Landmark Publication from The American Journal of the Medical Sciences: The Clinical Pharmacology of Digitalis Glycosides: A Review

HISTORICAL ARTICLE

Landmark Publication from The American Journal of the Medical Sciences The following contribution is one of a series of republications of original, highly-cited articles from the early years of The American Journal of the Medical Sciences. This original article was published in July 1968, Volume 255, pp. 382– 414. Tinsley R. Harrison, MD

A historical perspective by Dr. John R. Feussner and Mr. Derek J. Feussner follows this original landmark article.

THE CLINICAL PHARMACOLOGY OF DIGITALIS GLYCOSIDES: A REVIEW BY J. E. DOHERTY, MD DEPARTMENT OF MEDICINE, UNIVERSITY OF ARKANSAS SCHOOL OF MEDICINE; AND MEDICAL SERVICE, LITTLE ROCK VETERANS ADMINISTRATION HOSPITAL, LITTLE ROCK, ARKANSAS

Abstract: The metabolic half-times and excretion rates of labeled cardiac glycosides are related to pharmacologic or therapeutic activity. Digoxin, a short-acting glycoside, is excreted primarily in the urine and has a serum half-time of 34 hours. This function is related primarily to digoxin excretion. Digitoxin, a long-acting glycoside, has a serum half-time of 50⫹ hours and a physiologic half-time of 51/2 days. It is excreted primarily in the urine as metabolites of the parent compound. Renal failure compromises digoxin excretion and prolongs the digoxin serum halftime and thereby the pharmacologic activity of the drug. As excretion is related to creatinine clearance, dosage should be reduced in proportion to reduction in creatinine clearance in renal insufficiency. Hyper-thyroid patients have lower digoxin serum levels and hypothyroid patients higher digoxin serum levels when compared to euthyroid patients. This finding confirms the clinical impression of digitalis resistance in hyperthyoidism and sensitivity in myxe-dema. Appropriate alterations in digoxin dosage in thyroid disease are justified. Patients with liver disease and cor pulmonale have normal digoxin serum levels, half-times and excretion rates. Increased digitalis sensitivity in these patients is related to other factors. Knowledge of the serum half-time, route of excretion, and rate of excretion of the cardiac glycosides is now available. Application of this information to the patient receiving digitalis assists in avoiding over- and under-digitalization— both common clinical problems. If doubt exists regarding digitalis dosage, smaller, rather than larger, doses are suggested to avoid toxicity.

This work was supported in part by the National Institutes of Health (Grant HE 06642-06); the Arkansas Heart Association, Division of Research Facilities (Grant FR-49) and the Bur-rouglis-Wellcome and Co. (USA) Inc., Tuckahoe, New York.

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Key Indexing Terms: Digitalis glycosides; Radioactive digitalis; Lanatoside C; Digoxin; Excretion of digitalis glycosides; Metabolism of digitalis glycosides; Digitoxin; Ouabain [Am J Med Sci 2010;339(5):462–481.]

THE clinical pharmacology of digitalis glycosides has received great impetus in the last 20 years through studies with radioactive compounds. Because of the importance of these studies as related to the clinical management of the patient requiring digitalis, this review is, in large part, restricted to studies obtained through the use of a radioactive label. The findings complement the many other biologic, biochemical, and clinical studies of digitalis. A brief historical resume followed by a survey of radiochemical compounds and techniques will assist in orientation for the discussion of the clinical pharmacology of digitalis glycosides in this paper.

HISTORY Digitalis glycosides in many different forms have been used for thousands of years with little knowledge of their actual potency, metabolism, or rates of excretion. The introduction of the bioassay of digitalis for potency answered the need for a preparation of known potency for clinical use, as dosage by weight of crude leaves proved to be extremely variable. This technique was refined in its ultimate form by Friedman and his associates1 in an embryonic duck heart preparation, sensitive enough to measure digitalis activity in excreta, sera and tissue, after digitalis was given in several different forms. Through such studies much meaningful information was secured and additional studies were stimulated. Other methods of assay include chromatographic2,3 which is rather sensitive but painstaking, and polarographic4 which is also a sensitive but difficult technique and reportedly

The American Journal of the Medical Sciences • Volume 339, Number 5, May 2010

Clinical Pharmacology of Digitalis Glycosides

quite variable. Recently, clinical human assay has been described employing the alterations in the systolic time intervals induced by the digitalis glycosides.5,6 The most accurate method of assay yet devised is that of the radioactive tracer compound. The glycoside, tagged with a radioactive element, can be measured in minute quantities in serum, body fluids, excreta and tissues. The accuracy of the method is possible because a known quantity of radioactivity per milligram of glycoside in the sample originally given, the amount of recovery, and degradation products corrected for radioactive decay (when necessary) approaches 100%. The sensitivity and reproducibility of the results are excellent. A random labeled tracer may be used which enables the specific metabolites to be separated from serum and excreta by column, thin-layer, or paper chromatography.

RADIOACTIVE DIGITALIS LABELING Chemically pure digitalis glycosides of known chemical structure and which could be assayed by weight were in general use in 1947 to 1950. Although artificial synthesis of cardiac glycosides was not possible or practical, the availability of radioactive isotopes for medical and investigational use was becoming widespread. Geiling and his associates7–12 labeled digitoxin biosynthetically through the growth of digitalis purpurea in an atmosphere of radiocarbon dioxide. The plants utilized the compound in metabolic processes and yielded carbon-14 digitoxin of rather low but measurable specific radioactivity, presumed to be a random (or unknown) label.

In a slightly different application of the same principle used in the carbon-14 label of digitoxin, Doull, Dubois and Geiling13 labeled bufagin (a cardioactive toad venom) by feeding tropical toads algae grown in a radiocarbon dioxide environment. This was also presumed to be a random label. Wilzbach14 suggested the use of a radiohydrogen (tritium) label of compounds difficult to synthesize or label biologically, by exchange of radioactive (H3, tritium) with nonradioactive hydrogen (H) under specific conditions. Although many impurities resulted from exposure, usable random labeled tracer products could be prepared inexpensively by this method. Spratt, Okita and Geiling15 prepared tritiated digitoxin by this method and cautioned that much “scrubbing” or purification was necessary to obtain a stable, noncontaminated product. Doherty, Perkins and Mitchell16,17 with the cooperation of Searle, Ferry and Murphy of Burroughs-Wellcome and Co. (USA) Inc., had digoxin tritiated in 1958 with low but readily identifiable radioactivity in a stable compound. The use of this random labeled isotope enabled in vestigators to follow degradation of the labeled compound into its various metabolic by-products and study its metabolism and excretion. A third method of preparation of radioactive digitalis glycosides was introduced by Wartburg et al.18 in 1955. This method involved the breakdown and resynthesis of digoxin with a tritium (H3) label in the 12 ␣ position. Digitoxin has been labeled with tritium biosynthetically in the 7 ␣ position by Griffin and Burstein.19 As yet there have been no reports of the clinical use of these labeled compounds. Figure 1 illus-

FIGURE 1. RANDOM AND SPECIFIC LABELING OF DIGITALIS GLYCOSIDES. The compound shown is digoxin and three parts of the molecule are shown in this structural formula: (1) the sugar (digitoxose), (2) the steroid (cyclopentanoperhydrophenanthrene), and (3) the lactonering. A “random” label by the Wilzbach method with tritium (H3) might appear at any of the hydrogen positions in any portion of the molecule. A bio-synthetic carbon-14 label is an example of a random label of any of the carbon atoms. A specific label indicates incorporation of the radioactive element (carbon or hydrogen) in the molecule. This is shown in the enclosed circles for 12␣ position in digoxin, the 7␣ position in digitoxin, and the lactone ring in acetyl digitoxigenin. It should be remembered that appropriate changes in the molecule arc necessary to make the compound digitoxin or acetyl digitoxigenin. © 2010 Lippincott Williams & Wilkins

