The Tetracyclines

THE TETRACYCUNES CALVIN M. KUNIN, M.D.

The pharmacology of the tetracycline antibiotics was reviewed by Shwachman and Schuster36 in 1956. Since then more detailed pharmacologic studies have been conducted, and a series of additives purported to increase gastrointestinal absorption, new intramuscular preparations, combinations with other antibiotics-including antifungal agents-and an additional homologue, demethylchlortetracycline, have been introduced for clinical use. Despite these advances, the practicing physician's therapeutic armamentarium has not been significantly increased because the antimicrobial range of these drugs has not been extended, and there remains virtual cross resistance among all the available homologues. SOURCE

The initial tetracycline, chlortetracycline (CTC), was isolated from Streptomyces aureofaciens by Duggar9 in 1948, followed by oxytetracycline (OTC) in 1950, produced by Streptomyces rimosus,15 and tetracycline (TC) in 1952.3 Tetracycline was originally prepared by catalytic hydrogenation of the chlorine radical of CTC. Demethylchlortetracycline (DMCT), produced by a mutant of Duggar's original strain, was described in 195730 and introduced for clinical use in 1959. Many other homologues have been produced. These include a series of halogen-substituted compounds,7 various demethyl derivatives,30 and other modifications of the basic tetracycline molecule. 33 Some of these are presently under clinical investigation, but none as yet are available for therapeutic use. PHYSICAL AND CHEMICAL PROPERTIES

The structural formulas of the 4 tetracycline homologues currently in From the Departments of Preventive Medicine and Medicine, University of Virginia School of Medicine, Charlottesville. 1001

1002

THE TETRACYCLINES

use are presented in Figure 6. The pyrrolidinomethyl derivative of tetraR

CONH 2

o

H

o

o

o

H

RIO TETRACYCLINE CHLORTETRACYCLINE OXYTETRACYCLINE DEMETHYLCHLORTETRACYCLINE

R...

Ri

H CH 3 H CL CH 3 H H CH 3 0H CL H H

Fig. 6. Structural formulas of the tetracyclines.

cycline is formed by substitution at the -CONHz position.34 This derivative is much more soluble in aqueous solutions than tetracycline. Its principal use is in intramuscular preparations, but like the hydrochlorides of all the tetracycline homologues, it is administered together with a local anesthetic to minimize the pain following injection. 6 , 39 As a group the tetracycline antibiotics are amphoteric crystalline compounds soluble in glycol ethers, pyridine and dilute acid and alkali, very slightly soluble in water and in lower molecular weight alcohols, and insoluble in ether and hydrocarbons. The acid salts (used therapeutically) are well formed crystalline compounds with high solubilities in water. 33 METHOD OF ASSAY

A detailed account of the standard assay procedures for the commonly used antibiotics may be found in the excellent laboratory manual prepared by Grove and Randa1l1 6 of the Division of Antibiotics of the federal Food and Drug Administration. Assay methods for the tetracyclines may be classified into microbiologic and chemical. Of the microbiologic methods, the cylinder-plate assay utilizing Bacillus cereus, var. mycoides, as the test organism is most commonly used for accurate measurement of microbially active drug in biologic fluids. Serum concentrations are determined by diluting both the unknown and the simultaneous run standard solutions in human serum or solutions of bovine albumin. Results obtained by this method give total antibiotic concentration, and do not report bound versus free drug. A turbidimetric method utilizing a strain of Staphylococcus aureus is also available and is particularly useful in measuring concentrations of drug in urine. Chemical methods are generally reserved for assay of production

