NO802857L - ACTIVE SUBSTANCE COMBINATIONS AND MANUFACTURING THEREOF - Google Patents

Description

Peptide derivatives of the general formula

wherein R^" is hydrogen, methyl, hydroxymethyl, a mono-, di- or trihalomethyl group;

R 2 a residue that is characteristic of an α-amino acid that normally occurs in proteins or a lower alkyl or hydroxy-lower alkyl residue that is characteristic of an α-amino acid that does not normally occur in proteins;

R is lower alkyl, lower cycloalkyl, lower alkenyl,

aryl or aryl-lower alkyl;

R 4 means hydrogen or lower alkyl;

n means 2 or 3 and

the configuration at the C atoms (a), and (b) is R (when R<1>f H) and L respectively,

and their physiologically tolerable salts are known. These compounds have antibacterial activity.

It has now been found that peptide derivatives of formula I and salts thereof potentiate the activity of antibiotics.

The present invention therefore relates to active ingredient combinations containing a peptide derivative of formula I or a physiologically tolerable salt thereof as well as preparation

when using these.

The term "lower alkyl" shall in the present context mean a straight or branched alkyl group with preferably up to 8 C atoms, for example methyl, ethyl, propyl, iso-propyl, butyl, tert. butyl, pentyl, hexyl. Examples of hydroxy-lower alkyl groups are 2-hydroxyethyl, 3-hydroxypropyl, 4-hydroxybutyl. The term "lower cycloalkyl" means a cycloalkyl group with preferably 3-6 C atoms, for example cyclopropyl, cyclobutyl. The term "lower alkenyl" means a straight or branched alkenyl group with preferably 2 to 8 C atoms, for example allyl, butenyl. Examples of aryl groups are phenyl, tolyl and examples of aryl lower alkyl groups are benzyl, phenethyl. The term "halogen" means fluorine, chlorine, bromine or iodine. Examples of the halogen groups mentioned above are chloromethyl, dichloromethyl, trifluoromethyl. The expression "characteristic residues for an α-amino acid normally occurring in proteins" means the residue R of a natural α-amino acid with the general formula

which normally occurs in proteins. Thus, when the α-amino acid is glycine, R means hydrogen. When the α-amino acid is alanine, R means methyl. In methionine R means 2-methylthioethyl, in serine hydroxymethyl and in tyrosine p-hydroxybenzy1. R can too. be ring deleted together with the nitrogen atom of the amino group as in proline.

When R<1> in formula I is not hydrogen, the configuration at the C atoms indicated by (a) shall be (R), i.e. the configuration shall be such that upon replacement of the carboxyl group to a natural a- amino acid with a P03H2~ group must be maintained.

The residues R 2 in formula I may, within the scope of the given definitions, be the same or different from each other.

Preferred peptide derivatives of formula I are on the one hand those in which R<1> is hydrogen or methyl, on the other hand those in which the residues R 2 are a residue that is characteristic of an α-amino acid that normally occurs in proteins or is a lower alkyl residue that is characteristic of an α-amino acid that does not normally occur in proteins. Finally, such peptide derivatives of formula I are preferred in which R 3 is lower alkyl, especially methyl.

Examples of peptide derivatives of formula I are: (IR)-1-(N-sarcosyl-glycyl-L-alanylamino)-ethylphosphonic acid, (IR)-1-(N-sarcosyl-L-alanyl-L-alanylamino)-ethylphosphonic acid, (IR)-1-(N-sarcosyl-L-methionyl-L-alanylamino)-ethylphosphonic acid, (IR)-1-(N-sarcosyl-L-histidyl-L-alanylamino)-ethylphosphonic acid, (IR)-1- (N-sarcosyl-L-seryl-L-alanylamino)-ethylphosphonic acid, (IR)-1-(N-sarcosyl-L-tyrosyl-L-alanylamino)-ethylphosphonic acid, (IR)-1-(N-sarcosyl-L -arginyl-L-alanylamino)-ethylphosphonic acid, (IR)-1-(N-sarcosyl-L-alanyl-L-arginylamino)-ethylphosphonic acid, (IR)-1-(N-sarcosyl-L-alanyl-L-serylamino )-ethylphosphonic acid, (IR)-1-N-sarcosyl-L-alanyl-L-histidylamino)-ethylphosphonic acid, (N-sarcosyl-L-alanyl-L-alanylamino)-methylphosphonic acid, (IR)-1-(N- sarkosyl-L-norvalyl-L-alanylamino)-ethylphosphonic acid, (IR)-1-(N-sarcosyl-L-alanyl-L-norvalylamino)-ethylphosphonic acid, (IR)-1-(N-sarkosyl-L-norvalyl- L-norvalylamino)-ethylphosphonic acid,

