NO790512L - PROCEDURES FOR THE PREPARATION OF AN ENZYME FROM BACTERIAL SOURCES AND PHARMACEUTICAL COMPOSITIONS CONTAINING THIS - Google Patents

Description

The present invention relates to new pharmaceutical compositions which contain as active ingredient an enzyme or mixture

of enzymes of microbial origin.

More specifically, the purpose of the invention is to provide

new pharmaceutical compositions, namely intended for the treatment of viral diseases, the active ingredient of which is glycosidohydrolase obtained from cultures of a microbial strain.

In particular, the present invention provides pharmaceutical compositions which contain as active ingredient an N-acetylmuramine glycohydrolase produced from cultures of Streptococcus pneumoniae (genus micrococcaceae).

N-acetylmuramine glycohydrolase is an enzyme capable of

to cleave N-acetylmuramic acid from the glycoproteins when placed at the non-reductive end part of the chain of a polysaccharide.

Many N-acetylmuramine glycohydrolases are already known. They originate either from viruses or from microbes. N-acetylmuramine glycohydrolases from Vibrio cholerae, from Clostridium perfringens, from 'Vibrio comma, from Arthrobacter sialophilium are already mentioned in the literature. Furthermore, N-acetylmuramine glycohydrolase has been obtained by chemical treatment of different strains of Myxovirus influenzae.

Each of these glycohydrolases has one specific chemical structure that originates from different combinations. of the glycoprotein chains. It is clear from this that they have special physical and chemical properties.

I I

The N-acetylmuraminate glycohydrolase used here in the form of pharmaceutical compositions is obtained from a specific strain of 'Streptococcus pneumoniae deposited at the collection of the Pasteur Institute in Paris as No. 1-044 on 16 February 1978. It has physical properties which are completely demarcated from those already known to other N-acetylmuramine glycohydrolases. This enzyme or mixture of enzymes does not show any galacto-sidase, protease, N-acetylglucosaminidase, fucosidase and aryl sulfatase properties, contrary to what has been shown for the enzymes in the literature.

Furthermore, the Michaelis constant determined on bronchial glyco--4

peptides as substrate approx..Km = 2.3.10. which is a very high number which shows a strong affinity to the glycopeptides. . This isoelectric point determined by isoelectric focusing shows two active peaks in the enzyme which indicates the presence of two isoenzymes with an isoelectric point of 4.2 and 3.7. These two isoenzymes make the enzyme from Streptococcus pneumoniae strain 1.044 clearly different from that from Clostridium perfringens which is homogeneous by electrofocusing and the isoelectric point of this is 4.7.

The enzyme or mixture of enzymes from Streptococcus pneumoniae

cleaves N-acetylneuraminic acid from the acidic human -1 glycoprotein at a rate of 82% for 7 hours and releases near-quantitative (more than 90%) N-acetylneuraminic acid from a bronchial siolomucin after 6 hours of contact.

The chemical structure of N-acetylmuramine glycohydrolase from Streptococcus pneumoniae (strain 1-044) has already been determined by

P. Bienvenu et al. CR. Acad. Pollock. (Paris) ) 285 (section D) 837

(1977). The content of the different amino acids shows clearly

its "soecificity" in relation to the enzyme obtained by enzymatic hydrolysis of Myxovirus influenzae A (Hong Kong) 1/68.

Therefore, the purpose of the present invention is to provide pharmaceutical compositions with the enzyme or mixture of enzymes produced by cultures of Streptococcus pneumoniae (strain 1-044) as an active ingredient in a mixture or combination with an inert ! ■ ■■ ' non-toxic therapeutically acceptable carrier or excipient.

The pharmaceutical compositions according to the present invention

are those which are suitable for parenteral, percutaneous, permucous, perlingual or rectal administration. Preferably, the injectable solutions are divided into ampoules, multidose bottles, self-injectable syringes, sublingual tablets, pressurized. compositions ready for spraying on nasal or pharyngeal mucous membranes, solutions in a polar solvent suitable for per-, cutaneous use, sublingual.tablets and suppositories.

