- Email: [email protected]
Doxorubicin pharmacokinetics—The effects of protein deprivation
JOURNAL
OF SURGICAL
Doxorubicin DAVID
RESEARCH
30,331-337 (1981)
Pharmacokinetics
P. KAPELANSKI,
-The
Effects of Protein
Deprivation
M.D., JOHN M. DALY, M.D., EDWARD M. COPELAND, III, M.D., AND STANLEY J. DUDRICK, M.D.
University of Texas Medical School at Houston and University of Texas System Cancer Center. M.D. Anderson Hospital and Tumor Institute, Texas Medical Center, Houston, Te.ras 77030
Presented at the Annual Meeting of the Association for Academic Surgery, Birmingham, Alabama, November 5-8, 1980 Recent studies suggest that protein-calorie malnutrition decreases tumor response to chemotherapy. This study was designed to evaluate the effects of dietary protein deprivation on the pharmacokinetics and distribution of Adriamycin. Male Sprague-Dawley rats were assigned randomly to receive either a regular diet or a high-carbohydrate, protein-free diet for the duration of the experiment. Ten days after the initiation of the designated diet, all animals received a single dose (5.0 mg/kg) of Adriamycin intravenously. Animals were sacrificed at 0.5, 1, 2,6, 24, and 48 hr following drug administration, and the concentration of Adriamycin in serum, urine, and tissue was determined by fluorometric assay. Animals maintained on the regular diet continued to gain weight during the study interval, while animals provided the protein-free diet lost an average of 21% of their initial study weight (P < 0.01). Serum Adriamycin concentration was significantly lower in the protein-free diet animals at 0.5, 1, 2, 24, and 48 hr following administration; Adriamycin was not detectable in the serum of the protein-free diet animals at 48 hr, but was still present in the serum of the regular diet group (P < 0.01). The cumulative urinary excretion of Adriamycin, expressed as a percentage of the administered dose, was identical in both dietary groups during the initial 24 hr, but was significantly higher in the protein-free diet group by 48 hr after administration (P < 0.05). Significant differences were observed when liver, kidney, lung, and heart Adriamycin concentrations were compared. The elevated concentration of Adriamycin in the kidneys of the protein-free diet animals correlated with the increased urinary drug excretion, and indicated that accelerated renal clearance of Adriamycin was one possible mechanism for the more rapid serum elimination of the drug in the protein-free diet animals.
INTRODUCTION
Numerous clinical and experimental observations support the use of both enteral and parenteral alimentation techniques as an adjunct to the therapy of the patient with disseminated malignancy. Intravenous hyperalimentation (IVH) has permitted patients, at increased risk of complications secondary to inanition, to complete full courses of radiation therapy and chemotherapy successfully, with minimal morbidity [8]. Despite concurrent chemotherapy, many patients converted from a state of anergy to one of normal reactivity toward a panel of recall antigens; tumor regression was documented only in those patients with normal cutaneous delayed hypersensitivity [9]. In
that same study, no subject receiving radiation therapy developed or maintained delayed hypersensitivity. IVH was, however, thought to contribute to the general well being of the patients and to a conspicuous absence of therapeutic morbidity. The concomitant administration of IVH to patients receiving cyclic, multiple-agent chemotherapy was associated with an accelerated recovery from the gastrointestinal toxicity, but the incidence and severity of the hematopoietic toxicity were unaltered [13]. Although experimental tumor growth has been enhanced by IVH in some studies, other investigators have been unable to document tumor growth in excess of host weight gain during nutritional repletion
331
OO22-4804/81/040331-07$01 .OO/O Copyright 0 1981 by Academic Press, Inc. All rights of reproduction in any form reserved.
332
JOURNAL
OF SURGICAL
RESEARCH:
[5, 6, 10, 11, 161. Short-term IVH has not notably stimulated the growth of solid neoplasms in clinical experience, but it must be recognized that the subjects under investigation were receiving concurrent radiation therapy or chemotherapy [8]. The possibility of utilizing nutritional therapy to recruit quiescent tumor cells into an actively proliferating population as a prelude to the use of cell-cycle-specific chemotherapy has been examined; initial experimental results have demonstrated a substantial decrease in tumor growth when nutritional repletion was followed by methotrexate or hydroxyurea [7, 11, 121. The deleterious effects of malnutrition in the cancer patient include depression of systemic immunity, diminished tolerance for therapy, increased risk for therapeutic complications, and a diminished potential for therapeutic response. To our knowledge, no previous study has reported the effect of malnutrition on the distribution or metabolism of chemotherapeutic agents. We report here our investigation of the effects of dietary protein deprivation on the pharmacokinetics and distribution of Adriamycin in non-tumor-bearing rats. METHODS
All animals utilized in these experiments were male, Sprague-Dawley rats. All animals were housed in individual metabolic cages and were provided the designated diet and water ad libitum for the duration of the experiment. No attempts were made to prohibit copraphagy. The regular diet (Wayne Lab Blox, Allied Mills, Chicago, Ill,) is a nutritionally complete diet containing 24.5% protein, 4.1% soybean oil, 50% carbohydrate, minerals, and vitamins. The regular diet provides 3.97 kcal/g total energy. The protein-free diet (Teklad Test Diets, TD 70114, ARS/Sprague-Dawley, Div. of the Mogul Corp., Madison, Wise.) contains 70.8% sucrose, 15% cornstarch, 2% cod liver oil, 8% hydrogenated vegetable oil, 4% mineral mix (USP, XIV, Catalog
VOL. 30, NO. 4, APRIL
1981