463

464

13

105

103

36,103,104

102

91,92,93,94,95,96,97,98,99,100,101

Tritium Carbon-14 Tritium Tritium Carbon-14 Carbon-14

Specific (12␣) Random Random Random Random Random

1 C./mg (1965) 0.05 ␮c/mg (1962) 1 C./mg (1964) 1.5 C./mg (1963) 0.25 ␮c/mg (1962) 0.07 ␮c/mg (1951)

Breakdown, resynthesis Biosynthesis: digitalis lanata Hydrogen exchange Hydrogen exchange Biosynthesis: digitalis lanata Biosynthesis: Carbon-14 algae fed toads

18

74,75,76,77,78,79,80,81,82,83,84,85,86,87,88,89,90,

Digoxin Digoxin Ouabain Dihydro-ounbain Lanatoside C Bufagin

Carbon-14 Tritium Tritium Acetyl digitoxigenin Digitoxin Digoxin

Specific (lactone ring) Specific (7␣) Random

Carbon-14 Tritium Digitoxin Digitoxin

57,58,59,60,61,62,63,64,65,66,67,68,69,70,71,72,73,

16,17,36,40,43,44,45,46,47,48,49,50,51,52,53,54,55,56,

19

42

Breakdown, resynthesis Biosynthesis: digitalis purpurea Hydrogen exchange

15,28,36,37,38,39,40,41

Biosynthesis: digitalis purpurea Hydrogen exchange

0.48–0.65 ␮c/mg (1948–1958) 18 ␮c/mg (1962); 770 ␮c/mg (1965) ? (1954) 0.376 ␮c/mg (1967) 33–125 ␮c/mg (1958–1968)

Specific activity Location Label Glycoside

● Percent efficiency refers to the actual percent of disintegration of radioactive atoms identified as counts of radioactivity compared to the known number of disintegrations per minute. †Packard Instrument Co., Inc., Downers Crove, Illinois; Beckman Instruments, Inc., Fullerton, California.

TABLE 1. Labeled digitalis glycosides

The development of pure tracer digitalis compounds was only the first step in the study of the compounds and their metabolic by-products. Methods for analysis of the radioactivity present in biologic samples were necessary and early studies suffered not only from low specific radioactivity of the labeled digitalis compound, but also from relatively inefficient methods for analysis of the radioactivity representing digitalis which was present. The compounds labeled by all the methods described previously with the beta-emitting carbon-14 or tritium (H3) could be measured with only 1 to 2% efficiency for tritium and 20 to 40% for carbon-14 with the gas-flow counters then available. The development of the liquid scintillation counter greatly improved counting techniques; however, these were not available before 1954. Early liquid scintillation counters had an efficiency of 12 to 15% for tritium and 40% for carbon-14.● More 1recently developed models achieve 25 to 40% for tritium and 60 to 90% for carbon-14.† Counting rates may be corrected for “quenching” of counts (suppression of counts by color of the solution or the presence of substances which would otherwise suppress the counting rate) by addition of a known amount of radioactivity to the sample (internal standard) or an external source of radiation (external standard). The counts before and after the addition of an internal standard or the application of an external standard are compared and correction for the impurities or color present in the biological sample may be made in the final determination of radioactivity actually present. Incineration methods for analysis of tritium radioactivity have been developed20,21 in which the sample is subjected to complete combustion, leaving a residue of tritiated water vapor which is condensed and counted as tritiated water, with good levels of efficiency. These methods identify total tritium radioactivity, however, and will not allow separation of metabolic products from the parent compound. The more recently completed studies utilize the more efficient and sophisticated methods for analysis of radioactivity together with labeled digitalis glycosides of higher specific radioactivity. More than half of the studies referred to in this review have been performed since 1960, and these primarily concern tritiated digoxin. Once radioactivity is characterized and counted, the problem of the identity of the material counted is important. Digitalis glycosides may be metabolized by the body to a large extent (digitoxin) or very little (digoxin). Some metabolites are more cardioactive

Source

IDENTIFICATION OF DIGITALIS RADIOACTIVITY

Random Random

References

trates the principle of the random and specific label of a digitalis compound. The choice of radiocarbon or radiohydrogen provides a compound with a very long half-life (carbon 5,568 years, tritium 12.26 years) which makes correction for radioactive decay unnecessary in shortterm biologic experiments.

7,8,9,10,11,12,22,23,24,25,27,28,29,30,31,32,33,34,35

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FIGURE 2A. CARBON-14 DIGITOXIN WHOLE BLOOD TURNOVER. Micrograms percent of digitoxin is shown on a semilogarithmic scale on the vertical axis, time in hours on the horizontal. Figures represent nearly identical turnovers for (---) 1.2 to 1.5 mg of digitoxin and for (—) 0.5 mg of digitoxin. The dominant turnover line represents a half-time of 44 to 52 hours. A shorter exponential function with a half-time of 15 to 30 minutes is also shown to the left of the graph. See text for explanation.

than others, some not cardioactive at all, yet they may be counted as total radioactivity. Metabolic by-products are most often identified by chromatographic methods, usually paper, thin-layer, or column partition. An effort will be made to identify the metabolites of the glycosides discussed in each section to follow.

DIGITALIS COMPOUNDS LABELED Table 1 lists the digitalis glycosides labeled thus far and the nature of the label applied. All references on radioactive digitalis are included in this table to simplify the task of a literature search in this field.

CLINICAL PHARMACOLOGY OF DIGITALIS This discussion segregates the various glycosides by the radioactive compounds studied and tends to emphasize the differences in serum half-times, excretion and metabolic degradation, together with their clinical applicability as suggested by these studies. DIGITOXIN. Carbon-14 labeled digitoxin was first biosynthesized by Geiling and McIntosh in 1949.● Purity of the compound was established by Okita et al.12 in 1954. Sjoerdsma and Fisher33 reported in 1951 that digitoxin uptake by the isolated perfused hearts or guinea pigs, rats, cats and rabbits was relatively small compared to total dose administered. Washing of the tissues before analysis indicated that metabolites were more firmly bound to cardiac tissue than was digitoxin. This study was done with digitoxin of low specific activity and relatively inefficient counting techniques. In 1953 Okita et al.11 reported the first studies with radioactive carbon-14 digitoxin in human subjects relating to excretion in the urine. This study established the kidney as the major organ of digitoxin excretion, 60 to 80% being excreted by this route in the urine. Burdette24 detected no carbon-14 radioactivity in human myocardial (atrial) biopsies obtained at cardiac surgery after giving carbon-14 digitoxin prior to the surgical procedure. The low specific activity of the digitoxin and counting techniques of low efficiency were undoubtedly responsible for these negative results. Okita et al.31 reported in 1955 on blood levels in human subjects given carbon-14 digitoxin in cardiac failure. These investigators reported two ex© 2010 Lippincott Williams & Wilkins

ponential functions to be present in the whole blood disappearance curve of digitoxin after intravenous administration. Figure 2A illustrates these findings. Note that the early disappearance curve has a halftime (the time required for one-half of the radioactivity originally present to disappear) of 15 to 30 minutes and the dominant (late) curve 44 to 52 hours. These figures are nearly identical for both digitoxin and its metabolites. The early exponential half-time of 15 to 30 minutes was felt to be associated with tissue equilibration, the dominant half-time of 50 hours to represent release of “loosely” bound glycoside from tissues. The authors suggested the presence of another component to explain the prolonged renal excretion of digitoxin which could not be identified on the published graphs. Figure 2B illustrates the excretion of digitoxin in the urine (Okita et al).27 Note that only about 10% of the initial dose was eliminated in the first 24 hours, and the urine digitoxin half-time was nine days, a finding much more in keeping with the physiologic half-time of digitoxin observed clinically and measured objectively in normal human subjects by Weissler et al. (Figure 3).6 The amount of glycoside excreted appeared to be dependent upon that present in the body. Most of the radioactivity was again noted to be in the form of metabolites, which were unidentified in these studies. Tissue studies were performed on three patients given carbon-14 digitoxin by Okita et al.32 These revealed greatest concentrations of digitoxin and its metabolites in the liver, bowel and kidney, and less in the heart (Figure 4). The intracellular distribution of carbon-14 digitoxin in the heart was studied in guinea pigs by Harvey and Pieper in 1955,26 with most of the radioactivity being in cellular debris, next in nuclear material, mitochondria, and finally the aqueous fraction. It should be noted that certain species are “insensitive,” and studies with any digitalis glycoside in rats or rabbits are not necessarily applicable to man. Clinical applicability of animal studies depends upon biotransformation and the mode of excretion, which may be related to the species differences. Hence, one cannot readily transpose animal studies to the human without some reservation. Kaihara and Leland78 report that the species difference observed for digitalis glycosides may be related

