Journal of Medicinal and Pharmaceutical Chemistry VOL. 2, No. 1 (1060)
The Effect of plDiethylaminoethy1 diphenylpropylacetate Hydrochloride (SKF 525-A) on the Therapeutic Action of a Variety of Antibacterial, Antiviral or Antiprotozoal Drugs J. M. THORP,E. WESTONHURSTand A. R. MARTIN, Imperial Chemical Industries Limited, Pltarmaceuticals Division, Alderley Park, Macclesjield, Cheshire Introduction The enhancing effect of fl-diethylaminoethyl diphenylpropylacetate hydrochloride (referred to as SKF 525-A), which in the experimental animal prolongs the period of activity of a diversity of drugs, has been shown by Axelrod, Reichenthal and Brodiel to result from an inhibition of the metabolic transformation of these drugs. Using liver slices and homogenates, Cooper, Axelrod and Brodie2 demonstrated that SKF 525-A inhibits the enzyme systems which bring about oxidation of alkyl side-chains, dealkylation of alkylamines, deamination of sympathomimetic amines, ether-cleavage and phenol-conjugation. Metabolic hydroxylation of aromatic nuclei is also blocked by SKF 525-A, though a t a relatively high concentration of the compound. The whole subject has been reviewed by Brodie. s Diphenylpropylacetic acid (SKF-acid) is equally active a t a cellular level, though only feebly so in the intact animal. A number of studies with the two compounds have confirmed the general nature of the phenomena, and it is apparent that SKF 525-A may sometimes be used to indicate whether the unchanged drug or a metabolite is responsible for a therapeutic or pharmacological response in treated animals. We ourselves have used SKF 625-A in conjunction with a number of therapeutic agents, and report here our observations of its influence on the therapy of four bacterial, a viral and two protozoal infections. 15
16
J. &I. THORP, E. WESTON HURST AND A. R. MARTlN
1. A Streptococcal Infection in Mice
Methods Groups of 12 or more mice received intraperitoneally 0 2 ml of a suitable dilution to of Streptococcus agahctiae (Kriiger strain), grown for 24 h in serum-broth or blood-broth. In earlier experiments the culture was derived from a stock conserved in Robertson's meat medium; later, owing to highly inconstant results, we passed the stock culture weekly in mice, re-isolated the organisms from the heart-blood, and used the culture so obtained to initiate that for infecting the experimental groups. Except where otherwise stated, treatments began 15 min after infection and continued twice daily for 3 days.
Sulphoyuhide therapy Twice-daily oral dosing with 2 * 5 mg of sulphadiazine increased the mean period of survival of infected mice from approximately 1 day to over 4 - 5 days, and with 1 mg to nearly 4 days. Treated mice did not begin to die for several days after all the controls were dead. Dnses of 5 mg of sulphanilamide more than trebled the period of Burvival, and of 2 mg increased it by hound 50 per cent. None of these effects was modified by simultaneous intraperitoneal administration of 1 mg of SKF 525-A twice daily. These results accord with our knowledge of the metabolism of the sulphonamides and of the supposed action of SKF 525-A.
Bureornycin therapy As seen in Table I, doses of 0 25 mg chlortetracycline" slightly diminished mortality and more than doubled the period of survival of those mice which died. Doses of 0 . 1 mg exerted a lesser effect. The conjunction of SKF525-A did not influence mortality, but greatly increased the mean periods of survival. In a repeat experiment, in which a larger dose of chlortetracycline (0.4mg) was included, the effect of SKF was similar, except that a t the highest level of dosage there was in addition a significant reduction in the number of deaths among animals receiving the
*
Aureomyoin @
SKF 625-A AND THERAPEUTIC ACTION
17
combined treatment. Given by itself, SKF 525-A exerted no beneficial effect. Table I. Effect of SKF 525-A on the activity of chlortetracycline in streptococcal infection of the mouse Treatment
Deaths in 40 mice
None SKF 625-A 1 mg i.p. b i d . Chlortetracycline 0.25 mg orally b i d . Chlortetracycline 0.1 mg orally b.i.d. Chlortetraoycline 0.25 mg+SKF 525-A Chlortetracycline 0 . 1 mg+SKF 525-A
40 (1.1) 40 (1.1) 31 (2.4) 34 (1.5) 32 (4.4) 35 (3.9)
We gave streptococciintraperitoneally. Treatments began 16 min after infection and continued twice daily for 3 days. The Agures in parentheses are the mean periods of survival (in days) of the caaes proving fatal.