The pharmaceutical compositions may also contain one or more excipients, fillers, diluents, dispersants, surfactants, emulsifiers, flavorings or agents which enable a slow release of the active ingredient in the body. Depending on the chosen route of administration, the content of active ingredient can vary greatly. It varies from 300 U to 5,000 U per dosage unit, namely for the pharmaceutical compositions intended for human therapy. For children, the content of active ingredient in the pharmaceutical compositions according to the invention is reduced depending on the patient's age and weight.

The pharmaceutical compositions according to the invention must be given one to four times per day in humans.

When administered through mucous membranes in the nose or mouth

it is easy to use the pharmaceutical compositions according to the invention in the form of a pressurized spray in an aqueous aid three times a day for two days.

In parenteral administration, the pharmaceutical compositions are injected every day for a week and then once a week for a further two months.

The pharmaceutical compositions according to the present invention

is intended for the treatment of viral diseases, especially those where a virus that forms a capsule is the cause and the treatment of

in

microbial infections frequently associated with the viral infection.

They are particularly active against Myxovirus influenzae, Rhinovirus and other species of viruses that have a neuraminidase. This activity can be attributed either to a direct effect on the virus capsule of glycoprotein structure that induces a reduction in the number of cellular lesions on the reproduction of the virions, or a

specific immunostimulatory effect on species whose pathogenic effect is due to a neuraminidase with antigenic properties closely related to N-acetylneuraminidate glycohydrolase from Streptococcus pneumoniae such as Erysipellothrix.

Furthermore, they show a non-specific immunostimulant effect which makes it possible to use them as aids in the treatment of rhinitis, rhinopharyngitis, sinusitis, angina, amygdalytis, laryngitis, tracheitis, acute bronchitis, broncholites, bron-chpneumonias and chronic respiratory diseases.

In diseases where the body's defenses are reduced due to an inhibition, the compositions according to the invention can suppress the inhibition by hydrolysing the glycoprotein responsible for the inhibition and eliminating sialic acid from the surface of the cell membranes.

The pharmaceutical compositions according to the invention can be given prophylactically to prevent influenza on the first two days of each month during the six winter months to humans or animals exposed to infection in order to prevent the first stage of the viral infection or to avoid the clinical symptoms of infection due to influenza.

They can also be given after the first flu symptoms. They increase the speed of healing and the healing of the mucosae cells and they stop the complications due to over-infections. The pharmaceutical compositions according to the invention are very well tolerated because the N-acetylmuramine glycohydrolase from Streptococcus pneumoniae can be obtained in a very pure state and is neutral and free of foreign proteins. They therefore do not have the side effects of vaccines from neutralized or killed viruses.

Their activity can be attributed to an activation of the macrophages

which is shown in vitro by a greater spreading ability, an increase of phagocytosis, an increase in the number of E rosettes and EAC rosettes and an increase of the synthesis of DNA in the presence of lectins. In vitro, this activation is shown by an increase in humoral response and

•on stimulation, delayed hypersensitivity.

These immunostimulating and antiviral effects are surprisingly

end and not to be anticipated in light of the literature (namely P. Binder C R Acad.Sci. (Paris) 481 (1975) D, 1545) showing the increase in mortality in rats with a malpighian carcinoma.

Furthermore, other works have described that neuraminidase from bacteria is without any protective effect against viral infections caused by Myxovirus Influenzae.

Therefore, the surprising therapeutic properties of the pharmaceutical compositions according to the invention show a clear demarcation between the previously known neuraminidase and N-acetyl-nuramidate glycohydrase from the Streptococcus pneumoniae strain 1-044.

The following examples are only intended to illustrate the invention and show the best embodiment.

In EXAMPLE I

Production of N-acetylmuramine glycohydrolase from Streptococcus pneumoniae.

I_Tribe

The strain that produces this enzyme is a Streptococcus belonging to the genus Micrococcaceae deposited in the Pasteur Institute's collection in Paris under no. 1-044.