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FIGURE 2B. URINARY EXCRETION OF RADIOCARBON DIGITOXIN. Micrograms of digitoxin excreted per day are shown on the vertical axis on a semilogarithmic scale, days after administration on the horizontal. Note that the half-time of urinary excretion is 9 days with either 1.2 to 1.5 mg (—) or 0.5 mg (---) of digitoxin. (Reprinted with permission from: Okita, G. T., Ref. 27.)

to the cardiac myocardial uptake of the glycoside. In studies with tritiated digoxin and digitoxin, Okita40 has recently proposed that species differences in sensitivity to digitalis glycosides may be related to their retention and “recycling” in the enterohepatic circulation. This appeared to be a function of polarity of the glycosides, the nonpolar glycoside digitoxin having a large amount of the glycoside and metabolites recycled

(reabsorbed); whereas, digoxin (a polar glycoside) exhibited little recycling. These differences were felt to be related to the increased lipid solubility of the nonpolar glycoside (digitoxin) which leads to increased resorption in the bowel and increases the duration of digitoxin action when compared to digoxin. Okita et al. noted the placental transfer of digoxin and the deposition in fetal tissue in 195229 and in 1956.30 The protein binding of labeled digitoxin was studied by Spratt and Okita.35 Salt precipitation and membrane dialysis of serum protein revealed 0.01 ␮g of digitoxin was bound to 1.0 mg of serum albumin, however, much less was shown to be bound by electrophoretic techniques. The former finding is at variance with previous studies with nonlabeled material106 from a quantitative standpoint but agrees qualitatively with the previous experiments. Double isotope dilution studies by Lucas and Peterson39 reveal that digitoxin is 90% protein bound. The amount of protein binding in the serum probably contributes to the higher digitoxin blood levels (10 to 50 m␮g/ml) compared to digoxin (0.5 m␮g/ml),107, 108 and perhaps to the slow digitoxin excretion observed in most species. Only 5% of digitoxin present in the blood is recoverable from the red blood cell.108 Subcellular fractionation performed on rat liver slices and tumor cells34 reveal no specific distribution of labeled digitoxin in cellular compartments by this method. Digitoxin labeled with tritium has been reported,15,19 but as yet there are few published studies in human subjects,40 except by double isotope dilution techniques.39 Katzung and Meyer37 studied tritiated digitoxin excretion in dogs with biliary fistulae. Increased biliary and total digitoxin excretion was noted in these animals compared to that shown in sham operated dogs, which suggested that recycling of the glycoside and its metabolites in the gut may be important in its long half-time. The specific activity of the tritiated digitoxin used for these experiments was low. The excretory products (including metabolites) of carbon-14 digitoxin thus far demonstrated in a

FIGURE 3. PHYSIOLOGIC HALF-TIME OF DIGITOXIN. The ejection time index is plotted on the vertical axis on a semilogarithmic scale, time on the horizontal. Note the disappearance of one-half of the physiologic digitoxin activity at 102 to 112 hours. The physiologic halftimes for ouabain, deslanoside and digoxin are also shown. (Reprinted with permission from: Weissler, A. M, Snyder, J. R, Schoenfeld, C. D. and Cohen, S., Ref. 6.)

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TABLE 2. Metabolites of digitalis-labeled glycosides Glycoside

Metabolites recovered from excreta

Digitoxin

(Digitoxin 8%) Digoxin Digitoxigenin Digitoxigenin mono-digitoxiside Digitoxigenin bis-digitoxiside Digoxigenin Digoxigenin mono-digitoxiside Digoxigenin bis-digitoxiside (Four unidentified metabolites) (Digoxin 85%) Digoxigenin Digoxigenin mono-digitoxiside Digoxigenin bis-digitoxiside Dihydrodigoxigenin C-3 epidigoxigenin (Lanatoside C—none found) (Ouabain—none found) Not reported Not reported

Digoxin

Lanatoside C Ouabain Bufagin Acetyl digitoxigenin

FIGURE 4. DISTRIBUTION OF RADIOCARBON DIGITOXIN IN HUMAN TISSUES. Mean values of three patients arc indicated as micrograms, or microgram equivalent per 100 grams of tissue on the horizontal axis. The solid portion of the bar represents digitoxin and the cross-hatched area digitoxin metabolites. Note the relatively large portion of radioactivity representing metabolites. (Reprinted with permission from: Okita, G. T., Talso, P. J., Curry, J. H., Jr., Smith, F. D., Jr. and Geiling, E. M. K., Ref. 32.)

three week period may be characterized as follows:28 8% unchanged digitoxin, 70% metabolites. The metabolites are designated as chloroform-soluble (30%) and water-soluble (70%). One of the principal chloroform-soluble metabolites is digoxin (12-hydroxydigitoxin). Other metabolites include digoxigenin bis-digitoxiside, digoxigenin mono-digitoxiside, digoxigenin, digitoxigenin bis-digitoxiside, digitoxigenin mono-digitoxiside, digitoxigenin, and four unidentified metabolites. Metabolites thus far described in labeled glycoside studies are summarized in Table 2.23,28,37,38 Lucas and Peterson39 developed a technique of double isotope dilution for the study of digitoxin. The patients are not given the labeled glycoside but the method requires radioactive digitoxin for the analysis. Only digitoxin can be measured by this method. Okita et al.28,31,32 and Ashley et al.22 have shown that the major portion of the identifiable drug or its breakdown products in the human subject is in the form of metabolites, suggesting that the therapeutic activity of digitoxin may reside in its metabolic by-products. Lucas and Peterson39 demonstrate high serum levels of digitoxin and decreased renal excretion in renal failure, but conclude that the digitoxin is metabolized and excreted. No measurement of metabolites was done. Digoxin is a major chloroform-soluble metabolite of digitoxin28 © 2010 Lippincott Williams & Wilkins

and has been shown not to be excreted normally in renal failure.47,55,62,64,90,91 As the method described measures only digitoxin and not metabolites, these findings should be viewed with some reservation. In summary, digitoxin has been the subject of extensive radiochemical, pharmacological and clinical study. It has been labeled by biosynthesis, hydrogen exchange and breakdown resynthesis, with carbon-14 and tritium, both by specific and random label. It is excreted primarily in the urine as metabolic by-products. Its whole blood dominant half-time was 40 to 50 hours for both digitoxin and its metabolites. These half-times are at some variance with the physiologic halftime of digitoxin, reported to be 102 to 112 hours by Weissler et al.,6 and a longer radioactive exponential function which might explain this disparity has been proposed.31 Carbon-14 acetyl digitoxigenin has been prepared42 but has not been studied experimentally in either animals or human subjects. These findings are consistent with the long duration of action ascribed to digitoxin clinically. LANATOSIDE C. Lanatoside C was prepared with a carbon-14 label in Germany by Bretschneider et al.105 in 1962, by means of a biosynthetic technique similar to that reported by Geiling et al.40 A coronary arteriovenous difference was shown; as well as greater distribution to heart, liver and kidney than other tissues in dogs. No metabolites were detected in significant amounts in serum, urine or feces. No human studies are available with radioactive lanatoside C and this report is the only one published regarding its use. OUABAIN AND DIHYDRO-OUABAIN. Dutta and associates103 published a comparative report of the effect of these glycosides labeled with tritium in rats (in-sensitive species) and sheep (sensitive species). A very high specific radioactivity of the preparation was noted. Sheep excrete ouabain primarily in urine; rats in feces. The dominant serum half-time of ouabain in sheep was 45 minutes. The authors conclude that rats

