DE2262787A1 - ANTIBACTERIAL COMPOSITION - Google Patents

Description

2252787

Patent attorney
Dr. Ing.Walter Abitz

Dr. Dieter F. Morf December 21, 1972

Dr. Hans-A..Brauns 15365

Munich 86, Pienztneusr3tr. 28

MERCK & C0, ₃ INCORPORATED Rahway, New Jersey Ο? Οβ5, V.St.A,

Antibacterial composition

The invention relates to a novel composition having antibacterial activity, particularly, it relates to compositions containing a compound from 3 ~ fluoro-D-alanine-type in combination with a 3 ~ fluoro-D-alanine car antagonist inhibitor _s wherein the compositions by the fact distinguish that even in highly diluted concentrations they have the extraordinary antibacterial effectiveness

ability of the 3 "PlUOr-D-alanine type compounds to be expressed bring and which are further characterized by the fact that they cause a remarkable synergistic effect by the antibacterial effectiveness of the combination is considerably higher than that which can be attributed to the individual components.

The 3-fluoro-D-alanine-type compounds, such as 3-fluorine-D-alanine per se and its deutero analogs and lower alkyl ester

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and pharmacologically acceptable salts are valuable antibacterial agents that show effective activity against the growth of pathogenic bacteria, both gram-positive and gram-negative, including Streptococcus, Escherichia, Staphylococcus, Peeudomonas, Diplococcus, KlebEiella, Prote.us, Mycobacterium Serratia, Vibrio and Pasteurella and in particular those bacteria belonging to the species Escherichia coli, Proteus vulgaris, Proteus mirabilis, Proteus morganii, Streptomyces pyogenes, Include Pseudomonas aeruginosa, Staphylococcus aureus, and Aerobacter aerogenes.

The 3-fluoro-D-alanine type compounds, however, have the unusual Property of being autoantagonists at high concentrations, this property being a disadvantage there is where these antibacterial substances are used in high doses or where high, autoantagonistic substances Concentrations of the 3-fluoro-D-alanine-type compounds can arise during treatment, for example, of a patient with impaired kidney function. Because higher dosages of 3-fluoro-D-alanine-type compounds Can be safely handled, it is beneficial in treating infections the compounds of the 3-fluoro-D-alanine type in a sufficiently high dosage to be an effective antibacterial Achieve protection against the most resistant organisms that may cause the infection; at these high dosages the compound is of the 3-fluoro-D-alanine type however, an autoantagonist in terms of the sensitive microorganisms that may also be present can.

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It has now been found that the autoantagonistic mode of action, as shown by compounds of the 3-fluoro-D-alanine type at higher concentrations, can be completely suppressed by certain substances which are referred to below as FDA autoantagonist inhibitors. Cycloserine-like compounds are preferably used as PDA autoantagonist inhibitors, such as D-cyclöserin per se (hereinafter referred to as cycloserine), terizidone, D-cis-cyclothreonine or D-trans-cyclothreonine or compounds that are in vivo or simply when Dissolving in an aqueous medium releases cycloserine or cyclothreönin. Further preferred compounds are those which can be characterized by the fact that they themselves or the products released from them in vivo or when dissolved in an aqueous medium have the ability to have the effect of D-alanyl-D-alanine synthetase and / or from D-alanine: oC-keto-glutarate-transaminase to inhibit enzymes of microorganisms. For example, 3-fluoro-D-alanine per se in a concentration of 3 _S 2 mg / ml completely suppresses the growth of virulent cultures of Escherichia coli (strain 2017), but there is considerable growth at higher concentrations (i.e. at 50 mg / ml) of the organism, as it is characteristic of the autoantagonistic effect of 3-fluoro-D-alanine, instead. If 3-fluoro-D-alanine at the concentration of 50 mg / ml is combined with an FDA autoantagonist inhibitor, such as cycloserine, at concentrations as low as 0.39 mg / ml, terizidone at a concentration of 0.8 mg / ml or D-cis-cyclothreonine at a concentration of approximately 2 mg / ml, the growth of Escherichia coli is completely suppressed, surprisingly, the 0.39 mg / ml cycloserine concentration, which completely eliminates

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Sufficient as an FDA ^ autoantagonist inhibitor, less than 2 % of the minimum concentration of cycloserine (25 mg / ml), which is itself able to completely inhibit the growth of Escherichia coll organisms; the inhibitory concentration of terizidone and cyclothreonine is in the same way less than 2 % of their respective minimally effective antibacterial concentrations.