The streptococcus turns out to be gram-positive, two and two or

a very short chain, sometimes encapsulated cooks. They are facultatively aerobic. The optimum temperature for the culture is approx.

37°C

Storage

This strain can be maintained on agarose with 10% horse blood added with nalidixic acid.

Grafting

Grafting is carried out by streaking on sloping agarose.

Incubation of the inoculated test tubes is carried out for 18 to 24 hours at 37°C. They are then lyophilized on skimmed milk.

III_E£§m§tiiling_of_the ingculum

The contents of a lyophilized sample tube are suspended in approx. 2 ml of

the following medium:

The jpH value for this medium is adjusted to 7.8 and the medium sterilized

IN

In o

bake at 120 for 30 min.

This sterile medium is used for inoculation in a flask containing 4 liters of this medium. The incubation is carried out anaerobically at 37°C without stirring or air blowing for 24 hours.

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4 liters of the preceding sterile medium are used to inoculate the fermentation tank which contains 1000 liters of the following mixture:

The environment's pH value is adjusted to 7.8-8.0

It is sterilized at 120° for 20 min.

The fermentation is carried out at 37°C for 29 hours without stirring or air blowing. At the end of the fermentation, the medium is added

0.1 g per liter of lysozyme - to facilitate the breakdown of the microbe cells and improve the diffusion into the medium of the N-acetyl muraminate glycohydrolase thus produced. The medium is kept under stirring at 37°C for 3 hours and is then allowed to return to approx. 20°C.

The fermentation process is controlled by microscopic examinations, determination of the pH value and the neuraminidase

Activity.

The extraction of the enzyme is carried out by fractional precipitation.

in

aj__f ^rste_step

500 g/l ammonium sulphate and 3 g hyflo supercel are added for each liter of fermentate. The mixture is stirred for 1 hour and filtered. The residual cake is washed with a 40% aqueous solution of ammonium sulphate. The remaining paste is taken up in water at +4°C

3 times with 5 litres..

^i_<a>DD§t_step

Sufficient ammonium sulphate is added to the 15 liters of washing water to give a concentration of 250 g/l. 40 g/l hyflo is also added to the solution. supercel and stir for 1 hour. The entire mixture is filtered under suction.

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150 g/l ammonium sulphate and 10 g/l hyflo supercel are added to the previous filtrate and the mixture is stirred for 1 hour. The precipitate is separated by vacuum filtration and the insoluble materials are washed with a 40% aqueous solution of ammonium sulphate. The remaining paste is taken up with 2 times 2 liters of water at 4°C. The resulting solutions are combined and dialyzed against water at 4°C for 16 hours (water yield 20 litres/hour).

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A column with a diameter of 15 cm and a height of 100 cm is prepared for the chromatography. It is filled with 1.6 kg of CM 23 carboxymethyl cellulose and washed with 55 liters of 0.0025 M phosphate buffer solution (pH 6.8).

The dialyzed extracts are first clarified by filtration and adjusted to pH 6.8 by the addition of 1 M citric acid solution. They are then loaded onto the column and then applied to 15 liters of a 0.0025 M phosphate buffer solution. The fixed N-acetylmuramine glycohydrolase is eluted with 10-15 liters of 0.2 M citrate • phosphate buffer solution (pH 6.8) at a flow rate of 4 liters/hour

The main fraction has a volume of approx. 5 liters and then dialyzed against a flow of 1% sodium chloride solution for 16 hours at +4°C (outlet 4 litres/hour). The dialysate is lyophilized. The recovered residue consists of crude N-acetylmuraminate glycohydrolase with a specific activity of approx. 500.

