NO139004B - PREPARATION FOR TESTING AND DETERMINATION OF PROTOCOL AND BACTERIAL INFECTIONS - Google Patents

Abstract

Preparation for testing and determining protozoal and bacterial infections.

Description

The present invention relates to preparations for testing

and determination of protozoal and bacterial infections caused by microorganisms.

Tetrahydrofolate cofactors are essential metabolites

in all cells for the biosynthesis of purines, thymidylic acid, serine and other biologically important compounds. Most of these co-factors are one-carbon adducts of tetrahydrofolic acid. Humans and higher animals get these compounds via food that contains formed folates, usually in the form of vitamins.

In the microorganisms, these co-factors will be synthesized from simpler compounds. Typically, the biosynthetic process will first provide dihydropteridine (Pt), i.e., 2-amino-4-hydroxy-6-hydroxymethyl-7,8-dihydropteridine (HMPt) pyrophosphate ester, from its immediate precursor HMPt in the presence of the enzyme hydroxymethyldihydropteridine pyrophosphokinase (HMPPS). Pt is then condensed with p-aminobenzoic acid (pAB) in the presence of the enzyme dihydropteroate synthetase to dihydropteroic acid (DPtA). This intermediate product further condenses with glutamate to dihydrofolic acid (DFA or "Folate") which is then enzymatically reduced to the vital compound tetrahydrofolate, which e.g. takes place in bacteria and other microorganisms.

The synthesis of "folate" from the basic building blocks, ie: pteridine, pAB and glutamate, and the further conversion of these into tetrahydrofolate is known to be inhibited in two different ways. Thus, e.g. sulfonamides replace pAB in the above reaction core. Because of their close structural similarities to pAB, sulfonamides or similar other "competing compounds" enter the biosynthesis and prevent the formation of ay DPtA and of DFA, and are therefore antimetabolites for the metabolite pAB.

It is also known that compounds which are "inhibitors" or facilitators of the enzyme dihydrofolin reductase, block the synthesis

tical steps leading to tetrahydrofolate. A significant number of pyrimidine derivatives show a significant anti-microbial property on the basis of such blocking.

It has later been established that such facilitators or inhibitors can act synergistically together with the sulfonamide, i.e. there can be a double blocking in sequence and a strong mutual reinforcement or potentiation of the antibacterial effects of the two compounds. The range of antimicrobial action exerted by such combinations is considerably wider than would be expected from the activity of each individual compound, and organisms which are only marginally sensitive to the individual compounds may become highly sensitive to the combinations.

It has also been hypothetically proposed that the antimetabolites of Pt could inhibit the biosynthesis of DPtA (and DFA) (cf. Hitchings and Burchall Advances in Enzymology, 27, 417-468 (1965)), but the compounds that have so far been tested for this purpose , have been disappointing, being either inactive or too toxic or sometimes both (cf. the compounds which are not described in British Patent Nos. 981,506 and 987,916). It has also been established that for antimicrobial purposes it is a prerequisite for an effective antagonism of Pt that the compound should be an inhibitor or inhibitor of HMPPS without also acting as an antimetabolite for dihydropteridine, which serves as a co-factor for the hydroxylation of phenylalanine and tyrosine, precursors of catecholamines, such as norepinephrine, which are very important as regulators of the cardiovascular system.

It has now been found that certain pteridines fulfill the above requirements and these compounds not only inhibit the growth of microorganisms alone, albeit to a certain extent with certain bacteria such as Staphylococcus aureus, Streptococcus pyogenes, Streptococcus faecalis, Escherichia coli, Salmonella typhi, Proteus vulgaris , Pseudomonas aerugenosa, Pasteurella multocida among others, but have also been found to work with a highly significant synergistic effect when combined with a competitor to pAB, or with a selective inhibitor of dihydrofolin reductase, or with a combination of both these types of antimicrobial agents. ;

The various combinations of. competitive compounds, inhibitor and pteridine are suitable for the testing and determination of protozoal or bacterial infections caused by those microorganisms which synthesize at least a substantial part of their tetrahydrofolate co-factor requirements, such as Staphylocossus aureus, Pseudomonas aerugenosa and Pasteruella multocida. These infecting microorganisms are more particularly those which adequately absorb said combinations and against which these combinations have a synergistic effect in that they affect the re-synthesis of the necessary tetrahydrofolate co-factors.