(IR)-1-(N-sarcosyl-L-arginyl-L-arginylamino)-ethylphosphonic acid,

(IR)-1-(N-sarcosyl-L-norvalyl-L-arginylamino)-ethylphosphonic acid, (IR)-1-(N-sarcosyl-L-glycyl-L-norvalylamino-ethylphosphonic acid, (IR)-1-( N-sarcosyl-L-arginyl-L-norvalylamino)-ethylphosphonic acid, (IR)-1-(N-sarcosyl-L-valyl-L-norvalylamino)-ethylphosphonic acid, (IR)-1-(N-sarcosyl-glycyl- L-norvalyl-L-norvalylamino)-ethyl-phosphonic acid,

(N-sarcosyl-L-norvalyl-L-norvalylamino)-methylphosphonic acid, (IR)-1-(N-methyl-L-norvalyl-L-norvalyl-L-norvalylamino)-ethylphosphonic acid,

(IR)-1-(N-ethylglycyl-L-alanyl-L-alanylamino)-ethylphosphonic acid, (IR)-1-(N-n-propyl)-glycyl-L-alanyl-L-alanylamino)-ethylphosphonic acid,

(1R)-1-(N-allyl-glycyl-L-alanyl-L-alanylamino)-ethylphosphonic acid,

(IR)-1-(N-n-hexyl)-glycyl-L-alanyl-L-alanylamino)-ethylphosphonic acid,

(IR)-1-(N-cyclopropyl-glycyl-L-alanyl-L-alanylamino)-ethylphosphonic acid,

(IR) -1-(N-tert.butylglysyl'-L-alanyl-L-alanylamino)-ethylphosphonic acid,

(IR)-1-(N-benzyl-glysyl-L-alanyl-L-alanylamino)-ethylphosphonic acid, (IR)-1-(N-phenyl-glycyl-L-alanyl-L-alanylamino)-ethylphosphonic- acid,

((IR)-1-(N-methyl-L-alanyl-L-alanyl-L-alanylamino)-ethylphosphonic acid,

(IR)-1-(N-methyl-L-valyl-L-valyl-L-norvalylamino)-ethylphosphonic acid,

(IR)-1-(N-methyl-L-leusyl-L-norvalyl-L-norvalylamino)-ethylphosphonic acid,

(IR)-1-(N-sarcosyl-L-valyl-L-valyl-L-norvalylamino)-ethylphosphonic acid, and

(IR)-1-(N-methyl-L-valyl-L-norvalyl-L-norvalylamino)-ethylphosphonic acid,

Of the aforementioned peptide derivatives, (IR)-1-(N-sarcosyl-L-norvalyl-L-norvalylamino)-ethylphosphonic acid is preferred.

Physiologically tolerable salts form the peptide derivatives of formula I with physiologically tolerable strong organic and inorganic acids (for example HC1, HBr, H2SO4, methanesulfonic acid, p-toluenesulfonic acid) and bases (for example NaOH, KOH).

The antibiotic components in the active substance combinations according to the invention are preferably a 3-lactam antibiotic such as a penicillin, a cephalosporin or a monocyclic β-lactam antibiotic. As penicillins, in particular ampicillin, carbenicillin, penicillin G, sulbenicillin, mecillinam, pivmecillinam, pheneticillin, meticillin, pro-picillin, ticarcillin, amoxicillin and piperacillin come into consideration, as cephalosporins in particular cephalexin, cefazolin, cefoxitin, cefradin, cefsulodin, cefamandole, cephaloridine , cefaloglycin, cefatrizine, cefacetril, cefuroxime; cefaclor, cefotaxime and cefazedone, of which cephalexin is preferred. Furthermore, D-cycloserine, rifampicin, phosphonmycin, gentamycin, vancomycin and kanamycin come into consideration as antibiotics.

The weight ratio of a compound I or its salts to the antibiotic in the combination according to the invention can vary within wide limits. Generally, it is at 1:100 to 100:1, preferably at 1:64 to 64:1. Particularly preferred is a ratio from 1:16 to 16:1.

The active substance combination in the present application is prepared by mixing a compound of formula I or a physiologically tolerable salt thereof and an antibiotic with each other in the above-mentioned weight ratios.

