Gladys L. Hobby

Antimicrobial Action of Terramycin in Vitro and in Vivo.

Discussion and Summary The data reported in this and in a previous communication by Finlay et al.(1) emphasize the strong antimicrobial activity of terramycin and its salts against a wide variety of aerobic and anaerobic gram-positive and gram-negative bacteria, and against certain of the rickettsiae. In addition, attention is called to the possible tuberculostatic activity of this compound. Terramycin is absorbed readily following oral or parenteral administration and is a highly effective chemotherapeutic agent, active against a variety of experimental infections in mice. A comparative study of the chemotherapeutic activities of terramycin and its salts indicates that the highly insoluble amphoteric form of terramycin as well as its more soluble salts are all chemotherapeutically effective. In most instances, the CD50 of the hydrochloride has been less than that of the other forms of terramycin, especially when administered by the oral route. More satisfactory results, as indicated by lower CD50 values, have been obtained in some in fections, however, with terramycin (amphoteric) when therapy was carried out by means of a single injection. It is possible that this enhanced chemotherapeutic effect, observed in these infections when treatment is carried out by a single injection only, may be a reflection of the low solubility and slow absorption of terramycin in the amphoteric form. The toxicity of terramycin has been studied extensively by Pan and his associates (12). It is possible that the main virtue of terramycin resides in its remarkably low toxicity and low chemotherapeutic index. Terramycin, however, is a highly effective chemotherapeutic agent against experimental animal infections and it is believed that terramycin may prove of value in the treatment of certain human infections due to terramycin-sensitive organisms. A therapeutic evaluation of terramycin in human infections, therefore, has been initiated.

THE EVALUATION OF NEOMYCIN AND OTHER ANTIMICROBIAL AGENTS OF BACTERIAL AND FUNGAL ORIGIN, AND SUBSTANCES FROM HIGHER PLANTS

The extensive literature on antibiotics indicates the ease with which apparently new antimicrobial agents may be detected. The differentiation, however, between many of these readily detected and apparently new antibiotics and those already known to microbiologists is less readily accomplished. The evaluation of the toxicity and chemotherapeutic activity of new antimicrobial substances is often slow, and the inherent toxicity of many new products frequently becomes apparent only after intensive study. The complete evaluation of the toxicity of a potential chemotherapeutic agent is highly important prior to clinical trial. In a disease such as tuberculosis, in which prolonged administration is necessary, this is particularly essential. In the present report, methods for the evaluation of new chemotherapeutic agents will be discussed with reference to tuberculosis, and a few of the new antimicrobial agents will be mentioned.

Transmission of Penicillin Through Human Placenta.

Summary Penicillin was administered to 4 normal patients in active labor. Two patients received one hundred thousand units each. Both showed significant amounts of penicillin in the maternal and placental cord blood, 2 hours after administration. Two patients received twenty thousand units of penicillin each. The patient who received it one-half hour before delivery showed ample penicillin in the maternal and placental cord blood but not in the amniotic fluid. The patient who received penicillin one hour before delivery showed significant penicillin in the maternal blood but none in the placental cord blood.

THE ANTIMICROBIAL ACTION OF TERRAMYCIN

The isolation of terramycin and the chemical and physical properties, as well as certain of the pharmacologic properties, of this new antibiotic have been discussed previously.1-6 I t is apparent from these reports that terramycin is a substance of relatively low toxicity and is absorbed following either oral or parenteral administration in animals and man. Data reported in previous communications by Finlay and his associates,6 Hobby et d.,7 Welch et al.,B and others8- emphasize the strong antimicrobial activity of terramycin and its salts against a wide variety of aerobic and anaerobic microorganisms, Gram-positive and Gram-negative microorganisms, and certain of the rickettsiae. The species of bacteria which are susceptible to the action of terramycin in vilro are listed in TABLE 1. Of 109 strains tested by Hobby and her associates and by Welch el al.? 61 (56 per cent) were sensitive to < 1.0 mcg. of terramycin per ml. of medium, while 98 (90 per cent) were sensitive to <8 mcgs. per ml. of medium (TABLE 2). Only Proteus vulgaris has been uniformly resistant to its action. Terramycin is effective against Endamoeba histolytica, as well as against certain of the associated bacterial species.I6 Demonstration by experimental methods that terramycin is effective against the spirochaetes has been difficult. Nonetheless, observations by Hendricks el al.,O Schoch el al.; and others have indicated that terramycin is capable of reversing the darkfield in syphilis, suggesting an effect upon Treponema pdlidum. Loughlin and his associates,* furthermore, have demonstrated its effect against Treponema periunue, the causative organism of yaws. The activity of terramycinagainst the rickettsiae and larger viruses will be discussed in detail in subsequent reports by other investigators. Suffice it to say that experimental laboratory and/or clinical studies have indicated that terramycin is effective against many of the rickettsiae, including Ricketlsia tsalsugamushi, Rickettsia p~wazeki , ~ * 2o Rickettsia akari,21 Rickettsia riCkettsii,11 and Rickettsia bu~mti.~~ Terramycin is dective, furthermore, against the etiological agent of primary atypical pneumonia, and against the viruses of granuloma inguinale, lymphogranuloma venereum, and possibly other infections. There is no evidence to date that it is specifically active against the smaller viruses, and, in like manner, it is ineffective against the yeasts and fungi. It is a highly effective chemotherapeutic agent against a wide variety of experimental infections in animals. A comparative study of terramycin and three of its salts (the sulfate, hydrochloride, and sodium salts) in mice experimentally infected with Streptococcus hemolyticus, Diplococcus pneumoniae, Klebsiella pneumoniae, Hemophilus injuenzae and pertussis, and Salmonella lyphosa, typhi murium, cholwae suis, enteritidis, and newport has indicated that the highly insoluble