In particular, it has been found that such a pteridine compound,

hereinafter referred to as "potentiating compound", can be combined with an amount of the competing compound and/or inhibitor, which is usually not sufficient to be effective as an agent for testing microorganisms, to obtain a preparation which acts as an effective microbial agent. This is particularly evident when the quantity of the potentiating compound is so small that it essentially has no microbial effect, but nevertheless in combination has a certain effect, and in certain cases a very marked effect.

According to the present invention, there is thus provided a preparation for testing and determining protozoal and bacterial infections caused by microorganisms that synthesize at least a significant part of their tetrahydrofolate co-factor requirement, and this preparation is characterized by the fact that it comprises a potentiating amount of a pteridine compound that inhibits the enzyme hydroxymethyldihydropteridine pyrophosphokinase, and which has the general formula:

where R is hydroxymethyl and R.^ and R ? is lower alkyl, in combination with a competing compound to p-aminobenzoic acid, namely sulfamethoxazole, and/or an inhibitor to dihydrofolin reductase, namely 2,4-diamino-5~ ( 3' , 4 ' , 51 -trimethoxybenzyD-pyrimidine in that the preparation contains 1-30, preferably 5-15 parts of the pteridine compound, 1-30, preferably 5-15 parts of sulfamethoxazole,

and/or 1 part of the pyrimidine compound.

The amount of "competing compound" and "inhibitor" used will either (a) be effective to some extent to

provide an antimicrobial agent for the testing or determination of microorganisms, but which is enhanced by the use of a pteridine-potentiating compound, or (b) be ineffective in providing an antimicrobial agent for the above purpose, but which, combined with a pteridine- potentiating compound will give a preparation which is effective for the purpose.

The amount of potentiating compound, i.e. the pteridine compound,

will increase the activity of a competing compound and/or inhibitor and thereby provide a preparation with improved efficiency for the testing and determination of microorganisms.

It should be emphasized that the terms "competing compound", "inhibitor" and "potentiating compound" are ready-made and serve only as appropriate names for the appropriate type of components in the combination processes. The inhibition of the biosynthetic processes can be described as competitive antagonism in all three cases, and there can be potentiation between all three types of agents.

The potentiating or enhancing effect of a pteridine potentiating compound in combination with a competing compound and/or inhibitor can be demonstrated and utilized in vitro relatively easily for research and practical purposes. Such possibilities include diagnosis and characterization of the bacterial flora in individuals and a resulting choice of clinical treatment methods.

The preparation combination can e.g. incorporated into porous disks, (such as filter paper disks), or onto nutrient agar or other media for bacterial growth, for determination of sensitivity. These articles can then be distributed or sold to doctors, hospitals and clinics for the above-mentioned purposes. A typical test disc can be impregnated with a solution containing 5-50 µg/ml of a competing compound, 0.5-5 µg/ml of an inhibitor and about 10-100 µg/ml of a potentiating compound in a medium comprising a mixture of an aqueous infusion fluid.and papain-treated horse muscle.

Such tests involving the use of potentiating and competing compounds and/or inhibitors can also be useful for characterizing bacteria according to their sensitivity and their particular resistance, e.g. compared to a competing connection when this is used alone. Such investigations, which include a number of preparations according to the present invention, can also form the basis for determining preparations with special compositions for general treatment purposes.