The active ingredient combinations according to the invention are effective

against many gram-positive and gram-negative bacteria such as Escherichia coli, Proteus mirabilis, Pseudomonas aeruginosa, Staphylococcus aureus, Streptococcus pyogenes, Streptococcus faecalis, Streptococcus agalactiae, streptococcus dysgalactiae and Klebsiella aerogenes. The active substance combinations can therefore be used for the treatment and prophylaxis of bacterial infections. They can be given orally or parenterally.

The in vitro activity of the active substance combinations according to the invention was demonstrated in the following way: Concentrated solutions of mixtures of the compounds of formula I and the antibiotic were prepared in the desired weight ratios, which can be diluted as desired. Aliquots of the diluted solutions were mixed with a suitable nutrient agar in petri dishes. For comparison, similar agar plates were prepared which, in addition to the nutrient medium, contained the compound I or the antibiotic alone. After superficial inoculation with the microorganisms, the dishes were kept for 24 hours at 37°C and the minimum inhibitory concentration (M.I.C.) and the F.I.C. indices were then determined. The results using representative compounds of formula I, namely (IR)-1-(N-sarcosyl-L-norvalyl-L-norvalylamino)-ethylphosphonic acid, and (IR)-1-(N-methyl-L-leucyl -L-norvalyl-L-norvalylamino)ethylphosphonic acid and representative antibiotics, namely cephalexin and D-cycloserine, are summarized in the following tables I-III. Table I Activity of mixtures of cephalexin and (IR)-1-(N-sarkosyl-L-norvalyl-L- norvalylamino)-ethylphosphonic acid against Proteus mirabilis, Staphylococcus aureus, Streptococcus faecalis and Streptococcus pyogenes

Table II

Activity of mixtures of cephalexin and ( IR)- 1-( N- methyl- L- leucyl- L- norvalyl- L- norvalylamino)-ethylphosphonic acid against Staphylococcus aureus, Streptococcus pyogenes and Streptococcus faecalis

Table III

Activity of mixtures of D-cycloserine and

( IR)- 1-( N- sarkosyl- L- norvalyl- L- norvalylamino)-

ethylphosphonic acid against Escherichia coli,

Staphylococcus aureus, Streptococcus agalactiae and

Streptococcus dysgalactiae

The in vivo activity was demonstrated as follows:

Mice were infected intraperitoneally with 5-10 times LDgg of

a pathogenic organism. At specific times after the infection, groups of mice were treated subcutaneously with specific doses of the antibiotic, the peptide and a specific number of mixtures thereof. The number of mice that were still alive for each treatment 7 days after the infection was determined and CD^q was calculated from this. The F.I.C. indices were calculated for the mixtures in the usual way. The results obtained with (1R)-1-(N-sarcosyl-L-norvalyl-L-norvalylamino)-ethylphosphonic acid as a peptide of formula I and cephalexin and D-cycloserine respectively as antibiotics are summarized in the following tables IV

and V.

Table V

Chemotherapeutic activity (CD<-q mg/kg, s.c). of mixtures of D-cycloserine and (1R).-1-(N-sarkosyl-L-norvalyl-L- norvalylamino)-ethylphosphonic acid by

bacterial infections in mice

The active substance combinations can be given in the form of pharmaceutical preparations, which are likewise the subject of the present invention. The usual pharmaceutical carriers which are compatible with the compounds of formula I, respectively their salts and the antibiotics are used for the preparation of the preparations. Carriers for the enteral (eg oral) or parenteral application can be liquid or solid, organic or inorganic and include, for example, water, gelatin, mannitol, mineral oils, vegetable oils, gum arabic, propylene glycols or polyalkylene glycols.

The production of the pharmaceutical preparations can take place in a manner known per se by mixing the individual components with the suitable carrier material and bringing them into a suitable galenic form.

The pharmaceutical preparations can be in solid form (for example as lyophilisates) or in liquid form (for example as solutions, suspensions or emulsions). Optionally, they are sterilized and respectively or contain further auxiliary substances such as preservatives, stabilisers, wetting or emulsifying agents, agents for flavor improvement, salts for changing the osmotic pressure or buffer substances. When using buffers, the pH value of the preparations can vary within the usual limits.

The content of compounds of formula I or their salts and the antibiotic in the pharmaceutical preparations according to the invention and the dosage thereof can vary within wide limits within normal limits. The optimal conditions depend on the components used, the route of application, the type of infection and so on. Thus, for example, a suitable daily dosage for parenteral application is approx. 200-2000 mg of the mixture of the active components by oral application of 750-1500 mg. This dosage can be given as a single dose or in several divided doses and can be increased or decreased in special situations according to the doctor's prescription due to individual needs.