YII_B§D§Qi29_Y§^_]SE25}§É22ESli_El_§§El}§^§2_5_50 20 g of the previously obtained raw material (corresponding to 10 g of proteins determined by Lowry's method) are dissolved in 150 ml of 0.1 M phosphate citrate buffer solution (pH 6.8) and run on a column filled with sephadex G50 previously washed with a 0.1 M phosphate citrate buffer solution. After fixation, the column is washed with the same buffer solution and 20 ml fractions are collected. The fractions containing N-acetylmuraminidate glycohydrolase are collected, dialyzed against a 1% sodium chloride solution and the dialysate lyophilized. The specific activity of the resulting concentrate is approx. 20,000. It is determined by measuring the amount of sialic acid released in 1 min. at 37°C from N-acetyl. neuraminyl-lactose in mg protein and expressed in nanomoles.

The following table summarizes the cleaning process.

EXAMPLE II

Lack of immunological association between viral heuraminidases and N-acetyl muraminidate glycohydrolase from Streptococcus pneumoniae.

a) Attempts to decrease the neuraminidase activity of N-acetylmur-aminidate glycohydrolase from Streptococcus pneumoniae (strain 1-044)

with serum from rabbits previously immunized with neuraminidase of viral origin gives no results. The neuraminidases of viral origin come from human Myxovirus Influenzae Singapore 57, Hong Kong 68, England 72, from animal strains A equi I, A equi and

from human Myxovirus parainfluenzae types 1, 2 and 3, clumps, Sendai SV 5.

b) Attempts to neutralize the viral neuraminidase with rabbit sera previously immunized against N-acetylmuraminidate glycohydrolase remain

iinegative.

EXAMPLE III

Effect of N-acetylmuramine glycohydrolase on the multiplication of influenza virus strain A Port Chalmers 1/73 H^N2.

The different rates of N-acetylmuramine glycohydrolase

from Streptococcus pneumoniae (strain 1-044) has been attempted

against the egg bite method described by Fazekas. The egg pieces have been kept alive in small cups of Perspex sheets containing small

pieces of chloro-allantoin membrane. " The cells have been inoculated with A virus Port Chalmers (0.1 ml dilution + 0.9 ml culture medium which gives an infectious strength of virus equal to 10 ID /0.1 ml). After incubation for 6 hours at 33°C, N is added -acetylmuramine glycohydrolase from S.pneumoniae (0.1 ml dilution 10 -2 for a specific activity of 8000 and a concentration of 1 mg proteins per ml).

The effect of the enzyme on the propagation of the virus lasts approx. 7 2 hours. After this period, the infectious strength of the produced virus is measured by determining the hemagglutination strength on pieces of egg. The results obtained are summarized in Table I. All the experiments were carried out using the same dilution, the same times and the same protein concentration.

The effect of N-acetylmuramine glycohydrolase from S.pneumoniae has been tested by inoculation every six hours with the same dose of the enzyme. The virus grows for the first 6 hours on the egg pieces incubated at 33°C. After 6 hours, the N-acetyl-neuraminate glycohydrolase is added and the incubation is continued until the 72nd hour. Infectious strength is determined in a similar manner by hemagglutination using hen red blood cells in a 2% suspension.

The multiplication kinetics of the influenza virus with or without N-acetylmuramine glycohydrolase is expressed in Table I. The

shows that the N-acetylmuraminate glycohydrolase according to the invention plays a very important role in the propagation of the virus.

IN

1 IN EXAMPLE IV

Determination of action of N-acetylmuramine glycohydrolase

from S. pneumoniae on rats for propagation of Myxovirus (wild strains) in connection with the introduction period.

First try

The N-acetylmuramine glycohydrolase from S.pneumoniae has been inoculated into cultures of Myxovirus on egg pieces at a dosage of 2.7 U/ml/plate.

The propagation of the virus without N-acetylmuramine glycohydrolase from S.pneumonia is carried out for 72 hours at 33°C. After this period of time, part of the culture is taken out and the hemagglutination strength is determined, the remaining fractions are collected and combined into a group of fractions. The hemagglutination activity, the neuraminidase activity of this collection and the infecting strengths of embryonated eggs are determined. The collection is further reinoculated onto fresh pieces of egg. A further virus multiplication is thus obtained after 72 hours at 33°. Fractions of this are gathered for the same provisions. The remaining portions are re-inoculated to give a third virus multiplication under the same experimental conditions.