Observations on the Action of Streptomycin in vitro (I).

Summary 1. A standardized procedure for determination of sensitivity of microorganisms to streptomycin is described. 2. The sensitivity of an organism to streptomycin is influenced by age of culture, concentration of organisms, growth phase of culture, and constituents of medium used. Providing these factors are held constant, the sensitivity of a given strain will remain constant from day to day. 3. The action of streptomycin is bacterio-static rather than bactericidal. Its action is inhibited by certain growth stimulating substances such as peptone, as well as by certain reducing substances. 4. The sensitivity of 84 strains belonging to 7 species is described. Marked variation in sensitivity exists between different strains within a single species and at times between different cells within a given strain. 5. The sensitivity of 9 strains belonging to 8 species is essentially the same when tested against crude streptomycin sulfate (453 /xg/mg), against 3 preparations of the crys talline CaCl2 double salt of streptomycin (685 to 708 /xg/mg) and against a preparation of streptomycin sulfate (802 /xg/mg) prepared from a crystalline salt. 6. The sensitivity of 4 strains of E. typhosa to certain preparations of impure streptomycin sulfate is greater than to highly purified streptomycin sulfate.

Absorption and Excretion of Terramycin in Animals.

Discussion and Summary Studies on the absorption and excretion of terramycin in man will be reported by Werner and his associates(3) at an early date. The results obtained by these investigators agree, in general, with those reported herein. Terramycin and its salts are absorbed readily following either oral or parenteral administration. Terramycin in the amphoteric form is a highly insoluble compound. It is absorbed following oral or parenteral administration at an exceedingly slow rate. A comparative study of the chemotherapeutic action of terramycin and its salts in experimental infections in mice(l) have suggested that the slow absorption of this form of terramycin may be a valuable asset in the treatment of certain infections, provided a dosage form can be prepared which will permit its parenteral administration to humans. Studies are in progress, furthermore, to determine the extent to which this highly insoluble form of terramycin is excreted in the feces. Most satisfactory results have been obtained to date on oral administration of terramycin hydrochloride, a highly soluble salt. The high concentrations of terramycin which may be detected in the serum of animals following oral administration suggests a parallelism between terramycin and aureomycin and chloramphenicol. In contrast to aureomycin and chloramphenicol, however, a high percentage of the terramycin administered is excreted in a biologically active form. Terramycin hydrochloride in aqueous solution has a pH of 1.5; its administration, parenterally in aqueous solution, is not feasible. Preliminary observations suggest, however, that the intravenous administration of this form of terramycin in an appropriate buffer should be possible. The rate and degree of absorption of terramycin and its salts suggest that terramycin, a highly active antimicrobial agent, should be readily effective in the treatment of infections due to terramycin-sensitive microorganisms.

Biological Activity of Crystalline Procaine Penicillin In vitro and In vivo.

Conclusion Crystalline procaine penicillins G and dihydro-F are highly effective antibacterial agents in vitro and in vivo. Crystalline procaine penicillins G and dihydro-F, in oil, when injected intramuscularly in rabbits in a single dose of 30,000 units (0.1 cc) per kg body weight, in most instances produces blood levels lasting 24 to 30 hours or longer. Preliminary observations in man indicate that a single intramuscular injection of 300,000 units of crystalline procaine penicillin G in oil may produce detectable blood levels lasting from 24 to 48 hours while penicillin may be excreted in the urine for at least 48 to 60 hours. Preliminary observations suggest that the toxicity of crystalline procaine penicillin is probably low. Other water-insoluble salts of penicillin when suspended in oil also produce marked prolongation of blood levels.