SKF 525-A, therefore, enhances the therapeutic effect of chlortetracycline in a streptococcal infection in mice, without itself exhibiting therapeutic properties.
Penicillin therapy The effect of SKF 525-A on treatment wiLi penicillin G was unexpected. I n the experiment summarized in Table 11, it was Table 11. Effect of SKF 525-A on the activity of penicillin G in streptococcal infection of the mouse Treatment
Deaths in 40 mice
None SKF 626-11 1 mg i.p. b.i.d. Penicillin G 0.1 mg S.C. b.i.d. Penicillin G 0.02 mg S.C. b i d . Penicillin G 0.1 mg+SKF 525-A Penicillin G 0 * 02 mg SKF 625-A
+
Expefimental procedure as in Table J
40 (1.2) 40 (1.1) 8 (4.5) 13 (3.2) 32 (2.9) 38 (2.3)
18
J. M. THORP, E. WESTON HURST AND A. R. MARTIN
responsible for a very sharp diminution of the therapeutic effect, both when judged by the numbers of mice surviving and by the periods of survival of those ultimately succumbing to the infection. I n a further experiment in which the streptococci were rather more virulent and the doses of penicillin G were halved, the latter greatly increased the mean periods of survival without appreciably affecting mortality. On this occasion SKF 625-A almost completely abolished the effect of the penicillin, The phenomenon was confirmed in a t least 20 experiments. To explain the effect of SKF 525-A in diminishing the therapeutic action of penicillin, we considered the following possibilities. (a) Increased or reduced metabolic transformation of the antibiotic by the host or by the bacterium to, respectively, less or more active derivatives. (b) Reduced access of penicillin to the infecting organism by, (i) formation of an insoluble salt of penicillin in the host, (ii) enhanced excretion or modified distribution of penicillin in the host, or (iii)reduced permeability of the bacterial cell to penicillin. (0) Direct antagonism of fhe antibacterial action of penicillin within the bacterial cell. (d) A modified response of the host to infection, brought about in one of several possible ways. The results of experiments to test some of these hypotheses follow. Metabolism of penicillin. An extensive search of the literature failed to reveal any precise analytical study of the products of penicillin metabolism, with the single exception of the work of Ullberg, who demonstrated that 8SS-labelled benzyl penicillin was degraded (to the extent of about 10 per cent in 3 h) to 85spenicilloic acid by the cat and guinea-pig. Penicilloic acid is devoid of antibacterial activity. No suggestion that biologically active metabolites of penicillin may be formed in vivo has been made. That one such metabolite of penicillin G might be phydroxybenzyl penicillin (penicillin X) seemed possible, since SKF 625-A is known to block aromatic hydroxylation, and since penicillin X has been reported t o be two to three times as active 8s penicillin G (on a weight basis) against certain bacterial
19
SKF 525-A AND THERAPEUTIC ACTION
infections (Str. pyogenes and Str. pneurnoniae) in mice (Eagle,6 Hobby, Lenert and Hyman6). If penicillin X were the active metabolite formed from penicillin G, it would be expected from the experimental findings detailed above that its therapeutic effects would not be blocked by SKF525-A. As shown by the results presented in Table 111, neither penicillin X nor any Table 111. Effect of SKF 625-A on the antistreptococcal activity of a variety of penicillins
,
Treatment