Similarly, three subsequent multiplications of virus are carried out in the presence of N-acetylmuramine glycohydrolase from S.pneumoniae inoculated. the sixth hour in the multiplication step.

The remaining part of the pools of each propagation harvested after 72 hours is concentrated, then purified to determine the morphological aspects of the virus growth with or without N-acetyl muraminate glycohydrolase.

Some pieces of egg coated with a piece of echorio-allantoin membrane are taken out after 2 6 hours during the propagation step for each step.

They are fixed in 2% glutaraldehyde to show that the virus has moved away from the membrane by electron microscopy. Others are fixed in 1% glycaraldehyde to perform freeze-etching and to determine any morphological differences in the non-infected and infected, treated and untreated membranes.

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N-acetylmuramine glycohydrolase from S-pneumoniae is inoculated

in cultures of Myxovirus A2PC/7 3 and Myxovirus A2VIC /3/7.5 at a concentration of 0.5 U/ml/plate. The virological data of Myxovirus A2 VIC /3/75 are as follows:

The analytical constants of the N-acetylmuramine glycohydrolase used from S-pneumoniae are as follows:

The experiment is carried out with the usual techniques of the egg pieces, determination of hemagglutination strength, determination of the neuraminidase activity and determination of the infectious strength (EID^q) on embryonated eggs.

The preparation of the membranes is further carried out for inspection by electron microscopy and freeze etching.

Result

The results thus obtained are collected in the following histograms I, II and III. They show that the addition of the N-acetylmuraminate glycohydrolase from S-pneumoniae after 6 and 24 hours' cultivation of Myxovirus influenzae on egg pieces induces a growth

f

<I>rate which is 2 log 10 less than the untreated cultures. Addition after 30 hours of cultivation still has a positive effect. Addition after 48 to 54 hours of culture induces only one

weak effect.

EXAMPLE V

Determination of the growth rate of influenza virus in the presence of different amounts of N-acetylmuramine glycohydrolase from S pneumoniae.

Solutions of this enzyme are prepared which contain 0.1, 0.2, 0.4, 0.6, 0.8 and 1 mg of proteins per ml. A dose of higher solution (0.1 ml.10 2 ) is inoculated onto virus cultures on egg pieces with MCA and the effects of the enzyme on the rate of multiplication are tested.

Method

§l_uDd§rsearched_ strains

The cultivation technique is published by Fazekas (1958). After 6

hours of incubation at 33°C, a quantity of the solution of N-acetylmuramine glycohydrolase from S-pneumoniae in different concentrations is added to the cultures. Propagation is continued for 72 hours at 33°C. After this time, 0.1 ml of the medium is taken out in each plate and the multiplication rate of the virus is determined as a function of the varying concentration of N-acetylmuramate glycohydrolase (determination of hemagglutination and

neuraminidase activities). Furthermore, a hamagglutination test is performed directly in each plate with a 0.2% suspension of red blood cells from hen which allows a comparison between the infectivity of the virus with and without N-acetylmuramate glycohydrolase.

t

in Results

The results thus obtained are collected in tables II, III and

IV.

EXAMPLE VI

"In vivo" study of antiviral activity of N-acetyl muraminate-. glycohydrolase from S pneumohiae.

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It has been tested in vivo in batches of ferrets to which a specific dose of MRG 11 Myxovirus Influenzae has been inoculated intranasally. 6 or 18 hours later, a dose of N-acetylmuramine glycohydrolase is given by the same route.

The effect of the N-acetylmuramine glycohydrolase on the rate of multiplication of the virus is determined by measuring as a function of time the

--the disappearance of inoculated virus

- determination of hemagglutination activity.

- determination of neuraminidase activity.. • - determination of infection titers on eggs in swabs from the throat at different time intervals after inoculation of the virus (3 days, 6 days, 9 days, 15 days).