In addition, the combination according to the invention of compounds of the 3-fluoro-D-alanine type with FDA autoantagonist inhibitors exhibits notable synergism, especially when the FDA autoantagonist inhibitor is one Cycloserine-type compound is. The antibacterial effectiveness of such a combination is more than that 15 times that of the individual components. For example, complete inhibition of growth requires an existing one Culture of Escherichia coli (strain 2017) a minimum concentration from 25 mg / ml in the case of cycloserine and from 3.21 mg / ml in the case of 3-fluoro-D-alanine, while a complete Inhdbierung is achieved in the same way by a combination which only 1.56 mg / ml cycloserine in the mixture with 1.56 mg / ml contains 3 ~ fluoro-D-alanine, which is a »white Decrease in 3-fluoro-D-alanine concentration and a Over 15-fold reduction in the concentration of cycloserine means.

Cycloserine-type antibacterial agents, such as cycloserine itself, are used for the treatment of active pulmonary diseases or extrapulmonary tuberculosis (including kidney disease) used in cases where the

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infected organism has become resistant to drugs such as streptomycin and isoniazid. These antibacterial agents are also used for certain "acute urinary tract infections. However, their use has been limited even with these critical infections in view of the fact that at the relatively high doses (more than 1,000 mg daily) required for a _s are required effective therapy severe adverse reactions in the nervous system such as head dimerζen, dizziness, or cramps, occur. Therefore the 15-fold reduction in the cycloserine component 5, as achieved by the combination in which 1 _S 56 mg / ml of each individual compound is present, _{2 is} extremely important with regard to the negative reactions that usually occur with the usual high doses required for therapy with cycloserine. Therefore, the combination according to the invention (insofar as it relates to the contribution of the compounds of the cycloserine type) enables not only the use of high doses of compounds of the 3-fluoro-D-alanine type with a complete suppression of the autoantagonism but also (with regard to the contribution of the compound of the 3-fluoro-D-alanine type) the use of compounds of the oycloserine type in a dose reduced by one IS-fold «,

The new combination is administered in the form of pharmaceutical preparations for antibacterial purposes, which the compound of the 3-fluoro-D-alanine type in admixture with the FDA autoantagonist inhibitor and a pharmacologically acceptable carrier. Is the connection from the

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3-fluoro-D-alanine-type combined with a compound of the cycloserine-type, the ratio of the 3-fluoro-D-alanine-type component to the cycloserine-type component in the combination is generally about 1: to about 200: 1, with a range of about 1: k to about ¹ J: 1 being preferred.

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As the 3-fluoro-D-alanine type compound, is preferred uses "3-fluoro-D-alanine, 2-deutero-3-fluoro-D-alanine, 2,3-dideutero-3-fluoro-D-alanine, 2,3 »3 * -trideutero-3-fluoro-D-alanine, a lower alkyl ester thereof such as the methyl ester. or a pharmacologically acceptable salt such as sodium, Calcium or hydrochloride salt. As compounds of the cycloserine type Cycloserine per se are generally preferred, as are terizidone, D-cis-cyclothreonine or D-'trans-cyclothreonine.

As pharmaceutically acceptable carriers for the abovementioned combinations are the conventionally used carriers in question, such as are used for oral administration in the form of capsules, tablets or as liquid solutions or suspensions, or the combination can be in a carrier material which is suitable for injection administration to be resolved. Suitable formulations for oral use may contain diluents, granulating aids, stabilizers, binders, taste-improving substances ₄ and coating materials, they, as is generally used in this field, and the dosages of the products can vary in a wide range. A combination of a compound of the 3-fluoro-O-alanine type and the cycloserine type suitable for oral administration can be prepared by mixing the individual components in a dry, powdery state together with gelatin, starch, magnesium stearate and alginic acid , whereupon a tablet is pressed * You can also combine the individual components in an aqueous solution and evaporate the solvent in order to then obtain the combination in the form of a particularly good mixture of the components.

3 0 9 8 3 2? 1 ¹ I.