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TABLE 3. Serum half-times and excretion of tritiated digoxin

Oral Intramuscular Intravenous FIGURE 5. THIN-LAYER RADIOCHROMATOGRAM OF TRITIATED DIGOXIN. Radioactivity is plotted vertically and each closed circle represents radioactivity present in eluted scrapings from the thin-layer (silica gel) plate. The origin is at the left and solvent front at the right. Digoxin is identified as the dark spot. Note the radioactivity is confined to the area containing digoxin. (From: Doherty, J. E.,Flanigan, W. J. and Perkins, W. H.: Tritiated digoxin excretion in human subjects following renal transplantation. Circulation, to be published— by permission of the American Heart Association, Inc.)

are not good experimental animals for studies with digitalis because of differences in metabolism of the glycoside. Human studies were also reported with tritiated ouabain or dihydro-ouabain.104 This study revealed a dominant serum half-time of five hours, far less than the physiologic activity of the glycoside reported by Weissler et al.6 to be 22 hours in clinical human assay. No metabolites of ouabain were demonstrated by paper chromatographic techniques. A coronary arteriovenous difference was demonstrated, and atrial muscle (obtained at mitral valve surgery) was demonstrated to contain 5 to 10 times the blood concentration of ouabain. BUFAGIN. Carbon-14 labeled cardioactive toad venom was biosynthesized by Doull et al.13 This glycoside has not been used in a clinical or experimental situation other than by assay, which established the presence of a cardioactive principal of very low specific activity. DICOXIN. Radioactive (tritiated) digoxin was prepared in 1958. Scrubbing and purification resulted in a compound 99.5% chemically pure, cardioactive,

Seven day excretion of the total dose

Serum half-time●

Urine

Stool

Total

31 hours 37 hours 33 hours

42% 54% 80%

20% 10% 12%

62% 64% 92%

Time required for one-half of digoxin radioactivity originally present to disappear.

radiochromatographically pure (Figure 5), sterile and pyrogen-free. Gonzales and Layne69 reported blood levels, tissue distribution and excretion in dogs in 1960. These investigators found digoxin localized in kidney, heart and liver in acute animal experiments and suggested that studies with this glycoside in human subjects were possible and should prove informative. Doherty et al.16,17,63 reported studies of tritiated digoxin serum levels, serum half-time and urine and stool excretion in congestive heart failure in twelve human subjects who were given the drug by the oral route in an alcoholic solution. Excretion was noted principally in the urine, primarily as unchanged digoxin, although about 1% of metabolite “B” (later shown to be digoxigenin bis-digitoxiside)102 was present in the urine. An early peak serum level was demonstrated at 30 to 60 minutes after oral administration and digoxin radioactivity was demonstrable six minutes after administration. The serum turnover curve is reproduced in Figure 6A. Two exponential functions can be derived from the oral tritiated digoxin serum disappearance curve. The first of these is attributed to distribution and binding of the glycoside and has a half-time of 50 minutes; the second represents the dominant half-time and is attributed to metabolism and excretion (primarily the latter) of tritiated digoxin and has a half-time of 31.3 hours. It is of interest that the distribution and binding half-time correlates well with the beginning onset of action (inotropic effect) of digoxin seen in human subjects given

FIGURE 6A. SERUM TURNOVER OF TRTIATED DIGOXIN AFTER ORAL ADMINISTRATION. Radiohydrogen digoxin is shown on the vertical axis as percent of the 45 minute specimen on a semilogarithmic scale, time in minutes on the horizontal. Although the study continued for 8600 minutes (7 days), only the first 3000 minutes are indicated in order to demonstrate the early exponential functions more clearly. Curve A (X) represents the actual counting rates of radioactive digoxin. Line B represents the best straight line that can be drawn after the equilibration plateau is reached, extrapolated to zero time. This line represents the metabolism and excretion of digoxin and the dominant half-time is obtained by determining the extra polated quantity of digoxin present at zero time and identifying the time required for one-half of the radioactivity originally present to disappear. (Reprinted with permission from: Doherty, J. E., Perkins, W. H. and Mitchell, G. K., Ref. 63.)

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FIGURE 6B. SERUM TURNOVER OF TRITIATED DIGOXIN AFTER INTRAMUSCULAR ADMINISTRATION. Radioactivity representing digoxin is plotted on the vertical axis on a semilogarithmic scale, time in minutes on the horizontal. Curve A (●) represents the actual radioactive counting rate of digoxin plotted as percent of the total counts administered. Line B, an exponential function, is derived as explained in Figure 6A and represents metabolism and excretion of digoxin. It has a half-time of 37 hours. Line C, a second exponential, is derived as before and represents distribution and binding of digoxin to tissue. It has a half-time of 100 minutes. As in Figure 6A the total duration of this study was seven days; only the first 3000 minutes (two days) are shown. (Reprinted with permission from: Doherty, J. E. and Perkins, W. H., Ref. 58.)

digoxin orally. The dominant half-time appears to parallel the disappearance of pharmacologic activity as the drug is withdrawn and is consistent with the terms “short-acting” or “short duration of action” when applied to digoxin. Digoxin excretion after oral administration is shown in Table 3. Doherty and Perkins also studie digoxin metabolism after the intramus cular58 and intravenous57 routes of administration in human subjects. The serum turnover curve for intramuscular administration of tritiated digoxin is reproduced in Figure 6B. Intramuscular administration of the labeled glycoside to ten subjects revealed a similar serum half-time, although the peak serum concentration occurred later than in the oral study, as did the serum digoxin plateau (indicating a later serum tissue equilibration of digoxin by this route). It is noted that there are also two exponential functions observed after intramuscular administration which are similar to those seen after oral administration. The first exponential function is again related to tissue distribution and binding and has a half-time of 100 minutes. The second exponential function represents the dominant half time and is about twice as long by the intramuscular route compared to oral administration. This could be related to two factors: (1) the tritiated digoxin given orally was in an alcoholic

solution, facilitating more rapid absorption and subsequently more rapid distribution and binding to tissue, and (2) the intramuscular route (gluteal muscle) of administration is not as effective in absorbing digoxin (prepared in 40% propylene glycol, 10% ethyl alcohol and 50% distilled water) as the gastrointestinal tract. Excretion of tritiated digoxin by the intramuscular route was again noted to be primarily in the urine as the unchanged glycoside. Only 10% was recovered in the stool during a seven day study after a single intramuscular dose (Table 3). Intravenous tritiated digoxin was noted to produce an early high serum concentration and an earlier serum plateau than oral or intramuscular administration (Figure 6C). Note the presence of three exponential functions in this serum digoxin curve after intravenous administration. The addition to the oral and intramuscular exponentials is an earlier one representing serum distribution with a half-time of two minutes. This rapid early exponential is not meaningful because of the variability of the blood volume, injection time, cardiac output, etc. The second exponential function represents tissue distribution and binding and has a half time of 30 minutes, which is much faster than by oral or intramuscular routes. The third exponential function has a half-time of 33 hours

FIGURE 6C. SERUM TURNOVER OF TRITIATED DIGOXIN AFTER INTRAVENOUS ADMINISTRATION. Digoxin radioactivity is again plotted on the vertical axis on a semilogarithmic scale, time on the horizontal. Curve A represents the actual counting rate of tritiated digoxin. Line B is an exponential function, derived as in Figures 6A and 6B, and represents the metabolism and excretion of digoxin and has a half-time of 33 hours. Line C, a second exponential function, derived as before, represents tissue distribution and binding and has a half-time of 30 minutes. Line D, a third exponential with a half-time of two minutes, derived by subtracting Line C from Curve A, represents serum distribution and depends on the speed of injection, blood volume, cardiac output, etc. (Reprinted with permission from: Doherty, J. E. and Perkins, W. H., Ref. 57.)