For selection of a suitable pteridine compound, e.g. its inhibitory activity towards HMPPS is tested by controlling

32 the transfer of terminal phosphate in adenosine triphosphate ATP-γ-P to dihydropteridine. It was found that concentrations required for 50% inhibition of the formation of Pt (IC^q) in such samples were well correlated and within the margin of error obtained by other relevant samples in this respect, and which measure the inhibition of each of the two enzymes involved in the formation of HMPt and DPtA. Such inhibition can e.g. easy and on

simple way is carried out by incubating an extract of E.coli with

14

pAB-7-C, ATP, Mg and dihydropteridine. The formation of dihydro-

14

pteroate-C can be assessed quantitatively after separation of the unreacted pAB substrate, e.g. by chromatography. It has been found that compounds which with such tests have an IC[rn value of about 100 µM or less, usually below 50 µM, represent compounds which exhibit a useful potentiating effect.

The value is preferably 25 uM or less, e.g. in the range 2-12 yM. Generally, a value of less than 7 µM is desired.

It has been shown that the compounds 2-amino-4-hydroxy-6-hydroxymethyl-7,7~dimethyl-7,8-dihydropteridine, hereafter designated as DMHP, and 2-amino-4-hydroxy-6-hydroxymethyl-7, 7~diethyl-7,8-dihydropteridine has very good potentiating properties. These compounds and their analogs are described in British Patent No. 1,303,171, US Patent No. 3,635-978 and British Patent No. 1,454,165, and methods of their preparation are further described in an article by Pfleiderer and Zondler in Chem. Pray. 99, 3009 (1966)^,

in British Patent No. 1,363-064 and in Belgian Patent No. 770,577-

The methods mentioned above for the preparation of compounds of formula (I) generally comprise reacting a compound of formula (II) or a salt thereof, where R, R 1 and R 2 are as defined above, and X is a ketone oxygen atom or a protecting group thereof, such as an oxime or thiosemicarbazone group, with 2-amino-4-chloro-6-hydroxy-5-nitropyrimidine, followed by removal of the protecting group when appropriate, after which the resulting product is reductively ring-closed. If necessary, the substituent group can be converted into various other groups using standard techniques of a commonly known type, e.g. by brominating a 6-alkyl group whereby 6-bromoalkyl or the 6-dibromoalkyl derivative is obtained.

Several compounds are known which are competitive compounds to p-aminobenzoic acid and which are antimicrobial agents,

cf. e.g. Merck Index, 8th edition, 1968, pages 992-1007-

Likewise, many compounds are known which inhibit dihydrofolin reductase and act as antimicrobial agents, cf.

e.g. US Patent Nos. 2,658,897, 2,767-183, 3-021,332, 2,937-284, 3-332,765, 2,909-522, 2,624,732, 2,579-259, 2,945-859, 2-576-939, 2,926,166 , 2,697-710, 2,749-345 and 2,749-344.

On the basis of possible synergistic benefits of

use certain competitive compounds and inhibitors in combina-

tion for the testing and determination of microbial infections, and the potentiating effect of compounds of formula (I) on