CALVIN M. KUNIN

1003

lots and pharmaceutical dosage forms and studies of mixtures. These include a ferric chloride colorimetric, an acid colorimetric, ultraviolet spectrophotometric and fluorometric determinations. The last-mentioned method has also been adapted for determination of drug in biologic fluids. Concentration of drug is reported in micrograms per milliliter or gram. The foregoing methods are of great value in obtaining accurate assays for the performance of pharmacologic studies of the individual homoIogues. They are of considerably less value, and may at times be subject to erroneous interpretation, when used to compare the antimicrobial adivity achieved in biologic fluids by the various homologues. This is because the homologues differ in activity by weight against different microorganisms, and this frequently differs between strains of the same speciesY' 17 For this reason investigators interested in comparing homologues generally use a twofold broth dilution method of assay utilizing as the test organisms commonly encountered bacteria isolated from patients. All such studies must include a statement of the minimum inhibiting concentration of each homologue under study for the assay organism to be used. In comparative studies, specimens representing each of the homologues should be assayed on the same day because of the inherent variability of the twofold dilution method. Despite somewhat less accuracy than the cylinder plate method, and the failure of the dilution method to take protein binding fully into account, it has been found to be a reliable method for clinical studies.22 ANTIMICROBIAL SPECTRUM

The tetracycline antibiotics inhibit a broad range of microbial agents_ including both gram-positive and gram-negative bacteria, M. tuberculosis, rickettsiae, the psitticosis-lymphogranuloma venereum group of large viruses, and the agent of primary atypical pneumonia (Eaton agent). The most sensitive, clinically important organisms in the gram positive group include the pneumococcus, group A beta hemolytic streptococci, Str. viridans and, depending on the extent of utilization in a particular area, the staphylococcus. Among the gram-negative group sensitive organisms include most Escherichia coli, the meningococcus, gonococcus, Hemophilus inf/uenzae, pasteurella, brucella and shigella strains. Aerobacter and proteus species vary considerably in sensitivity to the tetracyclines. Only a few strains of Ps. aeru~inosa have been found to be sensitive. 14 ,17 MODE OF ACTION

The precise mechanism by which the tetracyclines exert their anti microbial action is unknown. An excellent review of the literature up

10°4

THE TETRACYCI.INES

to 1956 was prepared by Eagle and Saz.1° The following effects appear to have been established. The tetracyclines are active chelating com· pounds (vide infra) forming firm unions with divalent and trivalent cations, and may thereby interfere with enzymes requiring such cations as cofactors. They appear to interfere with the phosphorylation of glucose in both bacterial and mammalian cells. Their action is in part reversed by some vitamins and amino acids, particularly riboflavin, and they cause a derangement of cellular mechanisms leading to protein and nucleic acid synthesis. ANTIMICROBIAL RESISTANCE

One of the great difficulties encountered in the use of the tetracycline antibiotics had been the emergence of resistant bacteria. The over-all problem was summarized by Finland 12 in 1955, and has not changed much since that time. The problem separates itself into two distinct parts: the development of resistant strains during the course of therapy by means of mutation and selection, and the superimposition of strains of established resistance after suppression of the susceptible microflora. It is frequently difficult to distinguish between these two possibilities in clinical settings. The studies of Knight et aP1 on the appearance of antibiotic-resistant staphylococci in patients treated with tetracycline following admission to a medical service favor the concept of superimposition of antibiotic-resistant strains by continued exposure of patients and personnel to the drug. These authors found that within 4 to 5 days after institution of tetracycline therapy their population converted from about 18 to 90 per cent harboring tetracycline-resistant strains. A similar effect has been demonstrated with other antibiotics given prophylactically, erythromycin in particular. 26 Gram-negative organisms multiplying in complicated urinary tract infections, particularly in the presence of an indwelling catheter, tend to develop resistance to tetracycline rapidly, generally by replacement of sensitive species by naturally occurring resistant ones. Resistant strains of Str. viridans have been encountered during the course of treatment of subacute bacterial endocarditis with chlortetracycline. In contrast, group A beta hemolytic streptococci and pneumococci have remained sensitive to the tetracyclines, and only minor changes have been encountered with N. gonorrhoeae and N. meningitidis. 14 ,17 BACTERIOSTATIC VERSUS BACTERICIDAL ACTIVITY