1700880)and vitamins. The protein-free diet provides 4.27 kcal/g total energy. Serum Adriamycin kinetics. Sixty 200-g rats were randomly assigned to either regular diet or protein-free diet. Ten days after the initiation of the test diets, 5.0 mg/kg Adriamycin (Doxorubicin, Adria Laboratories, Columbus, Ohio) was administered intravenously to 25 animals from each dietary group. Five animals from each dietary group served as noninjected controls. Five animals from each dietary group were sacrificed at 0.5, 1, 2, 24, and 48 hr following drug administration. Blood was obtained by cardiac puncture and collected in plain glass tubes. Serum was separated by centrifugation for 10 min at room temperature, 4OOg,after clot formation and retraction. Serum specimens for Adriamycin determination were frozen at -4°C until analysis. Urinary Adriamycin excretion. Thirty 225-g rats were randomly assigned to either regular diet or protein-free diet. Ten days after dietary randomization, 5.0 m&kg Adriamycin was administered intravenously to 10 animals from each dietary group. Five animals from each dietary group served as noninjected controls. Urine for determination of quantitative Adriamycin excretion was collected in 24-h aliquots, centrifuged (10 min, room temperature, 400g) to remove debris, and frozen at -4°C until analysis. All animals were sacrificed 48 hr after drug administration. Blood was collected at sacrifice and processed as previously described for subsequent serum Adriamycin determination. Tissue Adriamycin concentration. Sixty 250-g rats were randomly assigned to either regular diet (Group I) or protein-free diet. In an effort to obtain a well-nourished dietary group of weight comparable to that of the protein-depleted group at 10 days, 30 rats weighing 140 g were also assigned a regular diet (Group II). Ten days after assignment to the designated diet, 24 animals from each dietary group received 5.0 mg/kg Adriamycin intravenously. Six
KAPELANSKI
ET AL.: DOXORUBICIN
animals from each dietary group served as noninjected controls. Six animals from each dietary group were sacrificed at 2, 6, 24, and 48 hr following drug administration. Blood was collected and processed as previously described for subsequent serum Adriamycin determination. The liver, heart, kidneys, and lungs were excised, weighed, and frozen at -4°C for determination of Adriamycin content. Urine for determination of quantitative Adriamycin excretion was collected in 24-hr aliquots from animals sacrificed at 24 and 48 hr, processed as previously described, and frozen until analysis. Adriamycin analysis. Adriamycin in serum and urine was determined using a modification of the method of Rosso et al. 1151.Onehalf milliliter of the sample was vigorously mixed with 2.5 ml n-butyl alcohol. The aqueous and organic phases separated overnight at 4°C in tightly capped tubes. The organic phase was removed and warmed to room temperature. Fluorescence of the organic phase was determined using a MK I spectrofluorometer (Farrand Optical Co., Inc., Valhalla, N. Y .). The excitation wavelength was 470 nm; the emission wavelength was 585 nm. Blank specimens were prepared from pooled urine or serum from noninjected control animals of the same dietary group, processed as described above. Adriamycin in tissue was determined using a modification of the acid alcohol method of Bachur et al. [2]. Tissue was homogenized with 5 vol cold 1.15% potassium chloride. Two milliliters of the homogenate was vigorously mixed with 3.0 ml cold acid alcohol (80% absolute ethyl alcohol, 20% concentrated hydrochloric acid, v/v). The mixture was refrigerated overnight at 4°C in tightly capped tubes. The extraction was completed by centrifugation for 15 min at 4°C 5000g. The supernatant was decanted into a clean tube and warmed to room temperature. Fluorescence of the supernatant phase was determined as noted above. Blank specimens were prepared from pooled tissue from noninjected control animals of the
333
PHARMACOKINETICS
same dietary group, processed as described. All Adriamycin determinations were completed within 1week of drug administration. Standard curves were prepared from Adriamycin dissolved in normal saline, extracted as above for serum, urine, and tissue. Statistical analysis was performed using Student’s t test. All values presented are the mean and standard deviation of the mean. RESULTS
The threshold for detection of Adriamycin was 0.01 pg/ml in serum and urine, and 0.1 (ug/gm in tissue. Fluorescence was linear with Adriamycin concentration throughout the range encountered in these experiments (data not shown). Serum Adriamycin kinetics. Animals provided a regular diet continued to gain weight during the study interval, while animals provided the protein-free diet lost an average of 21% of their initial study weight (Table 1). Although Adriamycin was administered to all animals on the basis of body weight, the serum Adriamycin concentration was significantly lower in the protein-depleted aniTABLE 1 THE EFFECTS OF DIETARY PROTEIN DEPRIVATION ON BODY WEIGHT AND SERUM ADRIAMYCIN CONCENTRATION
Initial weight (g) Final weight (g) Adriamycin dose (mg) Sacrifice time (W 0.5 1 2 24 48 * P < 0.05. **P < 0.01.
Regular diet
Protein-free diet
192 + 11 252 k 29 1.26 r 0.09
195 2 11 1.542 9** 0.76 -t 0.04**
Serum adriamycin concentration (&ml) 0.42 + 0.41 k 0.36 k 0.31 k 0.24 2
0.04 0.04 0.02 0.01 0.02
0.37 k O.Ol* 0.36 2 0.04* 0.28 2 0.03** 0.26 4 O.Ol** 0 -c o**
334
JOURNAL OF SURGICAL RESEARCH: VOL. 30, NO. 4, APRIL 1981 TABLE 2
THE EFFECTS OF DIETARY PROTEIN DEPRIVATION ON BODY WEIGHT AND URINARY ADRIAMYCIN EXCRETION
Initial weight (g) Final weight (g) Adriamycin dose (mg) Sacrifice time (hr) 24 48
Regular diet
Protein-free diet
222 5 13 295 -c 10 1.47 k 0.10
224 k 8 176 2 6** 0.86 k 0.04**
Cumulative urinary Adriamycin excretion (pg) 146 -c 26 230 -c 38
89 2 47 178 k 45
Cumulative urinary Adriamycin excretion as a percentage of administered dose 24 48
9.9 + 1.7 15.6 + 2.4
10.0 k 5.1 19.7 ” 5.2*
Serum adriamycin concentration Q.&ml) 48
0.07 % 0.01
0 ‘- o**
*P < 0.05. ** P < 0.01.
mals at all time intervals. Adriamycin was below the threshold for detection in the group fed the protein-free diet by 48 hr following administration, but was still present in the serum of the well-nourished animals. Urinary Adriamycin excretion. Animals
receiving the regular diet continued to gain weight during the experiment, while those animals receiving the protein-free diet lost an average of 22% of their initial study weight (Table 2). The cumulative urinary excretion of Adriamycin was greater at 24 and 48 hr in the animals fed the regular diet. The quantity of drug excreted as a percentage of the administered dose was identical in both groups during the initial 24-hr period, and was significantly higher in the proteindepleted animals by 48 hr. As in the previous experiment, Adriamycin was below the threshold for detection in the serum of the protein-depleted animals at 48 hr, but was still present in the serum of the regular diet group. Tissue Adriamycin concentration. Both regular diet groups continued to gain weight during the study period, with the largest proportional gain occurring in Group II animals (Table 3). The protein-free diet group lost an average of 22% of their initial weight. The concentration of Adriamycin in the liver was significantly lower in the proteinfree diet group when compared with either of the regular diet groups during the initial 6 hr after administration. These differences were no longer apparent at 48 hr (Table 4). The concentration of Adriamycin in the kidneys of the malnourished animals was significantly higher than the concentrations found in the kidneys of either regular diet group at all time intervals.