both of these types of antibacterial compounds, it has been preferred to work up three combinations. Thus, sulfamethoxazole/2,4-diamino-5-(3',4*,5'-trimethoxybenzyl)-pyrimidine ("Trimethoprim")/DMHP has been shown to have an improved efficacy when compared to the components alone or these in pairs together. The following examples illustrate the invention. ;Example 1 ;Potential pteridines which together with a competitor compound and/or an inhibitor are suitable for testing and determining microbial infections can be tested by examining their inhibitory effect on enzymes responsible for the biosynthesis of di-hydropteronic acid ( DPtA), i.e. hydroxymethyldihydropteridine pyrophosphokinase (HMPPS), and dihydropteroate synthetase, hereinafter referred to as "synthetase". ;1) HMPPS ;2-amino-4-hydroxy-6-hydroxymethyl-7 , 8-dihydropteridine (HMPt) +ATP Mg_2 ^+2-amino-4-hydroxy-6-pyrophosphomethyl-7,8-dihydropteridine (Pt) +AMP ;(Pt is the pyrophosphate ester of HMPt) ;2) Synthase ;Pt + p-aminobenzoic acid (pAB) Mg 2 +^ dihydropteroic acid (DPtA)+pyrophosphate. (a) A test was developed for HMPPS where the transfer of ;32 ;terminal phosphate from ATP-γ-P to Pt could be controlled and correlated with the degree of inhibition exerted on HMPPS by the compound to be tested. ;The compound to be tested, namely DMHP as a potential pteridine agonist, was incorporated into different preparations consisting of metabolites and enzymes in different samples, which is indicated in Table 1. ;The components of the mixture were the following: ;I 2-amino- 4-hydroxy-6-hydroxymethyl-7,8-dihydropteridine (HMPt) in a concentration of 800 uM, i.e. micromolar. ;II a source of HMPPS obtained from an extract of E.coli and separated from "synthetase" and "Sephadex G-100" by the method described by Richey and Brown in J.Biol.Chem. 244, 1582-1592 (1969). ;III 3 mM ATP-γ-P<32>;IV 0.10 M ATP neutralized (unlabeled) ;V 0.02 M MgCl2.6H20 ;VI 0.1 M MgCl2.6H20 ;VII source for HMPPS and "synthetase" ;VIII the test compound in a concentration of 0.93 x 10 3 M ; 1X 0.4 mM pAB-C<14>. ;As shown in Table 1, all tubes from 1 to 9 contained one source of HMPPS, labeled ATP and 0.02 M MgCl2.6H20, while tubes 2 to 9 additionally contained HMPt, while tubes 4 to 9 additionally contained the test compound (DMHP) . Control tubes 10 to 12 contained a source for both HMPPS and synthetase, unlabeled ATP, 0.1 M MgCl 2 .6H 2 O, and labeled pAB. Tubes 1 to 9 contained the amounts of the components shown in the table and the tubes were then filled to 200 µl with distilled water, incubated for 60 minutes at 37°C and then cooled on ice. Dextrose (20ul) containing 72.1 rag/ml<,> and hexbkinase (5ul containing 200 units/ml) were added to the solution which was then set aside at room temperature for 15 minutes. "Darco-G-60" (10 mg) was added to each tube and the contents stirred periodically for 10 minutes. The charcoal was removed through a "Millipore AP 2200" filter and the filter was washed with three 10 ml portions of cold water. The charcoal and the filter were then radioactively counted. The radioactive count from the contents of tubes 2 and 3 was taken as the maximum number, as these tubes did not contain any test compounds and thus gave 0% enzyme inhibition. The percentage inhibition produced by the contents of the remaining tubes could then be calculated by comparing their radioactive count with the maximum. ;The contents of tubes 10 to 12 were chromatographically analyzed as described under part (b) and used as a control, tubes 10 to 11 containing no test compound (and therefore giving 0% inhibition), ;and this control value was set as 100% . The percentage inhibition shown by the contents of the tubes in part (b) of the experiment could then be calculated in relation to this, by comparing the respective chromatograms. (b) The activity of the above test compound "synthetase" was determined as indicated below by examining how dihydropteroate-14 ;C was processed. ;A base solution of Pt was prepared from ATP (neutralized) ;(50 <y>l, 0.1 molar), MgCl2.6H20 (50 <y>l, 0.1 molar), dithiothreitol (100 µl, 0.1 M), tris buffer (100 <y>l, 0.4 M, pH 8.3), HMPt (25 VI, 876 VM) and 170 L of a solution containing HMPPS. The mixture was incubated for 60 minutes at 37°C, briefly cooled on ice, after which dextrose (100 μl containing 72.1 mg/ml) and hexokinase (20 VI containing 2000 units/ml) were added at room temperature, after which the solution was set aside at room temperature in 15 minutes. ;A solution of MgCl2.6H20 (10 µl, 0.1 M), pAB-C<11*> (10 µl, 0.4 mM), dithiothreitol (20 µl, 0.1 M) and Tris buffer (20 µl, 0.4 M, pH 8.3) was prepared in five different test tubes, after which 20 µl of the base solution was added to each tube together with synthetase and/or the test compound as indicated in Table 2. The solution was then diluted to 200 µl with distilled water.