In considering whether a drug is behaving as a bacteriostatic (suppressing multiplication) or as a bactericidal (sterilizing) agent, one must clearly distinguish between results obtained in tests in vitro and in vivo. Minimum inhibiting concentrations reported in the literature

If 'I CALVIN M. KUNIN

1,1 ;1/

!I "I

10°5

usually are based on bacteriostatic end-points; considerably more drug may be necessary to sterilize the test organism. In addition, since these end-points will vary, depending upon the pH and chemical constituents of the medium and upon inoculum size and the presence of serum, it is not always easy to predict what will occur in the treatment of specific infections in laboratory animals and in man. Although the tetracyclines are generally considered to be bacteriostatic agents, they may at times appear to exert a bactericidal effect in certain clinical situations. For example, they have been found to be as effective as penicillin in treatment of uncomplicated pneumococcal pneumonia, presumably owing to aid rendered by active host defense mechanisms. 27 Although of considerable value, they are less effective than penicillin in eradicating streptococci from the oropharynx and in inhibiting subsequent formation of antistreptolysin 0.19 In addition, the relation between time of initiation of therapy to onset of a disease may be of considerable importance, and this may be related to the bacteriostatic effect of these drugs. Thus Overholt et aPl found that in treating patients with laboratory-acquired tularemia, relapses frequently occurred when treatment was begun during the initial week of illness; relapses were fewer when treatment was begun approximately one week after onset, presumably after host defense mechanisms had begun to come into action. This phenomenon was not encountered when streptomycin, a bactericidal agent, was used. ABSORPTION

The tetracyclines are incompletely absorbed from the gastrointestinal tract. Large amounts can be recovered in the stools after oral administration. Incomplete absorption may be responsible for the profound changes in the fecal flora noted concurrent with their use. Shortly after the introduction of CTC, aluminum hydroxide gel was found to interfere with absorption, but most workers reported that food, milk and carboxymethylcellulose lacked this effect, and these substances were actually recommended to reduce gastric irritation resulting from oral doses. More detailed studies in experimental animals and man have demonstrated a depressing effect of divalent and trivalent cations on blood levels achieved with oral doses. 5 , 37 It is now recommended that, to obtain optimal absorption, the tetracyclines be administered during the fasting state. The subject of the formation of inactive chelates with metallic cations by the tetracyclines has been thoroughly reviewed by Weinberg. 38 Manufacturers have attempted to devise means to counter the effect. Of the many excipients tried, four have been studied in human beings and introduced into therapy: citric acid, a phosphate complex, sodium hexametaphosphate, and glucosamine hydrochloride.

1006

THE TETRACYCLINES

A critical analysis of their role in antibiotic blood level enhancement has been presented by Finland. 13 In brief, many of the studies purporting to demonstrate an enhancing effect upon TC absorption were hampered by one or more of the following: (1) the filler in the capsules used contained dicalcium phosphate, and some also contained small amounts of magnesium salts; (2) the actual content of the antibiotic at the time of the study was not stated; (3) data favorable to one or another preparation were inappropriately emphasized; (4) data obtained from other sources, some uncontrolled and others carefully controlled with cross-over studies, were uncritically combined and analyzed; and (5) small, clinically unimportant differences in antibiotic concentration in the blood were given undue emphasis. In other studies in man in which these defects were largely eliminated, the effects of various excipients, although in some cases statistically significant, were so small as to warrant the conclusion that for therapeutic purposes their addition was of no great importance. 2 , 23 The most detailed study of the site of absorption of TC was performed by Pindell and associates 32 in dogs. They prepared a series of isolated segments of the gastrointestinal tract, leaving the blood supply intact. Absorption of TC was determined by serial measurement of the concentration in the blood after instillation into the different segments. The drug was most rapidly absorped from the duodenum and ileum; peak levels in the blood were reached within a half hour, and were about as high from the duodenum as from the ileum. Absorption from the stomach was delayed so that the peak was more sustained than from the other sites. Relatively little drug was absorbed from the colon, in agreement with earlier studies in man. IS The observations of Pindell et al. are supported, in part, by those of Danopoulis et al.,4 who noted that a half hour after oral ingestion of OTC in dogs, blood levels were much higher in the left gastric than in the superior mesenteric vein, indicating gastric absorption at that time, but the latter authors did not study absorption beyond this brief period. DISTRIBUTION