TABLE 3 BODY WEIGHT AND ADRIAMYCIN DOSE IN ANIMALS MATCHED FOR WEIGHT AT THE START OF THE EXPERIMENT AND AFTER 10 DAYS OF DIETARY PROTEIN DEPRIVATION
Initial weight (g) Final weight (g) Adriamycin dose (mg)
Regular diet (I)
Protein-free diet
Regular diet (II)
252 2 15 338 k 18** 1.69 k 0.09**
248 k 17 193 + 15 0.97 f 0.08
142 k 11** 225 ? 21** 1.12 * 0.10**
Note. In Tables 3-6, regular diet (I) animals were matched for weight with the protein-free diet animals at the onset of the experiment. Since the protein-free diet animals were anticipated to lose 20% of their initial body weight, and Adriamycin was administered on a body-weight basis, a regular diet (II) group was also studied. The regular diet (II) group was anticipated to gain weight during the 10 days of the experiment to approximate the final weight of the protein-free diet animals, which was initially much higher. Thereby a regular diet group of the approximate final weight of the protein-free diet group could be evaluated. ** P < 0.01 compared with protein-depleted animals.
KAPELANSKI
ET AL.: DOXORUBICIN TABLE
335
PHARMACOKINETICS
4
TISSUE ADRIAMYCIN CONCENTRATION @g/g) IN ANIMALS MATCHED FOR WEIGHT AT THE START OF THE EXPERIMENT AND AFTER 10 DAYS OF DIETARY PROTEIN DEPRIVATION
Tissue
Sacrifice time (hr)
Regular diet (I)
Protein-free
diet
Regular diet (II)
Liver
2 6 24 48
18.2 12.9 5.4 2.4
k 2 + 2
2.8** 2.8** 1.2** 0.4
7.5 6.8 2.9 2.2
t k + k
1.5 0.5 0.5 0.7
13.4 8.9 3.0 1.7
k t 2 +
2.7** 0.4** 0.4 0.3
Kidney
2 6 24 48
16.2 12.7 8.3 5.9
2 2.1** -+ l.l** r 0.4** f 1.6**
22.8 17.4 13.8 8.9
k k + 2
2.1 2.6 1.1 1.4
14.5 11.5 7.1 3.4
” 2 ” +
2.2** 0.7** 0.5** 1.2**
Heart
2 6 24 48
10.9 10.8 7.4 7.1
2 2 + 2
2.3 2.9 2.0 1.5
8.5 9.3 6.6 5.6
+ 2 lr k
1.9 0.8 0.9 0.5
14.6 16.0 10.5 8.5
zt ” + k
1.4** 1.9** 0.5** 1.2**
Lung
2 6 24 48
18.1 15.8 10.4 8.3
2 f 2 k
1.5 1.3 1.0** 0.3
20.2 16.1 12.3 9.0
k f ” k
2.5 2.2 0.5 0.7
14.5 13.9 9.1 7.2
-c ” k +
3.8** 0.9* 0.9** 0.4**
* P < 0.05 compared with protein-depleted ** P < 0.01 compared with protein-depleted
animals. animals.
Adriamycin content was lowest in the hearts of animals receiving the protein-free diet. The differences were not significant when Group I regular diet animals were compared with the protein-free diet group. Animals receiving the protein-free diet had higher lung Adriamycin concentrations than were found in either regular diet group. These differences were significant at all times for Group II animals, but were statistically significant for Group I animals only at 24 hr following drug administration. The serum concentration of Adriamycin was significantly higher in Group I animals at all times, when compared with the proteinfree diet group (Table 5). With a single exception (6 hr after administration), the serum Adriamycin level was also significantly higher in Group II animals, compared with the protein-depleted group. As in the previous two experiments, Adriamycin was no longer detectable in the serum of the proteindepleted animals 48 hours following drug administration.
The cumulative urinary excretion of Adriamycin was highest in Group I animals and lowest in Group II animals (Table 6). The differences between Group II animals and the protein-free diet animals were not significant. The proportion of drug excreted in the urine as a percentage of the administered dose was greatest in the protein depleted animals. These differences were not statistically significant. TABLE
5
SERUM ADRIAMYCIN CONCENTRATION (&ml) IN ANIMALS MATCHED FOR WEIGHT AT THE START OF THE EXPERIMENT AND AFTER 10 DAYS OF DIETARY PROTEIN DEPRIVATION Sacrifice time (hr)
Regular diet (0
Protein-free diet
Regular diet (II)