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FLGURE 6D. SERUM TRITIATED DIGOXIN TURNOVER BY ORAL, INTRAMUSCULAR AND INTRAVENOUS ADMINISTRATION. This is a composite graph showing the comparative digoxin serum levels after oral (䡲), intramuscular (⌬), and intravenous (●) administration. Radiohydrogen digoxin is plotted on the vertical axis as the comparative digoxin concentration, time on the horizontal. Note the difference in the early portions of the radioactivity curves, the dominant (late) half-times being very similar. The serum level is higher with intravenous administration and lowest after oral administration. These differences are not, however, statistically significant for the small groups of patients studied. (Reprinted with permission from: Doherty, J. E. and Perkins, W. H., Ref. 58.)

and represents metabolism and excretion of digoxin, which is not significantly different from that observed after oral and intra muscular administration. Urine excretion in seven days was noted to be 80% (Table 3). Unchanged digoxin was again demonstrated to be the principal excretory product although small amounts of metabolites were present. The relative lack of metabolic break down of digoxin by the body is of interest as it is somewhat unusual in biologically active pharmaceutical products. The limited metabolic degra dation of digoxin may be a product of the relatively short half-time of the compound and the fact that it is one of the major chloroform-soluble meta bolic breakdown products of digitoxin, perhaps resisting further metabolism to some degree. The increased polarity of the compound (compared to digitoxin) also probably explains the large quantities of digoxin which are recovered unchanged in the urine.40 The dominant serum half-time by all the routes of administration was about 34 hours and is shown in

Figure 6D.This figure also illustrates the differences in the early portion of the digoxin turnover curves given by different routes of administration and the serum level of digoxin. Higher serum levels are present after intravenous administration, followed by intramuscular and oral, for comparable doses of tritiated digoxin. The differences in serum concentration, however, are not statistically significant for the number of patients studied, but are probably real when applied to larger numbers of subjects. These serum half-times closely approximate the physiologic halftime of digoxin determined by Weissler et al.6 (Figure 3) after intravenous administration of unlabeled digoxin. The lack of correlation in physiologic response and radioactivity curves in the early response to digoxin may be due to the dose of digoxin given in this study, 3.25 mg in a single dose, or to the form of the oral preparation (alcoholic solution vs solid pill). This dose of digoxin may be excessive because absorption has been shown to be greater when determined by tracer tech-

FIGURE 7A. SERUM DIGOXIN CONCENTRATION COMPARED TO DIGOXIN TISSUE LEVELS IN MONGREL DOGS GIVEN A SINGLE INTRAVENOUS DOSE OF TRITIATED DIGOXIN. The digoxin serum level in micrograms percent of digoxin (●) is shown on the vertical axis on the left, and tissue concentration in micrograms per gram of tissue as indicated on the figure for kidney, heart and liver is shown on the right. Time in hours (not plotted to scale) is shown horizontally. Note the rise in tissue concentration as the serum level falls, and the high digoxin concentration in the kidney, heart and liver respectively.

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FIGURE 7B. TRITIATED DIGOXIN TISSUE CONCENTRATION AT 12 HOURS. Concentration of digoxin is shown on the horizontal axis as micrograms of digoxin per gram of tissue X 10⫺8. Animals were sacrificed at 12 hours after being given 0.5 mg of tritiated digoxin intravenously. Although not shown on the figure, gallbladder tissue contains a significant amount of digoxin. (Reprinted with permission from: Doherty, J. E. and Perkins. W. H., Ref. 59.)

niques (80%)63 than originally proposed by Gold (50%).109 Friedberg110 feels that the usually recommended oral digitalizing doses of digoxin are excessive. It has been demonstrated that doses of digitalis required for control of the ventricular rate in atrial fibrillation (A-V block) are frequently greater than required for optimum inotropic effect and the doses of digoxin required for control of atrial fibrillation are often excessive with normal sinus rhythm. Overt digitalis tox-

icity with as little as 1.0 mg of digoxin given orally has been observed, without other factors being present which would alter digitalis sensitivity. A minimal dose is sometimes effective in the treatment of congestive heart failure or arrhythmia when digoxin is given intravenously. Tritiated digoxin turnover and excretion times in patients with pulmonary heart disease (cor pulmonale) appear to be “normal” (approach those of other patients with congestive heart failure).61 This disease state, frequently associated with digitalis sensitivity, has other factors present which might alter the tissue response to digitalis; namely: (1) pH and electrolyte changes, (2) red cell mass, and (3) hypoxia and hypercapnea. These are probably of greater importance than digitalis as such in the production of digitalis intoxication in these patients. Marcus87 has studied tritiated digoxin serum levels, turnover rates and excretion in normal subjects and demonstrated no important differences between normal individuals and those with congestive heart failure. Doherty and Perkins59 and Brown et al.48 studied the distribution of tritiated digoxin in tissue of dogs by a serial sacrifice technique and showed disappearance from tissue parallels the serum disappearance rate, in spite of differences in tissue concentration. The largest amount of digoxin was present in the kidney (the major organ of excretion), heart and liver in normal mongrel dogs. These data are reproduced in part in Figures 7A and B. Doherty et al.,55,62,64 Marcus et al.91 and Bloom and Nelp47 have studied tritiated digoxin turnover in human subjects with renal failure and demonstrated reduced renal excretion of digoxin, increased serum levels of digoxin, and prolonged serum half-times of digoxin, without a compensatory increase in stool excretion, and propose that overt renal failure is a major factor in digoxin toxicity. Figures 8A and B illustrate these findings. Digoxin doses should be reduced by onethird to one half when blood urea nitrogen (BUN) levels reach 70 to 80 mg%. Clearance ratios for creatinine and digoxin are near unity.47,54 This does not suggest that a creatinine clearance determination is necessary for every patient to be digitalized or receiving digitalis, but that caution should be exercised in giving digoxin to patients with a reduced creatinine clearance or an ele-

FIGURE 8A. TRITIATED DIGOXIN SERUM TURNOVER IN RENAL FAILURE COMPARED TO NORMAL RENAL FUNCTION. The composite serum turnover curve of 12 patients with renal failure (blood urea nitrogen—mean 80 mg%) is compared to 13 patients with normal renal function given digoxin intravenously. Digoxin radioactivity is plotted on the vertical axis as percent of the five minute specimen on a semilogarithmic scale against time in minutes on the horizontal axis. Note that serum levels are higher and the serum half-time is longer— 82 hours with renal failure, 33 hours with a normal blood urea nitrogen and congestive heart failure—indicating prolongation of digoxin excretion in renal failure. (Reprinted with permission from: Doherty, J. E., Perkins, W. H. and Wilson, M. C, Ref. 64.)