The tetracyclines appear in the milk of lactating patients, pass the placenta into the fetus, and appear in the saliva, cornea, sclera, iris and vitreous humor. The concentrations of all the homologues are lower in the cerebrospinal fluid than in the blood. Wood and Kipnis40 found that penetration into the cerebrospinal fluid was best obtained with TC, the poorest with CTC, while OTC was intermediate. CTC appears in the bile at a concentration 8 to 16 times that observed in the serum; DMCT is also concentrated in the liver and excreted in the bile, with bile-serum ratios of 2: 32. Similar high biliary concentrations have been noted with the other homologues.

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Detailed studies of tissue distribution in experimental animals and man have also been conducted using sensitive fluorometric and radioisotope labeling methods. The drugs appear to be sequestered by fastgrowing tissues, liver, tumors, and areas of new bone formation. DuBuy and Showacre8 have recently found that tetracycline fluorophores are concentrated in the mitochondria of both mammalian and bacterial cells. Small amounts of tetracycline remain tightly bound by chelation to bone calcium long after disappearance from all other organs. BINDING TO PLASMA PROTEINS

The fraction of active drug bound to plasma proteins differs among the various homologues. 24 • 42 As shown in Table 8, CTC and DMTC are TABLE

8. Distribution and Excretion of 4 Tetracycline Homologues after Intravenous Injection of 500 Mg. of Each in 4 Normal Young Men 24

RATES

TC

OTC

CTC

DMCT

Half-life in serum (hours) ................... . Renal clearance (ml./min./1.73 mY) .......... . % of dose excreted in 96 hours .............. . % serum protein binding ................... .

8.5 74 60 31

9.2 99 70 20

5.6 32 18 48

12.7 35 39 51

most highly bound, followed by TC and OTC. This binding appears to be reversible, and is in all likelihood responsible for differences noted in the rate of renal clearance. EXCRETION AND FATE

The principal mechanism of excretion is by the kidneys, probably by means of simple glomerular filtration. The rate of excretion is not altered by rate of urine flow, nor by the urine pH. The excretion rates, as determined in man, are listed in Table 8. 24 OTC is most rapidly and completely excreted, followed by TC, DMTC and CTC. The over-all nonrenal excretion rate (nonrenal clearance) is similar for all the homologues except CTC. This homologue is rapidly converted to an inactive product on exposure to alkaline solutions at body temperature. It is of some interest that despite stability of DMCT in vitro, it is rendered inactive or eliminated from the body at the same rate, as determined by nonrenal clearance rates in man, as the remaining homologues. 24 The prolonged blood levels achieved with DMCT are due to its relatively slow renal clearance and stability in vivo greater than CTC. TC and OTC (and undoubtedly DMCT as well) tend to accumulate in the blood of patients with renal failure when given continuously. 'Vood and co-authors41 observed a serum concentration of 80 mg. of TC per milliliter in an oliguric patient on the fourth day of therapy. The half-life of single doses of TC and CTC, as shown in Table 9.

1008 TABLE

THE TETRACYCLINES

9. Effect of Acute Renal Failure on Persistence of Tetracycline and Chlortetra· cycline in Blood 25 HALF-LIFE IN SERUM (HOURS)

TC

Oliguric patients .... 1 2 3 4 5 Normal patients. . . . .

CTC

108

> 2Yz

days

57 101 108 8.5

Oliguric patients ............ 1 2 3 4 5 6 Normal patients ............ .