2 6 24 48
0.19 k 0.01** 0.13 + 0.01**
0.16 2 0.01 0.11 + 0.01 0.03 2 0.01 Ok0
0.18 t 0.01** 0.11 -c 0.01 0.06 -c 0.01** 0.01 + 0.01**
0.06 f 0.01** 0.02 c 0.01**
** P < 0.01 compared with protein-depleted animals.
336
JOURNAL OF SURGICAL RESEARCH: VOL. 30, NO. 4, APRIL 1981 TABLE 6
URINARY ADRIAMYCIN EXCRETION IN ANIMALS MATCHED FOR WEIGHT AT THE START OF THE EXPERIMENT AND AFTER 10 DAYS OF DIETARY PROTEIN DEPRIVATION
Sacrifice time (hr)
Regular diet (1)
Protein-free diet
Regular diet (11)
Cumulative excretion (pg) 24 48
203 r 28 260 + 36
125 ” 35 175 k 33
101 k 36 156 k 60
Cumulative excretion as a percentage of administered dose 24 48
12.1 + 1.3 15.5 k 1.6
13.7 -c 3.9 19.3 ? 3.6
10.0 k 3.2 15.5 k 5.2
DISCUSSION
There has been extensive research on the pharmacokinetics, tissue distribution and metabolism of Adriamycin since the discovery of its broad-spectrum antineoplastic activity. Adriamycin exhibits a tri-phasic plasma disappearance curve following a single intravenous dose, with a prolonged tertiary phase half-life [3, 41. The drug is rapidly cleared from the intravascular and extracellular compartments, and accumulates in tissues in concentrations considerably in excess of plasma levels [ 15, 171.The extensive, intercalative binding of Adriamycin to nuclear DNA interferes with DNA synthesis, and provides a substantial intracellular reservoir of the drug [ 11. Adriamycin and related analogs undergo extensive metabolism; biliary excretion accounts for more than 60% of the recoverable drug, while urinary elimination is estimated at between 5 and 15% [4, 171. The prolonged tissue retention of Adriamycin provides the basis for the safety and efficacy of intermittent administration schedules. Although Adriamycin would be expected to interfere primarily with the S phase of cell replication, many neoplastic cells will pass through their most sensitive phase during the interval when tissue and
plasma drug concentrations remain elevated [4]. The fluorescent assay utilized in this study cannot discriminate Adriamycin from metabolites with an intact anthracycline ring. Although Adriamycinol, a prominent metabolite of Adriamycin, exhibits activity similar to that of the parent compound, the role of the various metabolites in either the antineoplastic activity or toxicity of the drug remains speculative [ 1, 31. Further investigation will be required to determine if Adriamycin metabolism is significantly altered by malnutrition. The concentration and distribution of Adriamycin observed in this study are in general agreement with the results reported by other investigators [15, 171. Animals maintained on the protein-free diet exhibited significant alterations in drug concentration and distribution when compared with the well-nourished groups. The most important of these differences was the complete absence of detectable drug in the serum of the protein-depleted animals within 48 hr of administration, when Adriamycin was still detectable in the serum of the wellnourished animals. One plausible mechanism for the more rapid serum elimination might be accelerated renal clearance. Adriamycin is approximately 50% bound to plasma proteins [ 141. A diminished plasma protein concentration in the protein-deprived animals may allow a greater proportion of nonbound drug in plasma following administration, which would facilitate glomerular filtration of the drug. The elevated Adriamycin concentration in the kidneys of the protein-free diet animals tends to corroborate this hypothesis, as does the increased proportion of drug found in the urine of the malnourished animals. The role of hepatic elimination in the more rapid serum clearance of Adriamycin is unknown; hepatic drug concentrations were initially lower in the malnourished animals, but there were no significant differences by 48 hr after drug administration. Direct biliary cannulation would be necessary to resolve the possibility of altered hepatic excretion, since in-
KAPELANSKI
ET AL.: DOXORUBICIN
testinal flora destroy the anthracycline ring of Adriamycin and render the spectrofluorometric assay invalid. Since the principal, dose-limiting toxicity of Adriamycin is progressive cardiac dysfunction, it is of interest that the proteindeprived group exhibited lower myocardial drug concentrations at all times. The significance of this observation to clinical drug use is uncertain, as malnutrition may alter myocardial performance and cellular repair mechanisms. This area is under current investigation. Pathologic alteration in the pharmacokinetics of Adriamycin is not without precedent. Hepatic dysfunction results in delayed elimination of the drug and its metabolites; failure to reduce the dosage of Adriamycin in patients with severe hepatic derangement resulted in a four- to fivefold elevation in the plasma drug concentration, and was associated with a prohibitive morbidity and mortality [4]. Renal impairment appears to exhibit minimal effect on drug toxicity, presumably a function of the relatively minor role of the kidneys in drug elimination. Malnutrition is a frequent finding in individuals with malignant disease, and is a preventable complication of oncologic therapy. Although we are unable, on the basis of this study, to speculate on either the toxicity or therapeutic efficacy of Adriamycin in the malnourished cancer patient, our findings in non-tumor-bearing rats indicate that further investigation of the effects of malnutrition on the pharmacokinetics, metabolism, and distribution of chemotherapeutic drugs is warranted. REFERENCES 1. Bachur, N. R. Anthracycline antibiotic pharmacology and metabolism. Cancer Treat. Rep. 63: 817, 1979. 2. Bachur, N. R., Moore, L. E., Bernstein, J. G., and Liu, A. Tissue distribution and disposition of Daunomycin in mice: Fluorometric and isotopic methods. Cancer Chemother. Rep. 54: 89, 1970. 3. Benjamin, R. S., Riggs, C. E., and Bachur, N. R. Plasma pharmacokinetics of Adriamycin and its
PHARMACOKINETICS
331
metabolites in humans with normal hepatic and renal function. Cancer Res. 37: 1416, 1977. 4. Benjamin, R. S., Wiemik, P. H., and Bachur, N. R. Adriamycin chemotherapy-Efficacy, safety and pharmacologic basis of an intermittent, highdosage schedule. Cancer 33: 19, 1974. 5. Cameron, I. L., Ackley, W. J., and Rogers, W. Responses of hepatoma-bearing rats to total parenteral hyperalimentation and to ad libitum feeding. J. Surg. Res. 23: 189, 1977. 6. Cameron, I. L., and Pavlat, W. A. Stimulation of growth of a transplantable hepatoma in rats by parenteral nutrition. J. Natl. Cancer Inst. 56: 597, 1976. 7. Cameron, I. L., and Rogers, W. Total intravenous hyperalimentation and hydroxyurea chemotherapy in hepatoma-bearing rats. J. Surg. Res., 23: 279, 1977. 8. Copeland, E. M., Daly, J. M., and Dudrick, S. J. Nutrition as an adjunct to cancer treatment in the adult. Cancer Res. 37: 2451, 1977. 9. Copeland, E. M., MacFadyen, B. V., and Dudrick, S. J. Effect of intravenous hyperalimentation on established delayed hypersensitivity in the cancer patient. Ann. Surg. 184: 60, 1976. 10. Daly, J. M., Copeland, E. M., and Dudrick, S. J. Effects of intravenous nutrition on tumor growth and host immunocompetence in malnourished animals. Surgery 84: 655, 1978. 11. Daly, J. M., Reynolds, H. M., Dudrick, S. J., and Copeland, E. M. Effects of nutritional repletion on host and tumor response to chemotherapy. Curr. Surg. 2: 138, 1979. 12. Daly, J. M., Reynolds, H. M., Rowlands, B. J., Dudrick, S. J., and Copeland, E. M. Tumor growth in experimental animals-Nutritional manipulation and chemotherapeutic response in the rat. Ann. Surg. 191: 316, 1980. 13. Dudrick, S. J. Artificial Feeding Techniques. Presented at the Workshop on Diet and Nutrition in the Therapy and Rehabilitation of the Cancer Patient, March 26, 1975. Bethesda, Md: National Institutes of Health. 14. Harris, P. A., and Gross, J. F. Preliminary pharmacokinetic model for Adriamycin. Cancer Chemother. Rep. 59: 819, 1975. 15. Rosso, R., Esposito, M., Sala, R., and Santi, L. Distribution of Daunomycin and Adriamycin in mice-A comparative study. Biomedicine 19: 304, 1973. 16. Steiger, E., Oram-Smith, J., Miller, E., Kuo, L., and Vars, H. M. Effects of nutrition on tumor growth and tolerance to chemotherapy. J. Surg. Res. 18: 455, 1975. 17. Yesair, D. W., Schwartzbach, E., Shuck, D., Denine, E. P., and Asbell, M. A. Comparative pharmacokinetics of Daunomycin and Adriamycin in several animal species. Cancer Res. 32: 1177, 1972.