Similar determinations have been made on controls that received only dosage of the virus culture.

The enzyme's effect is also determined in ferrets by measuring the decrease in the production of virus antihemagglutin and antineuraminidase antibodies by searching for the local antibodies in swabs from the throat and searching in sera for circulating antibodies.

The batches have ferrets that have received only the virus cultures show

the characteristic flu symptoms. They sneeze a lot and six days after the infection their noses run profusely. In contrast... the ferrets that received N-acetylmuraminate glycohydrolase do not show

J /

"the tomes.

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Three batches of ferrets are nasally inoculated with a single dose

MRC 11 virus (1 ml allantoin mixture). The N-acetylmuramine glycohydrolase is injected in the same way 6 hours after the inoculation of virus in a single infection of a dose corresponding to 1 mg of protein in 0.5 ml of saline solution.

Washing of the throat was performed every three days after the inoculation. The absorption kinetics and virus propagation in the tracheal cells are determined with or without N-acetylmuramate glycohydrolase.

- one batch of ferrets only received the virus culture

- batches of ferrets received the virus culture and 6 hours later N-acetylmuramine-glycohydrolase. - batches of ferrets received the virus culture and 6 hours before this inoculation N-acetylmuramine-glycohydrolase from S-pneumoniae. The throat is washed every three days. The samples are then suspended in 1.5 ml of Parker's survival medium and the hemagglutination and hemagglutination inhibition activity (HA titer against MRC 11 virus is determined). A small cardiac puncture is performed on the 15th and 21st day after inoculation to determine the circulating amount of antibodies.

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Controls: batches of ferrets are inoculated with a single dose of MRC virus at zero time. 6 hours later they get a saline solution. All the ferrets show very clear symptoms of influenza from the sixth day after inoculation. A significant proportion of the ferrets died. The remaining surviving ferrets were completely healthy around day 9.

! .. in IHemagglutination strength - which shows the presence of the virus - shows

an increase until the 6th day with a maximum from the 3rd to the 6th day, then decreases and disappears completely on the 15th day.

The antihemagglutination antibodies appear from the 6th day and increase steadily until the 21st. day.

l§h§ndl§de_rates

The batches of ferrets that have been inoculated with the virus culture

and 6 hours later with N-acetylneuramine glycohydrolase from ' S.pneumoniae shows some sneezing after inoculation of the virus culture. The results thus obtained are collected in the tables

V and VI.

EXAMPLE VII

Clinical study of N-acetylmuramine glycohydrolase from S.pneumoniae.

Dé: pharmaceutical compositions according to the invention have been tested on 9 families consisting of 19 adults and 8 children.

gl_Dosage

They received an aqueous solution of the enzyme containing 333 units, per unit dosage 2 subsequent days. They each received a total of 4,000 units of N-acetylmuramine glycohydrolase from Streptococcus pneumoniae.

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17 patients received the solution of the enzyme after infection. The

showed a significant subjective improvement. No side effects have been detected.

I Of the 12 volunteers who received the enzyme solution as a preventive measure •only one became ill. In contrast, 4 out of 6 controls were poor.

Claims (4)

'". 1. Process for the production of pharmaceutical compositions containing as active ingredient N-acetyl muraminate glycohydrolase from Streptococcus pneumoniae in mixture or connection with an inert non-toxic therapeutically tolerable pharmaceutical carrier or excipient. 2. Process for producing pharmaceutical compositions according to claim 1, characterized in that the active ingredient is present in a dosage of approx. 300 to 5000 units per unit dosage. 3. Method for producing pharmaceutical compositions according to claim 1, characterized in that the carriers or auxiliaries are those which are suitable for parenteral permucous, percutaneous or rectal route of administration. 4. Process for the production of pharmaceutical compositions according to claim 1 characterized by . that N-acetylmuramine glycohydrolase from Streptococcus pneumoniae is produced by strain 1-044 deposited in the Institut Pasteur's collection in Paris.