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FIGURE 8B. TRITIATED DIGOXIN EXCRETION IN RENAL FAILURE. Digoxin excretion is shown in this figure for the same patients whose serum turnover curve is reproduced in Figure 8A. Note that total seven day digoxin excretion is less than onehalf that observed in normal renal function in these patients. Stool excretion is somewhat increased with renal failure, but does not compensate for prolonged renal excretion. (Originally published in Excerpta Medica International Congress Series, ICS 137. Proc. of the IX International Congress of Internal Medicine, Amsterdam 1966, Ref. 52.)

vated BUN. As the latter is more frequently determined clinically and Doherty et al.64 have shown that digoxin excretion is inversely related to the level of the BUN (Figure 9), smaller doses of digoxin are therefore recommended when the BUN reaches 70 to 80 mg%. Renal failure (overt or occult) is responsible for much of the digitalis toxicity observed in patients with acute or chronic renal failure with attendant lack of digoxin excretion in the urine. Studies in anephric patients54 and patients with renal transplants55 reveal similar findings. Potassium retention doubtless protects many patients from overt toxicity and may actually inhibit myocar dial digoxin uptake.86,100 D. C. Harrison et al.76 have shown that potassium depletion and renal failure lead to increased tritiated digoxin myocardial concentration in mice, an insensitive species but one which does not metabolize digoxin and in this respect resembles the human. Although these findings were obtained in mice, they appear to support the studies of Ebert,65 Marcus et al.,84,86,91 Doherty et al.,55,62,64 and Bloom and Nelp47

relating to digoxin retention and sensitivity in renal failure. C. E. Harrison et al.75 report that myocardial tritiated digoxin uptake in potassium deficient cardiomyopathic rats is inhibited incomparison to normal control animals. This finding indicates an alteration in digoxin metabolism in potassium deficient rats with myocardial lesions (a “digitalis insensitive” species). An objective comparison to studies in dogs and human subjects was not attempted. Goldsmith et al.68 report that potassium infusion in the dog given tritiated digoxin in nontoxic doses does not affect the myocardial digoxin concentration compared to control animals. He suggests the data indicate that a mechanism other than myocardial digoxin concentration is responsible for the effect of potassium in digitalis-induced arrhythmias. It is well documented that hemodialysis or peritoneal dialysis44,111 may precipitate digitalis toxicity in the digitalized patient as serum levels approach normal figures. This appears to occur with digitoxin111 as well as with digoxin, although reduced renal excretion of digitoxin has not been well documented in spite of higher levels of blood digitoxin and reduced renal excretion.39 Peritoneal dialysis and hemodialysis have been shown to be ineffective in removal of digoxin from the body44; only 1 to 4% of the total administered dose of tritiated digoxin being recovered in the dialysate baths from either six hours of hemodialysis or 36 hours of peritoneal dialysis. This technique is not recommended for the treatment of digitalis intoxication and may indeed be responsible for the production of digitalis toxicity if the potassium concentration in the serum is lowered by the procedure. Figures 10A and B illustrate the dialysis of tritiated digoxin and show the relatively low recovery of the glycoside by this method. Many patients are also protected from digitalis toxicity in renal failure by lack of absorption of digoxin52 or unusual metabolic pathways leading to largely cardioinactive metabolites. Prolongation of digoxin excretion is alnoted in anephric patients,55 and to a lesser degree in patients with renal transplants. A patient has been described by Gruber and Luchi70 who did not have hyperthyroidism or other recognized factors altering sensitivity to digoxin, but required large doses of digoxin for maintenance therapy. Digoxin was reported to be converted to a

FIGURE 9. THE RELATIONSHIP OF THE BLOOD UREA NITROGEN (BUN) TO DIGOXIN EXCRETION IN THE FIRST 24 HOURS AFTER INTRAVENOUS ADMINISTRATION. The BUN is plotted on the vertical axis on a semilogarithmic scale, percent of the total dose of digoxin excreted in the first 24 hours after administration on the horizontal axis. Note that the higher the BUN, the less digoxin is excreted. (Originally published in Excerpta Medica International Congress Series, ICS 137, Proc. of the IX International Congress of Internal Medicine, Amsterdam, 1966, Ref. 52.)

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FIGURE 10A. HEMODIALYSIS OF TRITIATED DIGOXIN. The tritiated digoxin serum turnover curve is shown in the upper portion of the figure plotted as counts per minute per ml on a semilogarithmic scale on the vertical axis; time is indicated on the horizontal axis. Note the “venous” counts (°) and the “arterial” counts (●) during application of a twin coil artificial kidney which show a modest arteriovenous difference across the kidney. Plotted on the vertical axis on the lower panel on the left is the dialysate concentration of digoxin as counts per minute per 10 ml, on a semilogarithmic scale against time. Note the increasing concentration of digoxin in the dialysate during three exchanges. Cumulative excretion is shown on the lower right as a cumulative percent of the total administered dose of digoxin. Only 2% of the dose was recovered in six hours of hemodialysis.

less cardioactive metabolite, thus increasing the maintenance requirement. Clinical confirmation of digitalis toxicity occurring with renal failure in patients receiving unlabeled ouabain, lanatoside C, digoxin and digitoxin is reported by Schelar et al.112 Tritiated digoxin excretion does not appear to be related to the volume of urine excreted, as urinary excretion rates of dogs with surgically induced diabetes insipidus are nearly the same as control animals.51

Marcus83 has also noted that tritiated digoxin excretion rates are not related to the volume of urine excreted in human subjects. Excretion rates of digoxin were no greater the day after the administration of the potent diuretic furosemide than the day prior to its administration, in spite of a twofold increase in urine volume obtained with the diuretic agent. Jelliffe and Blankenhorn113 have recently reported a method for determining dosage of digoxin in renal failure based upon the knowledge that the clearance of digoxin and creatinine is near unity. A maintenance dose is based upon the creatinine clearance and the known digoxin and digitoxin half-times derived from radioactive tracer and chemical studies; 41% of the usual dose of digoxin is recommended with anuria, to 100% of the usual dose with a creatinine clearance of 100 ml/min. A patient with a creatinine clearance of 40 ml/min would then appear to require a dose of about one-half of the usual daily maintenance dose of digoxin for optimum control. Studies have also been done at postmortem on tissue levels of tritiated digoxin given to human subjects.62 Patients with renal failure have higher myocardial digoxin content than those with a normal BUN. These findings correlate with the reduction in digoxin excretion in renal failure noted previously, and are shown in Figures 11 A, B and C. Al-though a relatively small amount is present in skeletal muscle, since it constitutes 40% of the body weight, it is a significant binding site for digoxin and may represent an important site of metabolism as suggested by Abel et al.43 Studies in anephric subjects54 and following renal transplantation55 indi-cate that total absence of renal tissue prolongs digoxin excretion in a similar fashion to renal insufficiency. Transplantation of a single kidney will proportionately restore the excretion of digoxin, depending upon its function. Marcus’ et al.92 studies in nephrectomized dogs reveal findings similar to those observed for anephric human subjects.54 The mechanism of tritiated digoxin urinary excretion has been studied in the dog by the stop-flow technique of Malvin114 and reveals that digoxin is filtered by the glomerulus and partially resorbed in the tubules.53,96 Figures 12 A and B demonstrate filtration and absorption of tritiated digoxin.

FIGURE 10B. PERITONEAL DIALYSIS OF TRITIATED DIGOXIN. The serum tritiated digoxin turnover curve is shown as in Figure 10A. Peritoneal dialysis was started near the equilibration time of the serum curve of digoxin, and the cumulative total excretion in percent of the total dose of digoxin recovered in the dialysate baths is shown on the lower panel on the right. Note that only 2.6% of the dose of digoxin was recovered in 26 hours of peritoneal dialysis. (Reprinted with permission from: Ackerman, G. L., Doherty, J. E. and Flanigan, W. J., Ref. 44.)

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FIGURE 11A. TISSUE CONCENTRATION OF TRITIATED DIGOXIN IN A 43-YEAR-OLD CAUCASIAN MALE PATIENT WITH PULMONARY HEART DISEASE (BLOOD UREA NITROGEN 13 MG%), WHO RE-CEIVED 1.0 MG DIGOXIN INTRAVENOUSLY 31⁄2 HOURS BEFORE DEATH. Micrograms of digoxin per gram of tissue are shown on the horizontal axis of a bar graph. Note the high renal concentration followed by heart and other organs as indicated.

FIGURE 11B. TISSUE CONCENTRATION OF TRITIATED DIGOXIN IN A 71-YEAR-OLD NEGRO MALE PATIENT WITH RHEUMATIC HEART DISEASE, CARCINOMA OF THE PROSTATE AND PYELONEPHRITIS (BLOOD UREA NITROGEN 86 MG%), WHO DIED 32 HOURS AFTER RECEIVING 1.0 MG OF TRITIATED DIGOXIN INTRAVENOUSLY. Digoxin concentration is shown as indicated on Figure 11A. Note the greatest concentrations of digoxin in the heart muscle followed by kidney, liver and other organs. (Reprinted with permission from: Doherty, J. E., Perkins, W. H. and Flanigan, W. J., Ref. 62.)