OF SURGICAL
Doxorubicin DAVID
RESEARCH
30,331-337 (1981)
Pharmacokinetics
P. KAPELANSKI,
-The
Effects of Protein
Deprivation
M.D., JOHN M. DALY, M.D., EDWARD M. COPELAND, III, M.D., AND STANLEY J. DUDRICK, M.D.
University of Texas Medical School at Houston and University of Texas System Cancer Center. M.D. Anderson Hospital and Tumor Institute, Texas Medical Center, Houston, Te.ras 77030
Presented at the Annual Meeting of the Association for Academic Surgery, Birmingham, Alabama, November 5-8, 1980 Recent studies suggest that protein-calorie malnutrition decreases tumor response to chemotherapy. This study was designed to evaluate the effects of dietary protein deprivation on the pharmacokinetics and distribution of Adriamycin. Male Sprague-Dawley rats were assigned randomly to receive either a regular diet or a high-carbohydrate, protein-free diet for the duration of the experiment. Ten days after the initiation of the designated diet, all animals received a single dose (5.0 mg/kg) of Adriamycin intravenously. Animals were sacrificed at 0.5, 1, 2,6, 24, and 48 hr following drug administration, and the concentration of Adriamycin in serum, urine, and tissue was determined by fluorometric assay. Animals maintained on the regular diet continued to gain weight during the study interval, while animals provided the protein-free diet lost an average of 21% of their initial study weight (P < 0.01). Serum Adriamycin concentration was significantly lower in the protein-free diet animals at 0.5, 1, 2, 24, and 48 hr following administration; Adriamycin was not detectable in the serum of the protein-free diet animals at 48 hr, but was still present in the serum of the regular diet group (P < 0.01). The cumulative urinary excretion of Adriamycin, expressed as a percentage of the administered dose, was identical in both dietary groups during the initial 24 hr, but was significantly higher in the protein-free diet group by 48 hr after administration (P < 0.05). Significant differences were observed when liver, kidney, lung, and heart Adriamycin concentrations were compared. The elevated concentration of Adriamycin in the kidneys of the protein-free diet animals correlated with the increased urinary drug excretion, and indicated that accelerated renal clearance of Adriamycin was one possible mechanism for the more rapid serum elimination of the drug in the protein-free diet animals.
INTRODUCTION
Numerous clinical and experimental observations support the use of both enteral and parenteral alimentation techniques as an adjunct to the therapy of the patient with disseminated malignancy. Intravenous hyperalimentation (IVH) has permitted patients, at increased risk of complications secondary to inanition, to complete full courses of radiation therapy and chemotherapy successfully, with minimal morbidity [8]. Despite concurrent chemotherapy, many patients converted from a state of anergy to one of normal reactivity toward a panel of recall antigens; tumor regression was documented only in those patients with normal cutaneous delayed hypersensitivity [9]. In
that same study, no subject receiving radiation therapy developed or maintained delayed hypersensitivity. IVH was, however, thought to contribute to the general well being of the patients and to a conspicuous absence of therapeutic morbidity. The concomitant administration of IVH to patients receiving cyclic, multiple-agent chemotherapy was associated with an accelerated recovery from the gastrointestinal toxicity, but the incidence and severity of the hematopoietic toxicity were unaltered [13]. Although experimental tumor growth has been enhanced by IVH in some studies, other investigators have been unable to document tumor growth in excess of host weight gain during nutritional repletion
331
OO22-4804/81/040331-07$01 .OO/O Copyright 0 1981 by Academic Press, Inc. All rights of reproduction in any form reserved.