Two. control test tubes were prepared, each containing_ATP

(10 µl, 0.1 M), MgCl2.6H20-(10 µl, Q.1 M), dithiothreitol (20 µl, 0.1 M)

tris buffer (20 µl, 0.4 M, pH 8.3), pAR-C<1>^ (10 µl, 0.4 mM) and 20 µl sv-ene solution containing HMPPS and "synthetase" of known activity. The test compound was added to the second of these two tubes to a final concentration of 10 J molar, and both tubes were then diluted with distilled water to 200 µl.

All seven tubes were then incubated for 30 minutes at 37°C, cooled on ice, after which these, together with the control tubes of 10 to 12 from part (a), were analytically chromatographed in the following manner: 100 µl of the contents of each of the tubes was deposited on Whatman No. 3MM chromatographic paper (2 x 20 cm) at the starting point, the experiment being carried out in a Sørensen's buffer of potassium and sodium phosphates (0.1 M, pH 7.0) at distances of 10 to 15 cm. From the relative positions of the spots obtained from the different tubes, one could calculate the varying percentage inhibition of synthetase with reference to the control tubes 10 and 11, which gave 0% inhibition.

Column (X) of Table 1 and fourth column of Table 2 give percent inhibition shown with DMHP as the test compound.

The compounds which, according to these tests, gave a 50% inhibition at a concentration of 10 µM or less, are those which show a usable potentiating effect, so they can be used in pharmaceuticals.

The results of the inhibition exhibited by preferred potentiating compounds are shown in Table 3.

Example 2

The antibacterial activities against Staphylococcus aureus of sulfamethoxazole and trimethoprim combined with DMHP, either alone or as a triple combination, were investigated. A typical experiment was carried out using "Wellcome Nutrient Agar" as a growth medium by incorporating sulfamethoxazole and trimethoprim and varying amounts of DMHP from 3.1 µg/ml to 25 µg/ml, inoculating the microorganism on the surface of the medium and incubating the culture system in 18 hours at 37°C. Table 4 shows that the addition of DMHP potentiated the individual activities of sulfamethoxazole and trimethoprim and further promotes the synergy of these substances when they work together.

Example 3

The percentage inhibition of the growth rate of Staphylococcus aureus N 491 by -sub-effective doses of sulfamethoxazole and trimethoprim alone or each in combination with 5 or 10 µg/ml of DMHP or triple combinations of these components was examined after a 7 hour incubation period in "Wellcome Nutrient Brotn" at 37°C. The inhibitory effects on growth rate, which were accentuated by the presence of DMHP, were measured turbidometrically by comparing the opacity of the suspensions with those of a series of 10 standard tubes containing different dilutions of suspended barium sulfate, which were suitably calibrated for the particular organism and weight condi- the others. The results from this experiment are shown in table 5.

Example 4

The results of a similar experiment to that described

in example 2, is shown graphically in table 6, the growth rate being measured spectrophotometrically by measuring the light lost from a beam by scattering of particulate matter using a spectrophotometer and calculating the bacterial growth using Beer's law. In said table, the percentage growth achieved is represented by the black bar when 10 µg/ml DMHP was also present, while the unfilled bar when said compound was absent.

From the above-mentioned experiments, it can be concluded that DMHP not only promotes the effects of trimethoprim and sulfamethoxazole when these substances act alone, but the presence of said compounds generally improves and potentiates the activity of combinations containing the compound. cf

Example 5

In this experiment, the effects of DMHP on the bactericidal activity of rimethoprim and sulfamethoxazole against Staphylococcus aureus were determined, whether the compounds were administered individually or in combination.