6.9 11.0

6.8 8.4 10.1 8.9

5.6

differs in oliguric patients (less than 400 ml. of urine excreted per day). The half-life of microbially active tetracycline increases from values in normal persons of about 8 hours up to 108 hours in patients with renal shutdown, while that of CTC increases from about 6 hours to only 7 to 11 hours. 25 These profound differences between homologues differing only in the presence or absence of a chlorine atom are due to the lability in vivo of CTC referred to above. Thus, in treatment of uremic patients, TC used sparingly will produce therapeutic blood levels which can be attained with CTC given only in usual doses. TOXICITY AND UNTOWARD EFFECTS

The most fre~ent and distressing complications of tetracycline therapy have been nausea, vomiting and diarrhea, the last at times severe when accompanied by overgrowth of resistant strains of S. aureus in the feces. In general, these side effects have been infrequent and mild when minimum effective doses (1 gm. daily of TC, CTC and OTC, or 600 mg. of DMCT) in divided doses in adults were used, and their liability to produce diarrhea has been slight with moderate doses. The severe diarrheas, including staphylococcal enteritis, usually subside promptly if recognized early, and if the drug is discontinued and proper hydration along with antidiarrheal therapy is instituted. These complications are generally absent when moderate doses are given intravenously. Although the gastrointestinal complications of therapy with the tetracyclines are the most frequent and so have received the most attention, certain other more subtle complications are of interest. Each of the three earlier homologues has been shown to produce, during prolonged therapy, morphologic and functional changes in the liver, a negative nitrogen balance and increased riboflavin excretion into the urine. Elevation of the nonprotein nitrogen level in the blood during OTC therapy has also been reported in man. 1 Rabbits given large doses of tetracyclines exhibit rising nonprotein nitrogen in the blood, anorexIa, weight loss, lethargy, convulsions and respiratory failureY

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1009

Another indirect effect of tetracycline therapy, peptic ulceration and bleeding in uremic patients, has been reported by Lieber and Desneaux. 28 This effect was studied in detail by Lieber and Lefevre,29 who found the low level of free and total acid in the gastric juice of uremic patients to be associated with high gastric ammonia content; when OTC (or other antibiotics) was administered, the gastric ammonia was stoichiometrically replaced by urea, presumably owing to suppression of formation of bacterial urease in the gastric mucosa, resulting in an increase in gastric acid. Thus the protective effect upon the gastric mucosa afforded by ammonia released from high concentrations of urea in the uremic patient was abolished by antibiotic therapy and, in some cases, resulted in hemorrhage and ulceration. Drug fevers and various rashes have been observed with each of the tetracycline drugs, but have been relatively infrequent. There have been essentially no blood dyscrasias clearly attributable to them. Pruritus ani and vulvae have been encountered with varying frequency in some groups of patients. As with all antibiotics, particularly those with a wide spectrum, superinfections caused by overgrowth of resistant organisms, including staphylococci, enteric bacteria, yeasts and fungi, have been reported with varying frequency. Superinfections are particularly frequent and severe in hospitalized patients treated for long periods or with large doses and in patients with other serious and debilitating diseases, especially those receiving therapy with other toxic agents such as radiation, antitumor drugs and adrenocortical hormones. Strains of Candida, in particular, are frequently increatJed in the feces during treatment with tetracyclines, and their numbers can be reduced by concomitant administration of nystatin or amphotericin B. The clinical significance of monilia in most cases is questionable. Nutritional deficiencies, some of specific types and allegedly reversible or preventable by administration of the specific vitamins, have also been noted. These also are difficult to assess. Of particular interest recently has been the occurrence of an exaggerated sunburn reaction resulting from direct exposure of patients to sunlight during therapy with moderate or large doses of DMCT; such reactions have been only rarely noted with the other homologues. A recent review35 of untoward reactions ascribed to DMCT reports photosensitivity in about 1.1 per cent of recipients, but the exact incidence is unknown. THERAPEUTIC INDICATIONS