332
JOURNAL
OF SURGICAL
RESEARCH:
[5, 6, 10, 11, 161. Short-term IVH has not notably stimulated the growth of solid neoplasms in clinical experience, but it must be recognized that the subjects under investigation were receiving concurrent radiation therapy or chemotherapy [8]. The possibility of utilizing nutritional therapy to recruit quiescent tumor cells into an actively proliferating population as a prelude to the use of cell-cycle-specific chemotherapy has been examined; initial experimental results have demonstrated a substantial decrease in tumor growth when nutritional repletion was followed by methotrexate or hydroxyurea [7, 11, 121. The deleterious effects of malnutrition in the cancer patient include depression of systemic immunity, diminished tolerance for therapy, increased risk for therapeutic complications, and a diminished potential for therapeutic response. To our knowledge, no previous study has reported the effect of malnutrition on the distribution or metabolism of chemotherapeutic agents. We report here our investigation of the effects of dietary protein deprivation on the pharmacokinetics and distribution of Adriamycin in non-tumor-bearing rats. METHODS
All animals utilized in these experiments were male, Sprague-Dawley rats. All animals were housed in individual metabolic cages and were provided the designated diet and water ad libitum for the duration of the experiment. No attempts were made to prohibit copraphagy. The regular diet (Wayne Lab Blox, Allied Mills, Chicago, Ill,) is a nutritionally complete diet containing 24.5% protein, 4.1% soybean oil, 50% carbohydrate, minerals, and vitamins. The regular diet provides 3.97 kcal/g total energy. The protein-free diet (Teklad Test Diets, TD 70114, ARS/Sprague-Dawley, Div. of the Mogul Corp., Madison, Wise.) contains 70.8% sucrose, 15% cornstarch, 2% cod liver oil, 8% hydrogenated vegetable oil, 4% mineral mix (USP, XIV, Catalog
VOL. 30, NO. 4, APRIL
1981
1700880)and vitamins. The protein-free diet provides 4.27 kcal/g total energy. Serum Adriamycin kinetics. Sixty 200-g rats were randomly assigned to either regular diet or protein-free diet. Ten days after the initiation of the test diets, 5.0 mg/kg Adriamycin (Doxorubicin, Adria Laboratories, Columbus, Ohio) was administered intravenously to 25 animals from each dietary group. Five animals from each dietary group served as noninjected controls. Five animals from each dietary group were sacrificed at 0.5, 1, 2, 24, and 48 hr following drug administration. Blood was obtained by cardiac puncture and collected in plain glass tubes. Serum was separated by centrifugation for 10 min at room temperature, 4OOg,after clot formation and retraction. Serum specimens for Adriamycin determination were frozen at -4°C until analysis. Urinary Adriamycin excretion. Thirty 225-g rats were randomly assigned to either regular diet or protein-free diet. Ten days after dietary randomization, 5.0 m&kg Adriamycin was administered intravenously to 10 animals from each dietary group. Five animals from each dietary group served as noninjected controls. Urine for determination of quantitative Adriamycin excretion was collected in 24-h aliquots, centrifuged (10 min, room temperature, 400g) to remove debris, and frozen at -4°C until analysis. All animals were sacrificed 48 hr after drug administration. Blood was collected at sacrifice and processed as previously described for subsequent serum Adriamycin determination. Tissue Adriamycin concentration. Sixty 250-g rats were randomly assigned to either regular diet (Group I) or protein-free diet. In an effort to obtain a well-nourished dietary group of weight comparable to that of the protein-depleted group at 10 days, 30 rats weighing 140 g were also assigned a regular diet (Group II). Ten days after assignment to the designated diet, 24 animals from each dietary group received 5.0 mg/kg Adriamycin intravenously. Six
KAPELANSKI
ET AL.: DOXORUBICIN
animals from each dietary group served as noninjected controls. Six animals from each dietary group were sacrificed at 2, 6, 24, and 48 hr following drug administration. Blood was collected and processed as previously described for subsequent serum Adriamycin determination. The liver, heart, kidneys, and lungs were excised, weighed, and frozen at -4°C for determination of Adriamycin content. Urine for determination of quantitative Adriamycin excretion was collected in 24-hr aliquots from animals sacrificed at 24 and 48 hr, processed as previously described, and frozen until analysis. Adriamycin analysis. Adriamycin in serum and urine was determined using a modification of the method of Rosso et al. 1151.Onehalf milliliter of the sample was vigorously mixed with 2.5 ml n-butyl alcohol. The aqueous and organic phases separated overnight at 4°C in tightly capped tubes. The organic phase was removed and warmed to room temperature. Fluorescence of the organic phase was determined using a MK I spectrofluorometer (Farrand Optical Co., Inc., Valhalla, N. Y .). The excitation wavelength was 470 nm; the emission wavelength was 585 nm. Blank specimens were prepared from pooled urine or serum from noninjected control animals of the same dietary group, processed as described above. Adriamycin in tissue was determined using a modification of the acid alcohol method of Bachur et al. [2]. Tissue was homogenized with 5 vol cold 1.15% potassium chloride. Two milliliters of the homogenate was vigorously mixed with 3.0 ml cold acid alcohol (80% absolute ethyl alcohol, 20% concentrated hydrochloric acid, v/v). The mixture was refrigerated overnight at 4°C in tightly capped tubes. The extraction was completed by centrifugation for 15 min at 4°C 5000g. The supernatant was decanted into a clean tube and warmed to room temperature. Fluorescence of the supernatant phase was determined as noted above. Blank specimens were prepared from pooled tissue from noninjected control animals of the
333
PHARMACOKINETICS
same dietary group, processed as described. All Adriamycin determinations were completed within 1week of drug administration. Standard curves were prepared from Adriamycin dissolved in normal saline, extracted as above for serum, urine, and tissue. Statistical analysis was performed using Student’s t test. All values presented are the mean and standard deviation of the mean. RESULTS
The threshold for detection of Adriamycin was 0.01 pg/ml in serum and urine, and 0.1 (ug/gm in tissue. Fluorescence was linear with Adriamycin concentration throughout the range encountered in these experiments (data not shown). Serum Adriamycin kinetics. Animals provided a regular diet continued to gain weight during the study interval, while animals provided the protein-free diet lost an average of 21% of their initial study weight (Table 1). Although Adriamycin was administered to all animals on the basis of body weight, the serum Adriamycin concentration was significantly lower in the protein-depleted aniTABLE 1 THE EFFECTS OF DIETARY PROTEIN DEPRIVATION ON BODY WEIGHT AND SERUM ADRIAMYCIN CONCENTRATION
Initial weight (g) Final weight (g) Adriamycin dose (mg) Sacrifice time (W 0.5 1 2 24 48 * P < 0.05. **P < 0.01.
Regular diet
Protein-free diet
192 + 11 252 k 29 1.26 r 0.09
195 2 11 1.542 9** 0.76 -t 0.04**
Serum adriamycin concentration (&ml) 0.42 + 0.41 k 0.36 k 0.31 k 0.24 2
0.04 0.04 0.02 0.01 0.02
0.37 k O.Ol* 0.36 2 0.04* 0.28 2 0.03** 0.26 4 O.Ol** 0 -c o**
334
JOURNAL OF SURGICAL RESEARCH: VOL. 30, NO. 4, APRIL 1981 TABLE 2
THE EFFECTS OF DIETARY PROTEIN DEPRIVATION ON BODY WEIGHT AND URINARY ADRIAMYCIN EXCRETION
Initial weight (g) Final weight (g) Adriamycin dose (mg) Sacrifice time (hr) 24 48
Regular diet
Protein-free diet
222 5 13 295 -c 10 1.47 k 0.10
224 k 8 176 2 6** 0.86 k 0.04**
Cumulative urinary Adriamycin excretion (pg) 146 -c 26 230 -c 38
89 2 47 178 k 45
Cumulative urinary Adriamycin excretion as a percentage of administered dose 24 48
9.9 + 1.7 15.6 + 2.4
10.0 k 5.1 19.7 ” 5.2*
Serum adriamycin concentration Q.&ml) 48
0.07 % 0.01
0 ‘- o**
*P < 0.05. ** P < 0.01.