15365

example

Female mice with an average weight of 21.0 g are inoculated intraperitoneally with "0.5 ml of a dilution of a 16-hour broth culture containing the specified pathogen. (The severity of the infection is in each individual case as a multiple the dilution of the broth culture, which is fatal for 50% of _{the untreated animals, i.e. as LD 1} -Q, shown) Dosages of 3-fluoro-D-alanine (FDA) and of D-cycloserine are used individually and as a combination of the two compounds in a fixed ratio of 1: 1 in a 0.5 ml volume subcutaneously inoculated in the back of the animals, namely immediately after the infection and then in a two- and four-hour interval. The results of this experiment are reported as ED, - ₀ dosing expressed the ED, -Q dosage is 50% of the infected animals to protect the statistically interpolated dose required 7 days after infection and treatment.:

after 0, 2, k hours

LD 50 Individual! Components Cycloserine combination FDA + Cycloserine Infectious
organism 30th FDA 0.400 0.035 + Staphylococcus
aureus 29 * 19 > 100 0.801 > 5.0 0, ', Τ + O ₁ Proteus
morganii 3376 3 > 2.0 2.5 Ο, 18 + 0, Serratia
species 337 ^ 1.7 , 035 , 57 , 18

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The reduction of the dosage in the FDA cycloserine combination, compared to the dosage for the individual components is shown in the following table:

Q for combination, expressed as an approximate percentage of the ED ™ for the single

50

Infekt10nsOrganism components Cycloserine FDA 9 %
10 %
■ 7 % Staphylococcus aureus
Proteus morganii
Serratia species 5 %
25 %
10 % ■

Example 2

Groups of five female mice with an average weight of 21.0 g are inoculated intraperitoneally with 0.5 ml of a diluted meat broth culture of Escherichia coli 2017, which ^{corresponds to 3 LD} trn of ^the bacterial pathogen. Subsequently, the doses of 3-fluoro-D-alanine (PDA) and D-cycloserine given in the table below are injected subcutaneously into the back _{in a volume of 0 s 5 nil.}

In the first row of the following table the number of surviving mice (from each group of 5 infected mice) for the indicated dosage of 3 ~ pluor-D-alanine given alone (in mg). The first vertical column shows the number of surviving animals for the

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given dosages of cycloserine alone (in, mg) are shown. In the second vertical column is the number of survivors Mice indicated when one combined 5.0 mg of 3-fluoro-D-alanine with the indicated amounts of cycloserine (in mg) applies.

Number of surviving mice in each group out of 5 infected Animals

mg 3-fluoro-D-alanine per dose

mg of D-cycloserine

per dose 0 0.078 mg 0.312 mg 1.25 mg 5.0 mg

0 0 0 3 ^ 1

0.019 mg 0

0.039 mg - 1st

0.078 mg 02

0.156 mg - 5th

0.312 mg 0 5

0.625 mg - 5th

1.25 mg 3 5

2.5 mg - 5

5.0 mg 5 5

It is noteworthy that with a 1.25 mg dose of 3-fluoro-D-alanine alone, 80 % of the animals survive, while with a dose of 5.0 mg (due to the autoantagonism) only a 20 % survival rate is obtained. A combination dosage of 5.0 mg 3 ~ fluoro-D-alanine and 0.156 mg cycloserine (with this amount cycloserine alone does not provide any protection) results in a survival percentage of 100, which means that 0.156 mg cycloserine results in a

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A 5.0 mg dose of 3-fluoro-D-alanine caused autoantagonism is completely prevented.

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Claims (8)

Claims (I- Antibacterial agent, characterized in that there, as active ingredients, a combination of a 3-fluoro-D-alanine type compound and a compound contains of the cycloserine type. 2. Composition according to claim 1, characterized in that it is in a mixture with a pharmaceutical carrier. 3. Composition according to claim 2, characterized in that the pharmaceutical carrier is for oral administration suitable is. 4. Means according to claim 2, characterized in that the pharmaceutical carrier for injection administration suitable is. 5. Means according to claim 1, characterized in that as active ingredients 3-fluoro-D-alanine or a pharmacologically acceptable salt thereof and D-cycloserine are included. 6. Agent according to claim 1, characterized in that 2 ~ or Deutcro-3-fluoro-D-alanine as active ingredients a pharmacologically acceptable salt thereof and D-cycloserine are included. - 12 0 9 8 3 2/1158 7. Means according to claim 1, characterized in that
as active ingredients 3-fluoro-D-alanine or a
pharmacologically acceptable salt thereof and terizidone are included. 8. Antibacterial agent, characterized in that It is a 3-fluoro-D-alanine type compound as the active ingredient and a 3-fluoro-D-alanine autoantagonist inhibitor. - 13 309832/115 8