Studies on antibiotics : Investigations on the mechanism of action of penicillin and pterygospermin

Abstract

A résumé of the historical development and present status of research in the field of antibiotics is given under the headings: (i) Early investigations on the treatment of infectious diseases, (ii) Sulphonamides, (iii) Antitoxins, (iv) Definition of antibiotics, (v) History of antibiotics, (vi) Sources of antibiotics, (vii) “Chemotherapy” and mechanism of action of antibiotics. A clear understanding of the way in which the antibiotic effect is exerted is undoubtedly desirable, in that it will not only rationalise the application of antibiotics in therapeutics on a scientific basis, but also may provide scope for the selection and preparation of simpler active substances synthetically as substitutes for the costlier antibiotics. However, owing to the fact that in spite of concerted endeavour to unravel the mechanism of action of many antibiotics, information collected has provided an inconsistent and heavy picture. Many of the theories advanced involve the effect of antibacterial agents on the fertility, growth, and multiplication of other metabolic processes such as protein and carbohydrate metabolism of the susceptible organisms. Some theories involve the concept of interference in certain enzyme systems or systems by acting competitively with an essential metabolite or in some other ways as yet unknown. Some hypotheses are also advanced to explain their action on physico chemical grounds. Considering the diverse chemical nature of antibiotics, any generalised theory encompassing the explanation for the action of all antibiotics and other inhibitory substances appears impossible, and as yet little headway has been made to explain the relation between chemical constitution and antibacterial action. The work described in this thesis forms a part of the programme in this laboratory dealing with the mechanism of action of antibiotics, and concerns an effort to understand the mechanism of action of penicillin on a biological as well as a structural basis, and a comparison of its action with that of pterygospermin, the antibiotic principle present in the roots of Wrightia tinctoria (the common Indian “dye stick tree”). Pterygospermin was isolated in these laboratories. ________________________________________ Chapter I The study of the mechanism of inactivation of penicillin by many micro organisms has been undertaken. Various types of bacteria such as Bacillus cereus, B. anthracis, B. subtilis, Streptococcus pyogenes var. aureus, have been screened for their resistance to penicillin and formation of “penicillinase.” The amount of “penicillinase” formed depends not only on the adaptive capacity of the organism to produce the enzyme under the stress of added penicillin, but also on cultural conditions in which the organisms are grown. Organisms belonging to the spore forming bacilli produce large quantities of the enzyme under the stress of added penicillin. In this connection, the cultural conditions under which good yields of penicillinase could be obtained, the purification and biochemical characteristics, thermal stability, effect of dialysis, effect of pH and metal ions, etc., from two typical organisms-B. subtilis and B. cereus-are studied. In conformity with earlier observations, evidence has been adduced to show that apart from the formation of “penicillinase,” there are other ill defined factors responsible for the resistance of organisms to the action of penicillin. To demonstrate the process of inactivation of penicillin by penicillinase, a circular paper chromatographic method has been developed to identify the inactivation product-penicilloic acid. Such a method demonstrates qualitatively the presence of penicillinase in bacterial culture fluids. A method of estimation of penicilloic acid formed by the penicillin-penicillinase reaction has been described for the first time, based on chromatographic separation and spectrophotometric estimation. This affords a simple and accurate way of determining penicillinase activity. The existing methods of assay of penicillinase and the principles of paper chromatography are also described. By a comparative study of bacterial resistance to penicillin in relation to the formation of penicillinase and other factors, evidence has been produced to show that resistance can be brought about not only by the acquisition of capacity to form penicillinase, but also by increased synthesis of cysteine and other probably related sulphur containing compounds-a fact that has escaped previous workers. ________________________________________ Chapter II To obtain evidence as to the structural relation with the antibacterial activity of penicillin, and in view of its structural relation to pterygospermin, a number of substances related to them have been synthetically prepared and examined for their in vitro activity against standard strains of Staphylococcus aureus. The results bear out the fact that the grouping -N=C=S is of significance in bestowing high antibacterial activity in several known antibiotics such as penicillin, gliotoxin, and pterygospermin, since they can conceivably break down to isothiocyanate type compounds. Using this basis, it has been possible to show an altogether different mode of action of penicillin (pp. 134-135) in the presence of susceptible organisms, involving the formation of certain intermediates of the isothiocyanate type. This hypothesis explains many known facts about the action of penicillin. ________________________________________ Chapter III Evidence to implicate the group of substances analogous to isothiocyanates and their derivatives in the interference of transaminase systems has shown that these substances generally inhibit the transaminase specifically involving glutamic acid. There is also a relation between transaminase inhibition and antibacterial activity. Since the derivatives of isothiocyanates are not active against the transaminase system, the necessity for the liberation of the active isothiocyanates in vivo is suggested. ________________________________________ Chapter IV In view of the significance of such substances as possible sources of a new class of synthetic antibacterial agents, a few of them-namely pterygospermin, benzyl isothiocyanate oxide (B.I.T.C.O.), and the water soluble bisulphite addition compound of benzyl isothiocyanate (B.A.C.)-have been examined for their pharmacological and therapeutic properties. The study includes acute (oral and subcutaneous) and chronic toxicity in mice and rats; various pharmacodynamic effects on blood pressure, respiration, heart, intestines and smooth muscles; absorption and excretion of benzyl isothiocyanate in rabbits; and their actions in experimental staphylococcal, plague, and vaccinia infections. These studies indicate that P.P. and B.A.C. are not very toxic to mammals, do not have any untoward pharmacological reactions, protect animals from experimental staphylococcal infections, and offer no protection against experimental plague or vaccinia infections. Further, after oral administration of B.I.T.C. to fasting rabbits, it was observed that nearly all of it was excreted in urine within 6 hours-a finding of considerable therapeutic significance.