mals at all time intervals. Adriamycin was below the threshold for detection in the group fed the protein-free diet by 48 hr following administration, but was still present in the serum of the well-nourished animals. Urinary Adriamycin excretion. Animals
receiving the regular diet continued to gain weight during the experiment, while those animals receiving the protein-free diet lost an average of 22% of their initial study weight (Table 2). The cumulative urinary excretion of Adriamycin was greater at 24 and 48 hr in the animals fed the regular diet. The quantity of drug excreted as a percentage of the administered dose was identical in both groups during the initial 24-hr period, and was significantly higher in the proteindepleted animals by 48 hr. As in the previous experiment, Adriamycin was below the threshold for detection in the serum of the protein-depleted animals at 48 hr, but was still present in the serum of the regular diet group. Tissue Adriamycin concentration. Both regular diet groups continued to gain weight during the study period, with the largest proportional gain occurring in Group II animals (Table 3). The protein-free diet group lost an average of 22% of their initial weight. The concentration of Adriamycin in the liver was significantly lower in the proteinfree diet group when compared with either of the regular diet groups during the initial 6 hr after administration. These differences were no longer apparent at 48 hr (Table 4). The concentration of Adriamycin in the kidneys of the malnourished animals was significantly higher than the concentrations found in the kidneys of either regular diet group at all time intervals.
TABLE 3 BODY WEIGHT AND ADRIAMYCIN DOSE IN ANIMALS MATCHED FOR WEIGHT AT THE START OF THE EXPERIMENT AND AFTER 10 DAYS OF DIETARY PROTEIN DEPRIVATION
Initial weight (g) Final weight (g) Adriamycin dose (mg)
Regular diet (I)
Protein-free diet
Regular diet (II)
252 2 15 338 k 18** 1.69 k 0.09**
248 k 17 193 + 15 0.97 f 0.08
142 k 11** 225 ? 21** 1.12 * 0.10**
Note. In Tables 3-6, regular diet (I) animals were matched for weight with the protein-free diet animals at the onset of the experiment. Since the protein-free diet animals were anticipated to lose 20% of their initial body weight, and Adriamycin was administered on a body-weight basis, a regular diet (II) group was also studied. The regular diet (II) group was anticipated to gain weight during the 10 days of the experiment to approximate the final weight of the protein-free diet animals, which was initially much higher. Thereby a regular diet group of the approximate final weight of the protein-free diet group could be evaluated. ** P < 0.01 compared with protein-depleted animals.
KAPELANSKI
ET AL.: DOXORUBICIN TABLE
335
PHARMACOKINETICS
4
TISSUE ADRIAMYCIN CONCENTRATION @g/g) IN ANIMALS MATCHED FOR WEIGHT AT THE START OF THE EXPERIMENT AND AFTER 10 DAYS OF DIETARY PROTEIN DEPRIVATION
Tissue
Sacrifice time (hr)
Regular diet (I)
Protein-free
diet
Regular diet (II)
Liver
2 6 24 48
18.2 12.9 5.4 2.4
k 2 + 2
2.8** 2.8** 1.2** 0.4
7.5 6.8 2.9 2.2
t k + k
1.5 0.5 0.5 0.7
13.4 8.9 3.0 1.7
k t 2 +
2.7** 0.4** 0.4 0.3
Kidney
2 6 24 48
16.2 12.7 8.3 5.9
2 2.1** -+ l.l** r 0.4** f 1.6**
22.8 17.4 13.8 8.9
k k + 2
2.1 2.6 1.1 1.4
14.5 11.5 7.1 3.4
” 2 ” +
2.2** 0.7** 0.5** 1.2**
Heart
2 6 24 48
10.9 10.8 7.4 7.1
2 2 + 2
2.3 2.9 2.0 1.5
8.5 9.3 6.6 5.6
+ 2 lr k
1.9 0.8 0.9 0.5
14.6 16.0 10.5 8.5
zt ” + k
1.4** 1.9** 0.5** 1.2**
Lung
2 6 24 48
18.1 15.8 10.4 8.3
2 f 2 k
1.5 1.3 1.0** 0.3
20.2 16.1 12.3 9.0
k f ” k
2.5 2.2 0.5 0.7
14.5 13.9 9.1 7.2
-c ” k +
3.8** 0.9* 0.9** 0.4**
* P < 0.05 compared with protein-depleted ** P < 0.01 compared with protein-depleted
animals. animals.
Adriamycin content was lowest in the hearts of animals receiving the protein-free diet. The differences were not significant when Group I regular diet animals were compared with the protein-free diet group. Animals receiving the protein-free diet had higher lung Adriamycin concentrations than were found in either regular diet group. These differences were significant at all times for Group II animals, but were statistically significant for Group I animals only at 24 hr following drug administration. The serum concentration of Adriamycin was significantly higher in Group I animals at all times, when compared with the proteinfree diet group (Table 5). With a single exception (6 hr after administration), the serum Adriamycin level was also significantly higher in Group II animals, compared with the protein-depleted group. As in the previous two experiments, Adriamycin was no longer detectable in the serum of the proteindepleted animals 48 hours following drug administration.