I

The tetracyclines have a wide antimicrobial spectrum, are readily administered, and are relatively free from hazardous toxic reactions. They have been widely used since CTC was first introduced in 1948. Principal drawbacks are expense (when compared to injectible penicillin

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THE TETRACYCLINES

and sulfonamides), general bacteriostatic action (of considerable importance in infections in which the host response is either limited or relatively ineffective, such as subacute bacterial endocarditis, brucellosis, tularemia, typhoid carriers), the superimposition of resistant S. aureus or naturally resistant coliforms, particularly when the drugs are used prophylactically, and the propensity of S. au reus and coliform organisms to develop resistance during therapy. They are effective against the organisms generally found in the respiratory and urinary tracts. This group of drugs is particularly favored by many physicians for infections of uncertain etiology. In many instances, however, these infections may be viral, or other drugs such as penicillin may be more effective. Most of the childhood fevers of unknown origin are of viral etiology, and are not responsive to any of the presently available antimicrobial agents. Most uncomplicated infections of the urinary tract respond well to the relatively inexpensive sulfonamides. Tetracyclines and other antibiotics are of only limited value in complicated urinary tract infections. The tetracyclines are effective in amebiasis, and very useful in rickettsial infections and disease produced by the lymphogranuloma-psitticosis group of viruses; their usefulness in the treatment of primary atypical pneumonia due to the Eaton agent has been recently re-emphasized in the elegant studies of Kingston et a1. 20 They are of no value as antifungal agents, and of limited value in tuberculosis. DMCT shares almost all the advantages and disadvantages of the other homologues. The lower dose form (150 mg. as opposed to 250 mg. per capsule of the others) diminishes the advantages of more prolonged action and greater antimicrobial activity of this homologue; photosensitivity is occasionally troublesome. Its main advantages, as currently compounded, are that the daily regimen may be divided into doses given every 8 to 12 hours rather than the customary 6-hour intervals used for the other tetracyclines, and that there is a tendency for more prolonged antimicrobial activity after cessation of therapy. CONCLUSION

It would appear from this brief account that the tetracyclines, although

of great clinical value, are by no means the drugs of choice for many infections. They are poor substitutes for good bacteriologic evaluation, and are potentially hazardous when used as prophylactic agents. They, like all other antibiotics, are most helpful when the physician, first, considers the probable nature of the disease he is attempting to treat, the most likely organisms to be dealt with, and the most suitable drug for those organisms; and, second, has taken time to obtain smears and cultures before instituting therapy to confirm or deny his initial impression.