None SKF 525-A 1 mg i.p. b i d . Penicillin G 0.05 mg 8.0. b.i.d. Penicillin G SKF 525-A Penicillin X 0.025 mg 5.0. b i d . Penicillin X SKF 525-A Penicillin K 0.25 mg S.C. b i d . Penicillin K 0 * 1 mg 8.0. b i d . Penicillin K 0.25 mg+SKF 625-A Penicillin K 0 * 1 mg SKF 626-A Penicillin D 0.04 mg S.C. b.i.d. Penicillin D +SKF 525-A Penicillin V 0.05 mg S.C. b i d . Penicillin V SKF 525-A Penicillin F 0.05 mg 5.0. b.i.d. Penicillin F +SKF 525-A
+
+
Deaths in 20 mice -7
Exp. 1
Exp. 2
20 (1.0) 20 (1.0) 0 16 (3.9) l(6.0) 6 (4.8)
20 (1.0) 20 (1.2) l(7.0) 16 (3.9) l(7.0) 13 (3.6) 2 (4.0)
-
15 (3.9) 17 (3.2)
+
-
11 (4.4)
-
0 14 (4.5) 0 16 (3.4)
+
-
-
0 15 (3.6) l(4.0) 12 (4.0)
Experimental procedure as in Table I. The chemical constitution of the above penicillins and the potency in u/mg (assayed against Staph. aweus-Oxford 6571) of the samples tested are as follows:
kHCO--R
G; R=benzyl (1660), X ; R=p-hydroxybenzyl (700), K; R=n-heptyl (2661), D; R-n-amyl (1713), V ; R-phenoxymethyl (1680)and F; R=d*-pentenyl(1784).
20
J. M. THORP, E. WESTON HURST AND A. R. MARTIN
of the other penicillins available to us escaped the dystherapeutic effect of SKF 525-A. Since the therapeutic activity of all the penicillins tested was affected by SKF 525-A, it appeared that the variable side-chain R- was unlikely to be the 5 0 k point of SKF 525-A-blocked metabolism, if this were the explanation of the phenomena observed. Other sites of the penicillin molecule might of course be subject to metabolic attack. Oxidation of the sulphur atom of the thiazolidine ring gives rise to the sulphone or sulphoxide, but once again the antistreptococcal activity of methyl penicillinate sulphone and methyl penicillinate sulphoxide was blocked by SKF 525-A. Conjugation with the carboxylic acid group of penicillin has been suggested as a stage in the renal tubular excretion of penicillin, involving conjugation with glycine. We have found, however, that no detectable glycine conjugate of penicillin occurs in the urine of penicillin-dosed animals (Thorp and Pickstock, unpublished). Nor has it been shown that penicillin occurs in the urine in a form other than the free acid, or that SKF 525-A blocks conjugation. Thus this would seem an unlikely metabolic explanation. Oxidation of a methyl group. We have shown (Francis et aZ.)7 that 2,3-dimethylquinoxaline-di-N-oxide is rapidly and extensively metabolized to the corresponding 2-hydroxymethyl derivative. Such oxidation of penicillin would give rise t o (I),from which the lactone (11)could be formed. Bachhawat et a1.8 have recently demonstrated the enzymatic carboxylation of /I-hydroxyisovaleric acid (111) to p-hydroxy-/I-methyl glutaric acid (IV). A similar carboxylation of penicillin would give rise to the dicarboxylic acid (V). Since none of the metabolites postulated were available for direct test, evidence for their occurrence was sought by other means. Paper chromatographic analysis, by our colleague Dr. R. R. Goodall, of the urine of mice treated with penicillin G disclosed the presence of a bacteriostatic substbnce, not present in the original penicillin or in normal urine ; this took up an intermediate position between penicillin G and penicillin X in the system of Goodall and Levi.9 The amount of the metabolite was decreased in mice given SKF 525-A, and its excretion was blocked similarly to that of penicillin by a renal blocking agent related to
SKF 625-A AND THERAPEUTIC ACTION
21
probenecid. Penicillin V gave two derivatives, the appearances of which were completely abolished by simultaneous administration HOCH, \
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