NEWER ANTIBIOTICS: FACTORS INFLUENCING THEIR ANTIMICROBIAL ACTIVITY

The mechanisms by which antimicrobial agents function has long been of interest to microbiologists. This interest, in part, has been due to the belief that an understanding of the factors responsible for the mechanisms of action of these substances may result in the development of better chemotherapeutic agents. To date, essentially all studies on their mode of action have concerned themselves with penicillin and streptomycin. Innumerable other antimicrobial agents have been described. Many more have been observed but not reported in the scientific literature. Few attempts have been made, however, to determine the mechanisms by which they function. Sulfanilamide, although not an antibiotic in the true sense, was one of the first antimicrobial agents to receive widespread attention from the chemotherapeutic standpoint. Sulfanilamide and the sulfonamide group of compounds in general are bacteriostatic agents which exert a strong antibacterial action against certain of the Gram-positive group of organisms. I t is well recognized that the sulfonamides act by inhibiting growth rather than by killing, that their action is influenced by the number of organisms present, and that they are relatively ineffective against large numbers of cells. The sulfonamides, furthermore, exert their effect only after a lag period lasting from five to seven hours. Many of the sulfonamides have a structure essentially similar to para-aminobenzoic acid and are inhibited by the presence of this substance. Although it is believed by some investigators that the sulfonamides exert their effect by inhibiting bacterial respiration, others believe that they act by virtue of their similarity to the essential metabolite, para-aminobenzoic acid. The weight of evidence perhaps favors the latter theory. Penicillin, in contrast to the sulfonamides, may exert either a bactericidal or bacteriostatic effect depending upon the experimental conditions? Penicillin is an organic acid which is active in low concentrations against many microorganisms. Early studies indicated that, under the influence of high concentrations of penicillin, the number of organisms decreases a t a constant rate until 99 per cent of the organisms have been destroyed. The rate of killing varies with different organisms and with the concentration of penicillin used. Its action is influenced only slightly by the number of organisms present and by environmental conditions. Its action is specifically inhibited, however, by penicillinase, an enzyme capable of destroying penicillin. Penicillin is effective only when active multiplication takes place. These observations have been confirmed abundantly in recent years: and, in view of the fact that penicillin approaches the perfect chemotherapeutic agent more nearly than any other substance known, they may be used to advantage as a basis for comparison of other compounds. Streptomycin, in contrast to penicillin, is an organic base which likewise

Chemotherapeutic Action of Various Forms of Penicillin on Hemolytic Streptococcal Infections in Mice.

Summary The comparative efficacy of 4 forms of penicillin and random samples of “commercial” penicillin has been studied in experimentally-produced hemolytic streptococcal infections in mice. On a unitage basis the relative chemotherapeutic efficacy of penicillin X, G, F and K under the experimental conditions used, was on the order of 500, 100, slightly less than 100, and 60 respectively. On a gravimetric basis, the relative order of efficacy was 270 for X, 100 for G, slightly less than 92 for F and 82 for K. The values for penicillin F were obtained with a preparation which contained impurities, the action of which is not known. All of the samples of “commercial” penicillin were 3 to 5 times as effective as crystalline penicillin G in protecting the animals against the infection. Moreover, the protective action exerted by these impure penicillins was of the same order regardless of the unitage per mg or the value of the Bacillus subtilis-Staphylococcus aureus differential ratio. The protective power of pure X is as great as that observed following the administration of the impure “commercial” penicillins. As the latter were manufactured by a process which presumably excludes the presence of X, it would not seem that the superiority of the impure penicillins over crystalline G could be attributed to the presence of X in the impure material.

Biological Activity of a Residual Form of Streptomycin against Eberthella typhosa.

Summary The sensitivity of E. typhosa to a residual fraction of streptomycin obtained during the purification process is at least 2 to 5 times greater than to the crystalline CaCl2 double salt of streptomycin. By the Oxford cup plate method, the E. typhosa-Be. subtilis (or E. colt) differential ratio of this material is approximately 2.0-3.0. This residual streptomycin is active in vivo as well as in vitro. Its activity in vivo is not as great, however, as might be anticipated from the in vitro results.