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REFERENCES

1. Bateman, J. C., Barberio, J. R., Grice, P., Klopp, C. T., and Pierpont, H.: Fatal Complications of Intensive Antibiotic Therapy in Patients with Neoplastic Disease. Arch. Int. Med., 90:763, 1952. 2. Boger, W. P., and Gavin, J. J.: An Evaluation of Tetracycline Preparations. New England J. Med., 261:827, 1959. 3. Boothe, J. H., Morton, J., Petisi, J. P., Wilkinson, R. G., and Williams, J. H.: Tetracycline. J. Am. Chem. Soc., 75:4621, 1953. 4. Danopoulis, E., Angelopoulos, B., Zioudyou, C., and Amfra, P.: Experimental Study on the Absorption of Oxytetracycline in the Stomach and the Small Intestine and Its Excretion in the Bile. Antibiotics 6- Chemotherapy, 4:451, 1954. E. H., Litchfield, J. T., Jr., Eisner, H. J., Corbett, J. J., and Dunnett, 5. Dearborn, C. W.: The Effects of Various Substances on the Absorption of Tetracycline in Rats. Antibiotic Med., 4:627,1957. 6. Dimmling, T.: Experimental and Clinical Investigations with Pyrrolidinomethyl Tetracycline. Antibiotics Ann., 1959-1960, pp. 350--,57. 7. Doerschuk, A. P., and others: The Halide Metabolism of Streptomyces Aureofaciens Mutants. The Biosynthesis of 7-chloro, 7 chloro 36 and 7-Bromotetracycline and Tetracycline. J. Am. Chem. Soc., 78:1508,1956. 8. DuBuy, H. G., and Showacre, J. L.: Selective Localization of Tetracycline in Mitochondria of Living Cells. Science, 133:196, 1961. 9. Duggar, B. M., and others: Aureomycin-A New Antibiotic. Ann. New York Acad. Sc., 51:175, 1948. 10. Eagle, H., and Saz, A. K.: Antibiotics. Ann. Rev. Microbiol., 9:173,1955. 11. Farhat, S. M., Schelhart, D. L., and Musselman, M. M.: Clinical Toxicity of Antibiotics Correlated with Animal Studies. A.M.A. Arch. Surg., 76:762, 1958. 12. Finland, M.: Emergence of Antibiotic-Resistant Bacteria. New England J. Med., 253:909,1955. 13. Idem: Antibiotic Blood Level Enhancement. Antibiotic Med., 5:359, 1958. r 14. Finland, M., Hirsch, H. A., and Kunin, C. M.: Observations on Demethy1chlo tetracycline. Antibiotics Ann., 1959-1960, pp. 375-82. 15. Finlay, A. C., and others: Terramycin, a New Antibiotic. Science, 111:85, 1950. 16. Grove, D. C., and Randall, W. A.: Assay Methods of Antibiotics: A Laboratory Manual. New York, Medical Encyclopedia, Inc., 1955. 17. Hirsch, H. A., and Finland, M.: Comparative Activity of Four Tetracycline Analogues against Pathogenic Bacteria in Vitro. Am. J.M. Sc., 239:288, 1960. 18. Hoffman, M. S., Wellman, W. E., and Herrell, W. E.: Failure of Absorption of Aureomycin and Terramycin Administered as a Retention Enema. Proc. Staff Meet., Mayo Clin., 25:463, 1950. 19. Houser, H. B., and others: Effect of Aureomycin Treatment of Streptococcal Sore Throat on the Streptococcal Carrier State, the Immunologic Response of the Host, and the Incidence of Acute Rheumatic Fever. Pediatrics, 12: 593, 19 53 . 20. Kingston, J. R., and others: Eaton Agent Pneumonia. J.A.M.A., 176: 118, 1961. 21. Knight, V., White, A., Foster, F., and Wenzel, T.: Studies on Staphylococci from Hospital Patients. II. Effect of Antimicrobial Therapy and Hospitalization on Carrier Rates. Ann. New York Acad. Sc., 65:206, 1956. 22. Kunin, C. M., and Finland, M.: Demethylchlortetracycline. A New Tetracycline Antibiotic That Yields Greater and More Sustained Antibacterial Activity. New England J. Med., 259:999, 1958. 23. Kunin, C. M., Jones, W. F., and Finland, M.: Enhancement of Tetracycline Blood Levels. New England J. Med., 259:147, 1958. 24. Kunin, C. M., Dornbush, A. C., and Finland, M.: Distribution and Excretion of Four Tetracycline Analogues in Normal Young Men. J. Clin. Invest., 38:1950, 1959.C. M., Rees, S. B., Merrill, J. P., and Finland, M.: Persistence of Anti25. Kunin, biotics in Blood of Patients with Acute Renal Failure. 1. Tetracycline and Chlortetracycline. J. Clin. Invest., 38: 1487, 1959. 26. Lepper, M. H., and others: Epidemiology of Erythromycin-Resistant Staphylocci

