotometric tank, 1 ml of reagent and 20 ml of sample to be added are added,

• Incubate for 2 minutes then read the absorbance (A _mιtιa ι _e )

• Read the absorbance 1 minute later and 2 minutes longer (Ai and A)

• Calculate the slope ΔA per minute, then the activity according to the following formula:

ΔA / min x Nr 1000 x 1.25 activity (U / l) = -

£ ΝP X Vr £ X 1

with V _r: total volume in the tank = 1020 μl

VE: sample volume = 20 μl 1: tank width = 1 cm £ P P: millimolar extinction coefficient of PNP

either: Amylase activity (U / l) at 37 ° C = ΔA / min x 7005 Amylase activity (U / l) at 30 ° C = ΔA / min x 7244

R RESULTS

The measurements were carried out at two temperatures, and with two samples of different concentration: 0.0062 and 0.062 g / 1. Repeatability was tested by carrying out the activity measurement on each of these samples 10 times: Amylase activity U / g of powder

T = 30 ° C T = 37 ° C c = 0.0062g / L C = 0.062 g / l C = 0.0062 g / l C = 0.062 g / l

10968 13468 9839 16710

10806 12629 13548 18161

11129 13355 12742 17742

10806 12839 11290 18871

11452 12952 10323 19032

11129 13371 11290 19565

11613 14000 10645 18371

12581 14790 10645 19500

9032 14177 10000 18823

8871 14177 12258 194403

Standard variation%

7, 1 4, 2 8, 6 3, 7

Furthermore, the linearity of the method was checked using samples prepared from the composition according to the invention, of different concentrations. The standard variation is around 10%.

Note: The product used for the activity measurement, namely p-nitrophenol, is highly sensitive to the different operating conditions: temperature, ionic strength, etc. It is therefore very important that these parameters are always the same in a comparative study .

EXAMPLE 3

Kinetics of activation of alpha-amylase by sodium chloride

From a single batch of alpha-amylase powder, three solutions were prepared and three series of measurements were carried out.

- The solution As is a solution of amylase alone with a concentration of 60.25 g / l. - Solution AA1 is a solution of a composition containing alpha-amylase and calcium chloride, the alpha-amylase concentration of which is 60.95 g / l and that of calcium chloride is 10-5 mole / 1.

- The solution AA2 is a solution of a composition containing alpha-amylase and calcium chloride, the alpha-amylase concentration of which is 60.10 g / l and that of calcium chloride is 10-5 mole / 1. The three solutions were kept in airtight bottles in the dark and samples were taken after storage periods of a few hours to several days. The activity measurements were carried out on these successive withdrawals. The results are shown in Table C below.

TABLE C

The values obtained from 1 hour to 5 hours make it possible to calculate the average initial activity called activity at t, denoted A (to):

A (t ₀ ) s = 12,460 A / g

A (to) Al = 14 248 A / g

A (to) A2 = 14 259 A / g

Graph 1 represents the activity of the three solutions as a function of time. The activation is calculated at each instant t according to the formula (I) indicated above, from the average of the activities measured on AAl and AA2.

Graph 2 shows activation as a function of time.

Claims

R E V E N D I C A T I O N S

1. Composition containing an amylolytic enzyme and a calcium salt characterized in that it has an enzymatic activity at to + 7 days at least equal to 60%, and preferably at least equal to 70% of the enzymatic activity at t , and / or an enzymatic activity at to + 27 days at least equal to 40%, and preferably at least equal to 50% of the enzymatic activity to to, t ₀ being counted from the dissolution of said composition .

2. Composition according to claim 1, characterized in that said amylolytic enzyme has an activity from to to to + 4 days, of at least 13,000 A / g and preferably of at least 14,000 A / g, to being counted from the dissolution of said composition.

3. Composition according to claim 1, characterized in that the enzymatic activity at to + 7 days is at least equal to 9000 A / g, and preferably at least equal to 9 500 A / g, and / or that l the enzymatic activity at to + 27 days is at least equal to 5500 A / g, and preferably at least equal to 6000 A / g, to being counted from the dissolution of said composition.

4. Composition according to claim 1, characterized in that said amylolytic enzyme is an alpha-amylase produced naturally by Bacillus amyloliquefaciens (formerly assimilated to B. subtilis).

5. Composition according to claim 4, characterized in that said calcium salt is calcium chloride present in a ratio of 10-5 to 10 mole for a gram of enzyme and preferably from 0.125 to 2.5 mole for a gram of enzyme.

6. Composition according to claim 4, characterized in that it is used in solution with a concentration of amylolytic enzyme of 10- ² g / l to 10- ⁴ g / l, preferably 8.10-3 g / l.

7. Composition according to Claim 5, characterized in that it is used in solution with a calcium chloride concentration of between 10-2 mole / 1 and 10- ⁷ mole / 1, and preferably between 10-3 mole / 1 and 10-5 mole / 1.

8. Composition according to claim 5, characterized in that it is used in solution with a concentration of the amylolytic enzyme of 8.10-3 g / l and a concentration of calcium chloride of 10-5 mole / 1.

10. Composition according to one of the preceding claims, characterized in that it is used in admixture with other compounds having chemical or biological activity.

11. Use of the composition according to one of the preceding claims for the preparation of a product with enhanced amylase activity, in particular a product for therapeutic use.