The following examples illustrate the invention:

Example 1

Hard gelatin capsules containing the following components:

(IR)-1-(N-sarcosyl-L-norvalyl-L-norvalylamino)-ethylphosphonic acid was granulated with 10% corn starch paste containing the dioctyl sodium sulfosuccinate. The moist granulate was dried, sieved, mixed with cephalexin and stearic acid (pre-sieved). The resulting mixture was filled into hard gelatin capsules.

Example 2

Hard gelatin capsules containing the following components:

The production of the capsules was carried out in an analogous manner to example 1, during which, however, the microcrystalline cellulose was mixed with the other ingredients before filling the capsules.

Example 3

Powdered mixture for the preparation of injection solutions which contained the following components:

The component was ground individually and well mixed with each other. The mixture was filled under sterile conditions into suitable containers. To prepare an injection solution, the resulting mixture is dissolved in 2 ml of water for injection purposes.

Example 4

Hard gelatin capsules containing the following ingredients:

(IR)-1-(N-sarcosyl-L-norvalyl-L-norvalylamino)-ethylphosphonic acid is granulated with polyvinyl<p>yrrolidone, magnesium oxide and water. The moist granulate is dried, sieved, mixed with D-cycloserine and magnesium stearate (pre-sifted) and filled into hard gelatin capsules.

Example 5

An injection solution is prepared by dissolving 200 mg of freeze-dried D-cycloserine and 200 ml of freeze-dried (lR)-l-(N-sarcosyl-L-norvalyl-L-norvalylamino)-ethylphosphonic acid in a sterile buffer solution (pH 6.7). The solution obtained is filtered under sterile conditions and freeze-dried aseptically. Before use, the freeze-dried material is reconstituted with 10 ml of a buffer. A suitable puffer can have the following composition:

Claims (10)

1. Procedure for the production of active substance combinations with antibiotic properties, which contain a peptide derivative with the general formula wherein R <1> is hydrogen, methyl, hydroxymethyl, a mono-, di- or trihalomethyl group; R 2 is a residue characteristic of an α-amino acid which normally occurs in proteins or a lower alkyl or hydroxy-lower alkyl residue which is characteristic of an α-amino acid which does not normally occur in proteins; R 3 is lower alkyl, lower cycloalkyl, lower alkenyl, aryl or aryl-lower alkyl; R 4 means hydrogen or lower alkyl; n means 2 or 3 and the configurations at the C atoms (a), and (b) are R (when R1 ^ H) respectively L , or a physiologically tolerable salt of such a compound, characterized in that a compound of formula I or a physiologically tolerable salt thereof and an antibiotic are mixed together. 2. Method according to claim 1, characterized in that a peptide derivative of formula I in which R is hydrogen or methyl is used as starting material. 3. Method according to claim 1 or 2, characterized in that a peptide derivative of formula I is used as starting material, in which R 2 is a residue that is characteristic of an α-amino acid that normally occurs in proteins or a lower alkyl residue that is characteristic of an α-amino acid that does not normally occur in proteins. • 4. Method according to one of claims 1-3, characterized in that a peptide derivative of formula I in which R 3 is lower alkyl is used as starting material. 5. Method according to one of claims 1-4, characterized in that (IR)-1-(N-sarcosyl-L-norvalylamino)-ethylphosphonic acid is used as starting material in formula I. 6. Method according to one of claims 1-5, characterized in that a Ø-lactam antibiotic is used as antibiotic. 7. Method according to one of claims 1-5, characterized in that a penicillin, a cephalosporin or a monocyclic 3-lactam antibiotic is used as antibiotic. 8. Method according to one of claims 1-5, characterized in that ampicillin, carbenicillin, penicillin G, sulbenicillin, mecillinam, pivmecillinam, pheneticillin, methicillin, propicillin, ticarcillin, amoxicillin or piperacillin are used as antibiotics. 9. Method according to one of claims 1-5, characterized in that cephalexin, cefazolin, cefoxitin, cefradin, cefsulodin, cefamandole, cephaloridine, cephaloglycin, cefatrizine, cefacetril, cefuroxime, cefaktor, cefotaxime or cefazedone are used as antibiotics. 10. Method according to one of claims 1-5, characterized in that D-cycloserine, rifampicin, phosphonomycin, gentamycin, vancomycin or kanamycin are used as antibiotics.