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THE TETRACYCLINES

in Hospital Population-Effect on Therapeutic Activity of Erythromycin. Antl· biotics Ann., 1953~54, pp. 308-13. 27. Lepper, M. H.: Aureomycin (Chlortetracycline). Antibiotics Monographs No.7. New York, Medical Encyclopedia, Inc., 1956. 28. Lieber, C. S., and Desneux, J. J.: A propos de l'action ulcerogene gastrigue de l'oxytetracycline chez l'homme. Acta Gastro-enterologica Belgica, 20:738, 1957. 29. Lieber, C. S., and Lefevre, A.: Ammonia as a Source of Gastric Hypoacidity in Patients with Uremia. 1- Clin. Invest., 38:1271, 1959. 30. McCormick, J. R. D., Sjolander, N. 0., Hirsch, D., Jensen, E. R., and Doershuk, A. P.: A New Family of Antibiotics: The Demethyltetracyclines. 1- Am. Chern. Soc., 79 :4561, 1957. 31. Overholt, E. L., and others: An Analysis of Forty-Two Cases of Laboratory Acquired Tularemia. Am. 1- Med., 30:785, 1961. 32. Pindell, M. H., Cull, K. M., Doran, K. M., and Dickison, H. L.: Absorption and Excretion Studies on Tetracycline. 1- Pharmacol. & Exper. Therap., 125:387, 1959. 33. Regna, P. P.: Chemistry of Antibiotics of Clinical Importance. Am. 1- Med., 18:686,1955. 34. Seidel, W., and others: Zur Darstellung Pharmakologie und Chemotherapeutischen Anwendung des Pyrrolidino methyl-tetracyclins (Reverin). Munch. med. Wchnschr., 100:661, 1958. 35. Shapiro, J. L., and Philips, F. M.: Demethylchlortetracycline in Clinical Practice. l.A.M.A., 176: 596, 1961. 36. Shwachman, H., and Schuster, A.: The Tetracyclines: Applied Pharmacology. PEDIAT. CLIN. NORTH AMERICA, 3:295, 1956. 37. Sweeney, W. M., Hardy, S. M., Dornbush, A. C., and Ruegsegger, J. M.: Absorp. tion of Tetracycline in Human Beings as Affected by Certain Excipients. Antibiotic Med. & Clin. Ther., 4:642,1957. 38. Weinberg, E. D.: The Mutual Effects of Antimicrobial Compounds and Metallic Cations. Bacteriol. Rev., 21:46, 1957. 39 . Weinstein, H. J., Lidsky and Delahunt, C. S.: Preconstituted Oxytetracycline Intramuscular Solution, a New Preparation. Antibiotic Med. & Clin. Ther., 6:526, 1959. 40. Wood, W. S., and Kipnis, G. P.: The Concentrations of Tetracycline, Chlortetracycline, and Oxytetracycline in the Cerebrospinal Fluid after Intravenous Administration. Antibiotics Ann., 1953-1954. New York, Medical Encyclo. pedia, Inc., 1953. 41. Wood, W. S., and others: Tetracycline Therapy. Clinical and Laboratory Observations on One Hundred Eighty-Four Patients Treated with Tetracycline. A.M.A. Arch. Int. Med., 94:351, 1954. 42. Wozniak, L. A.: Studies on Binding of Tetracyclines by Dog and Human Plasma. Proc. Soc. Exper. BioI. & Med., 105:430, 1960. General References

Dowling, H. F.: Tetracycline. Antibiotics Monographs No. 13. New York, Medical Encyclopedia, Inc., 1955. Finland, M., and Garrod, L. P.: Demethylchlortetracycline. Brit. M.J., 2:959, 1960. Florey, M. E.: The Clinical Application of Antibiotics. Chloramphenicol and the Tetracyclines. London, Oxford University Press, 1957, Vol. III. Kunin, C. M., and Finland, M.: Clinical Pharmacology of the Tetracycline Antibiotics. Clin. Pharm. & Therap., 2:51, 1961. Lepper, M. H.: Aureomycin (Chlortetracycline). Antibiotics Monographs No.7. New York, Medical Encyclopedia, Inc., 1956. Musselman, M. M.: Terramycin (Oxytetracycline). Antibiotics Monographs No.6. New York, Medical Encyclopedia, Inc., 1956. University of Virginia School of Medicine Charlottesville, Va.