Tissue radioautography of tritiated digoxin has been studied by several investigators40,50,67,93,95,97,98 and most agree that there appears to be localization of digoxin radioactivity in association with the A band of the sarcomere. Fozzard and Smith67,95 feel there is also sarcotubular system localization which would be compatible with the primary effect of digitalis related to energy transfer through ATPase inhibition. Tubbs et al.97 also report findings consistent with this view. Marcus et al.89 have published data in abstract form indicating a reduction in digoxin uptake by the myocardium with reserpine administration in dogs. This suggests that larger doses of digoxin are necessary in reserpinized patients to attain optimal therapeutic effects. Gruber et al.71 have confirmed this work and have also shown a difference in uptake of the atrial muscle as well as the ventricular muscle, as was shown by Marcus.89 The reserpine effect was not mediated in these experiments through urinary excre-

tion, norepinephrine depletion, or poor myocardial perfusion. Another factor which may be important in this finding is suggested by the observation that reserpine administration to hyperthyroid patients, who are resistant to digitalis in the usual doses, will restore their sensitivity to digitalis.51 Tritiated digoxin excretion has been studied in several patients with biliary T-tube drainage following cholecystectomy.51 These patients excrete very little digoxin in the stool and demonstrate a proportionate increase in biliary excretion. There is no significant alteration in total seven day excretion (or stool plus bile excretion), indicating little “recycling” of digoxin in the bowel. Urinary excretion is relatively unchanged. Marcus et al. have studied tritiated digoxin blood levels in dogs83 and in human subjects88 with and without a loading dose (initial digitalizing dose). These studies reveal that blood digoxin concentrations are

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FIGURE 12A. FILTRATION OF TRITIATED DIGOXIN. Using the stop-flow technique of Malvin in dogs, sodium ferrocyanide (o) and tritiated digoxin (X) were injected simultaneously. Ureteral occlusion was released and serial samples were collected, earliest representing renal pelvis, then distal and proximal tubules and last, the glomerulus. Concentration of ferrocyanide is shown on the left vertical axis, digoxin in counts per minute per ml on the right. The virtual congruence of the curves indicates that digoxin is excreted in the same manner as the known filtered substance, sodium ferrocyanide.

FIGURE 11C. TISSUE CONCENTRATION OF TRITIATED DIGOXIN IN A 65-YEAR-OLD NEGRO FEMALE ANEPHRIC PATIENT, WHO DIED OF A DRUG REACTION 32 HOURS AFTER RECEIVING 1.0 MG OF DIGOXIN INTRAVENOUSLY (BLOOD UREA NITROGEN 100 MG%). Tissue digoxin concentration is plotted as shown in Figure 11A and B. Note the high concentration of digoxin in heart and liver. Kidneys were absent in this patient. (Reprinted with permission from: Doherty, J. E., Perkins, W. H. and Flanigan, W. J., Ref. 62.)

similar in patients given a maintenance dose (0.5 mg) of digoxin daily for 5 to 7 days and in those given 1.0 to 2.0 mg initially, followed by 0.5 mg daily for 5 to 7 days (Figure 13). It appears that therapeutic digitalization may be accomplished in this manner as effectively as with a large initial dose. Most clinicians will recall that this technique of daily maintenance dosage for slow digitalization was suggested by Gold and Degraff109 in 1929, and has been amply confirmed by this study. Harrison et al.72–74 administered tritiated digoxin to dogs with and without biliary fistulae and after analyzing tissue and excreta from the animals proposed a four-compartment system in a mathematical model in order to explain the distribution and excretion of digoxin. The greatest digoxin concentration was again demonstrated to be in liver, kidney and heart. Species differences between rats and dogs were shown with tissue studies: 0.2% of the total dose of digoxin (dose recovered per total organ) was present in the rat heart at 40 minutes; whereas, 4% of the © 2010 Lippincott Williams & Wilkins

total dose of digoxin was recovered from the dog heart at the same time interval. Butler and Chen115 have developed a technique for preparing a specific anti-body to digoxin utilizing tritiated digoxin and bovine serum albumin. It would be attractive to suppose that if digoxin antibodies could be produced they might be of value in the treatment of digoxin toxicity; however, as most of the digoxin resides in and is bound to tissue and less than 1% of the total glycoside in the body is in the vascular compartment (except for a very short time after administration), it would seem to be somewhat inaccessible to serum antibodies. The technique may afford a method of measuring digoxin serum levels for diagnosis of toxicity or inadequate digitalization which would be very useful. The ratio of digoxin in the heart to that present in the serum is 17 to 35:1 (mean 29:1), and the knowledge of increased digoxin serum levels would provide useful clinical information relative to myocardial digoxin content.62 The method of Lowenstein107 and its more recent modification108 have not proven satisfactory in determining the serum level of digoxin because of the very low serum levels of digoxin present in the digitalized patient.51,99 Abel et al.43 studied the role of the liver in the metabolism of tritiated digoxin and showed that hepatic circulatory by-pass increased digoxin and/or metabolic content of whole blood. Large amounts of metabolites of digoxin were recovered in the control state and there was little change noted in animals subjected to hepatic bypass. They suggest the presence of significant extrahepatic sites of metabolism. By thin-layer chromato-

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FIGURE 13.TRITIATED DIGOXIN SERUM CONCENTRATION WITH AND WITHOUT A LOADING DOSE. Tritiated digoxin serum concentration in mg X 10⫺4 is shown on the vertical axis, time on the horizontal axis. Two groups of patients are indicated on the figure. Note that serum concentrations are approximately the same on the 6th day with an initial loading oral dose of 2.0 mg followed by 0.5 mg daily maintanence dose and with no loading dose and 0.5 mg daily. (Reprinted with permission from: Marcus, F. I., Burkhalter, L., Cuccia, C., Pavlovich, J. and Kapadia, G. G.: Circulation 34:865, 1966 — by permission of the American Heart Association, Inc., Ref. 83.)

FIGURE 12B. TUBULAR RESORPTION OF TRI-TIATED DIGOXIN. USING THE STOP-FLOW TECHNIQUE OF MALVIN IN DOGS, DIGOXIN WAS GIVEN BY INTRAVENOUS DRIP CONTINUOUSLY, URETERAL FLOW STOPPED 3– 6 MINUTES AND THEN RELEASED. This figure shows analysis of the serial samples obtained for digoxin radioactivity, creatinine, digoxin-creatinine ratio, sodium (distal tubular marker) and glucose (proximal tubular marker). Note the trough in digoxin concentration in both the proximal and distal tubular areas indicating digoxin reabsorption.

graphic means these authors demonstrate 62% of the intravenously injected doses in dogs at 30 minutes to be digoxin, 23% in the form of metabolites, and 15% was not recovered. It should be recalled, however, that 30 minutes after intravenous administration of tritiated digoxin to the dog, only 1% of the dose of tritiated digoxin remains in the vascular compartment. The small amounts of metabolites of digoxin excreted also indicate the minor role of the liver and other organs in digoxin metabolism. Marcus and Kapadia85 reported that the metabolism of tritiated digoxin in patients with cirrho-