The results are shown in table 7 where the bactericidal activity is expressed as a percentage of the original inoculum, measured by the number of live cells that were present and alive after 24 hours of incubation at 37°C. The inoculum used gave a final concentration of 10^ organisms per ml.

In this test it could be demonstrated that although neither trimethoprim at a concentration of 1.0 ug/l nor sulfamethoxazole at a concentration of 10 ug/ml was bactericidal in the absence of DMHP, they were both very active when 10 ug/ml of DMHP was present . Furthermore, it could be demonstrated that the bactericidal activity of 0.1 ug sulfamethoxazole per ml increased significantly when 10 ug/ml DMHP was present.

Example 6

In this experiment, zone of inhibition data were determined to assess the synergistic activity of DMHP or its 7,7-diethyl analog on its combination with trimethoprim (TMP) and/or sulfamethoxazole (SMX) against Staphylococcus aureus and Pseudomonas aerguginosa.

The pteridine was included in a soy peptone medium with a low thymidine content ("Wellcotest Sensitivity Test Agar") in a Petri dish, and the other components were added to the hole obtained when a small plug of the medium was removed. The surface of the medium was inoculated with the test organism and then incubated. The degree of zone of inhibition is shown in Table 8, where the numbers represent complete zone inhibition (ie the number of cm from the edge of the hole after 6x magnification) and the numbers in parentheses include zones of partial inhibition.

The results show that both pteridine compounds show synergism with sulfamethoxazole and trimethoprim alone, and multiple synergism with both above Staphylococcus aureus, and the diethyl analogue was somewhat more active than DMHP. Against Pseudomonas aerginosa, on the other hand, DMHP showed the highest potentiating effect. One could thus provide a test system to distinguish between these two microorganisms.

Example 7

Sensitivity tests were carried out to show whether the selected microorganism was inhibited by certain combinations of DMHP, sulfamethoxazole and trimethoprim, and further to determine

a possible potentiation of sulfamethoxazole/trimethoprim combinations by means of DMHP. In these experiments, the selected microorganisms were spread over the surface of the "Wellcotest Sensitivity Test, Agar" using a cotton swab and then 6 mm filter paper discs were impregnated with a solution of a suitable preparation mixed with an aqueous infusion fluid and papain-horse muscle , placed at a great distance on said surface.

After the incubation, the degree of zone inhibition was produced

with the help of the preparations the growth of the microorganisms was measured and the results are indicated in table 9 for the microorganisms Staphylococcus aureus, Streptococcus faecalis, Escherichia coli, Proteus vulgaris cg Pseudomonas aeruginosa. An improvement in sensitivity could be observed when DMHP was present if the zone of inhibition around the triple-combination disk was larger than the zones around each of the double-combination or single-component disks. The usefulness of such sample discs for the identification of microorganisms has thus been demonstrated.

Claims (3)

1. Preparation for the testing and determination of protozoal and bacterial infections caused by microorganisms that synthesize at least a substantial part of their tetrahydrofolate co-factor requirement, characterized in that it comprises a potentiating amount of a pteridine compound that inhibits the enzyme hydroxymethyldihydropteridine-pyrophospho -kinase, and which has the general formula: where R is hydroxymethyl and R-^ and R^ is lower alkyl, in combination with a competitive compound to p-aminobenzoic acid, namely sulfamethoxazole, and/or an inhibitor to dihydrofolin reductase, namely 2,4-diamino-5-( 3',4',5'-trimethoxybenzyl)-pyrimidine, the preparation containing 1-30, preferably 5-15 parts of the pteridine compound, 1-30, preferably 5-15 parts of sulfamethoxazole, and/or 1 part of the pyrimidine compound. 2. Preparation according to claim 1, characterized in that the pteridine compound is 2-amino-4-hydroxy-6-hydroxymethyl-7,7-dimethyl-7,8-dihydropteridine. 3. Preparation according to claim 1, characterized in that the pteridine compound is 2-amino-it-hydroxy-6-hydroxymethyl-7,7-diethyl-7,8-dihydropteridine.