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sis of the liver was unchanged compared to normal patients. This study shows that the patient in hepatic decompensation should tolerate usual doses of digoxin, if these be required, without the danger of toxicity (provided renal function remains adequate). This study would also suggest that hepatic metabolism of digoxin is relatively unimportant. Lufkin et al.81 report that retinal concentrations of tritiated digoxin are high in cats. This finding correlates with the clinical observation of disturbances of color vision with digitalis toxicity and with objective measurements of color vision in digitalized subjects.116 The fate of digitalis during and after cardiopulmonary bypass has been the subject of a number of studies with tritiated digoxin.45,46,56,66,77 Doherty et al.56 report an alteration in the serum turnover curve of digoxin during the first 12 to 24 hours following cardiopulmonary bypass in dogs. It is during this period of time that most of the toxic problems following digitalis administration are noted. Because of conflicting reports regarding digitalis sensitivity following cardiopulmonary bypass, and in light of this observation, the author feels that the apparent differences observed are probably due to differences in “pump technique” employed by various surgical teams and the changes seen reflect these individual differences in technique rather than changes in digitalis concentration. It is abundantly clear that because of the minute amount of digoxin present in the vascular compartment (less than 1%), little would be “lost” in the pump through “dilution.” Protein binding of tritiated digoxin has been studied by Perkins and Doherty94 by salt fractionation, membrane equilibration dialysis, electro-phoresis and gel filtration. These studies reveal 0 to 30% Volume 339, Number 5, May 2010

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FIGURE 14A. COMPARATIVE SERUM LEVELS OF TRITIATED DIGOXIN IN THYROID DISEASE AFTER INTRAVENOUS ADMINISTRATION. Digoxin radioactivity is plotted on the vertical axis as percent of the 15 minute specimen, time in hours on the horizontal. Hyperthyroid patients are indicated (䡩), euthyroid (X), and hypothyroid (●). Note that levels are highest in myxedema, intermediate in euthyroid and lowest in hyperthyroid patients. These data are statistically significant.

protein binding of digoxin in the serum. As is the case with digitoxin, only minute amounts of digoxin can be recovered from the red blood cell. Serum levels of digoxin in the digitalized patient are much lower than those reported for digitoxin63,108 and are perhaps related to the polarity of the compound. The digoxin serum level is often well below 1 m␮g/ml and cannot be adequately measured by the technique of Lowenstein.107,108 Doherty and Perkins60 showed a statistically significant difference in the serum levels of digoxin in patients with thyroid disease compared to euthyroid patients. Patients with hyperthyroidism have low serum levels of digoxin and with hypothyroidism high levels of digoxin, compared to euthyroid patients (Figure 14A). These findings correlate with clinical evi-

dence of digitalis sensitivity in myxedema and digitalis resistance in hyperthyroidism. Digoxin excretion rates were similar in all groups. Tissue studies in hyperthyroid, euthyroid and hypothyroid dogs show similar levels of digoxin in the serum, yet fail to demonstrate significant cardiac tissue changes in digoxin concentration. There appears to be an alteration in tissue response to digoxin in thyroid disease, rather than a change in tissue concentration (Figure 14B). Metabolites of digoxin are usually excreted in small amounts (15 to 20% of the total administered dose), and include digoxigenin, digoxigenin mono-digitoxiside, digoxigenin bis-digitoxi-side, dihydrodigoxigenin and C-3 epidigoxigenin. These and other metabolites of glycosides are shown in Table 2.79,82,100,101 MISCELLANEOUS. Any discussion of digitalis glyco-

FIGURE 14B.TRITIATED DIGOXIN TISSUE CONCENTRATION IN HYPERTHYROID, EUTHYROID AND HYPOTHYROID DOGS. Four animals were studied in each group and were sacrificed eight hours after being given 0.5 mg of tritiated digoxin intravenously. Micrograms of digoxin per organ are shown on the vertical axis and the tissue concentrations for each group are shown in heart, kidney, liver and lung. Note the lack of important changes between the groups of animals. No significant difference was observed in digoxin radioactivity per gram of tissue between these groups in spite of changes in the serum concentration resembling those of Figure 14A. (Reprinted with permission from: Doherty J. E. and Perkins, W. H., Ref. 60.) © 2010 Lippincott Williams & Wilkins

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sides seems incomplete without reference to the splendid studies of Repke,117,118 whose work deals primarily with the mechanisms of digitalis action and is beyond the scope of the present discussion. Finally, a plea is extended for the use of the smallest effective dose of digitalis to accomplish the desired clinical effect. The prevalence of overt digitalis intoxication in as much as 20% of digitalized patients, together with the effectiveness of the newer diuretics, make overdigitalization inexcusable, unnecessary and unwise.

SUMMARY The clinical pharmacology of digitalis has been greatly enhanced through clinical studies and knowledge of the serum concentration, half-time, tissue distribution and excretion obtained through studies of the radioactive cardiac glycosides. The serum half-time of digoxin is about 34 hours, which closely approximates the physiologic half-time of 33 hours. The whole blood half-time of digitoxin is about 50 hours, and the urinary half-time is nine days, compared to a physiologic half-time of 51⁄2 days. Most cardioactive glycosides in human subjects are excreted primarily in the urine in the form of metabolites (digitoxin) or as the unchanged glycoside (digoxin, lanatoside C, ouabain). Renal failure compromises digoxin excretion and in the presence of oliguria or anuria digitalis dosage should be reduced in proportion to the reduction in creatinine clearance. Tritiated digoxin appears to be handled by the body in much the same way in patients with congestive heart failure and “normal” subjects without failure. Cirrhotic patients’ turnover and excretion of tritiated digoxin resemble the “normal” individual, suggesting a minor role of the liver in digoxin metabolism. Patients with cor pulmonale do not tolerate “normal” doses of digitalis and reduced dosage is recommended inspite of normal excretion and turnover times. Patients with myxedema are very sensitive to digoxin and lower than usual doses are recommended; patients with hyperthyroidism require larger doses than normal individuals with congestive heart failure. These findings are accompanied by increased digoxin serum levels in myxedema and lower serum levels in hyperthyroidism compared to euthyroid patients. Each commonly employed digitalis glycoside has been discussed with regard to its serum half-time, excretion site, and excretion rate. Application of this knowledge should lead to a more efficient, safe, and meaningful use of these valuable cardioactive biological in clinical medicine. A plea is made for use of smaller doses of digitalis to avoid the common and often serious complication of digitalis intoxication.

ACKNOWLEDGMENT The author wishes to express sincere thanks to: Dr. Carl Slaughter, who as a student research fellow of the Arkansas Heart Association helped devise the method currently used for tritiated digoxin recovery from biological samples; co-authors G. K. Mitchell, W. J. Flanigan, G. L. Ackerman, E. J. Towbin, C. H. Brown, M. L. Murphy, Carolyn Harvey, Florence Char, M. C. Wilson, W. Peternel, Stacey Stephens, Carl Ferrell, and the late Dr. W. H. Perkins who provided continuing support which was invaluable in performing these studies during the past ten years.

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The technical assistance from F. J. Gammill, Joyce Sherwood, and Carolyn Dodd, the encouragement and suggestions of Drs. R. V. Ebert, H. R. Hipp, O. W. Beard, W. H. Wilson, R. S. Abernathy and J. H. Bates, and the invaluable help of the house staff and the nursing staff of the University of Arkansas Medical Center (particularly those of the Clinical Study Center) and of the Veterans Administration Hospital in Little Rock, is greatly appreciated. The supplies of tritiated digoxin were made available through Drs. D. S. Searle, S. Bloom-field, C. C. Ferry, and Mr. James Murphy of the BurroughsWellcome & Co. (USA) Inc., Tuckahoe, New York, and are much appreciated and gratefully acknowledged. The generosity of the National Heart Institute (U.S. Public Health Service) in providing funds to support the study of tritiated digoxin, together with the Arkansas Heart Association, Burroughs-Wellcome & Co. (USA) Inc., the Veterans Administration, and the University of Arkansas Medical Center, was and continues to be very helpful in the studies of the author. The secretarial assistance of Mrs. Sondra Stone, Mrs. Gloria Breeding and Miss Bonnie Harris cannot be underestimated. For the help of these individuals, and the patients who consented to tritiated digoxin turnover studies, I owe a tremendous debt of gratitude. Without their help these studies with tritiated digoxin would not have been accomplished.

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