The cumulative urinary excretion of Adriamycin was highest in Group I animals and lowest in Group II animals (Table 6). The differences between Group II animals and the protein-free diet animals were not significant. The proportion of drug excreted in the urine as a percentage of the administered dose was greatest in the protein depleted animals. These differences were not statistically significant. TABLE
5
SERUM ADRIAMYCIN CONCENTRATION (&ml) IN ANIMALS MATCHED FOR WEIGHT AT THE START OF THE EXPERIMENT AND AFTER 10 DAYS OF DIETARY PROTEIN DEPRIVATION Sacrifice time (hr)
Regular diet (0
Protein-free diet
Regular diet (II)
2 6 24 48
0.19 k 0.01** 0.13 + 0.01**
0.16 2 0.01 0.11 + 0.01 0.03 2 0.01 Ok0
0.18 t 0.01** 0.11 -c 0.01 0.06 -c 0.01** 0.01 + 0.01**
0.06 f 0.01** 0.02 c 0.01**
** P < 0.01 compared with protein-depleted animals.
336
JOURNAL OF SURGICAL RESEARCH: VOL. 30, NO. 4, APRIL 1981 TABLE 6
URINARY ADRIAMYCIN EXCRETION IN ANIMALS MATCHED FOR WEIGHT AT THE START OF THE EXPERIMENT AND AFTER 10 DAYS OF DIETARY PROTEIN DEPRIVATION
Sacrifice time (hr)
Regular diet (1)
Protein-free diet
Regular diet (11)
Cumulative excretion (pg) 24 48
203 r 28 260 + 36
125 ” 35 175 k 33
101 k 36 156 k 60
Cumulative excretion as a percentage of administered dose 24 48
12.1 + 1.3 15.5 k 1.6
13.7 -c 3.9 19.3 ? 3.6
10.0 k 3.2 15.5 k 5.2
DISCUSSION
There has been extensive research on the pharmacokinetics, tissue distribution and metabolism of Adriamycin since the discovery of its broad-spectrum antineoplastic activity. Adriamycin exhibits a tri-phasic plasma disappearance curve following a single intravenous dose, with a prolonged tertiary phase half-life [3, 41. The drug is rapidly cleared from the intravascular and extracellular compartments, and accumulates in tissues in concentrations considerably in excess of plasma levels [ 15, 171.The extensive, intercalative binding of Adriamycin to nuclear DNA interferes with DNA synthesis, and provides a substantial intracellular reservoir of the drug [ 11. Adriamycin and related analogs undergo extensive metabolism; biliary excretion accounts for more than 60% of the recoverable drug, while urinary elimination is estimated at between 5 and 15% [4, 171. The prolonged tissue retention of Adriamycin provides the basis for the safety and efficacy of intermittent administration schedules. Although Adriamycin would be expected to interfere primarily with the S phase of cell replication, many neoplastic cells will pass through their most sensitive phase during the interval when tissue and
plasma drug concentrations remain elevated [4]. The fluorescent assay utilized in this study cannot discriminate Adriamycin from metabolites with an intact anthracycline ring. Although Adriamycinol, a prominent metabolite of Adriamycin, exhibits activity similar to that of the parent compound, the role of the various metabolites in either the antineoplastic activity or toxicity of the drug remains speculative [ 1, 31. Further investigation will be required to determine if Adriamycin metabolism is significantly altered by malnutrition. The concentration and distribution of Adriamycin observed in this study are in general agreement with the results reported by other investigators [15, 171. Animals maintained on the protein-free diet exhibited significant alterations in drug concentration and distribution when compared with the well-nourished groups. The most important of these differences was the complete absence of detectable drug in the serum of the protein-depleted animals within 48 hr of administration, when Adriamycin was still detectable in the serum of the wellnourished animals. One plausible mechanism for the more rapid serum elimination might be accelerated renal clearance. Adriamycin is approximately 50% bound to plasma proteins [ 141. A diminished plasma protein concentration in the protein-deprived animals may allow a greater proportion of nonbound drug in plasma following administration, which would facilitate glomerular filtration of the drug. The elevated Adriamycin concentration in the kidneys of the protein-free diet animals tends to corroborate this hypothesis, as does the increased proportion of drug found in the urine of the malnourished animals. The role of hepatic elimination in the more rapid serum clearance of Adriamycin is unknown; hepatic drug concentrations were initially lower in the malnourished animals, but there were no significant differences by 48 hr after drug administration. Direct biliary cannulation would be necessary to resolve the possibility of altered hepatic excretion, since in-
KAPELANSKI
ET AL.: DOXORUBICIN
testinal flora destroy the anthracycline ring of Adriamycin and render the spectrofluorometric assay invalid. Since the principal, dose-limiting toxicity of Adriamycin is progressive cardiac dysfunction, it is of interest that the proteindeprived group exhibited lower myocardial drug concentrations at all times. The significance of this observation to clinical drug use is uncertain, as malnutrition may alter myocardial performance and cellular repair mechanisms. This area is under current investigation. Pathologic alteration in the pharmacokinetics of Adriamycin is not without precedent. Hepatic dysfunction results in delayed elimination of the drug and its metabolites; failure to reduce the dosage of Adriamycin in patients with severe hepatic derangement resulted in a four- to fivefold elevation in the plasma drug concentration, and was associated with a prohibitive morbidity and mortality [4]. Renal impairment appears to exhibit minimal effect on drug toxicity, presumably a function of the relatively minor role of the kidneys in drug elimination. Malnutrition is a frequent finding in individuals with malignant disease, and is a preventable complication of oncologic therapy. Although we are unable, on the basis of this study, to speculate on either the toxicity or therapeutic efficacy of Adriamycin in the malnourished cancer patient, our findings in non-tumor-bearing rats indicate that further investigation of the effects of malnutrition on the pharmacokinetics, metabolism, and distribution of chemotherapeutic drugs is warranted. REFERENCES 1. Bachur, N. R. Anthracycline antibiotic pharmacology and metabolism. Cancer Treat. Rep. 63: 817, 1979. 2. Bachur, N. R., Moore, L. E., Bernstein, J. G., and Liu, A. Tissue distribution and disposition of Daunomycin in mice: Fluorometric and isotopic methods. Cancer Chemother. Rep. 54: 89, 1970. 3. Benjamin, R. S., Riggs, C. E., and Bachur, N. R. Plasma pharmacokinetics of Adriamycin and its
PHARMACOKINETICS
331
metabolites in humans with normal hepatic and renal function. Cancer Res. 37: 1416, 1977. 4. Benjamin, R. S., Wiemik, P. H., and Bachur, N. R. Adriamycin chemotherapy-Efficacy, safety and pharmacologic basis of an intermittent, highdosage schedule. Cancer 33: 19, 1974. 5. Cameron, I. L., Ackley, W. J., and Rogers, W. Responses of hepatoma-bearing rats to total parenteral hyperalimentation and to ad libitum feeding. J. Surg. Res. 23: 189, 1977. 6. Cameron, I. L., and Pavlat, W. A. Stimulation of growth of a transplantable hepatoma in rats by parenteral nutrition. J. Natl. Cancer Inst. 56: 597, 1976. 7. Cameron, I. L., and Rogers, W. Total intravenous hyperalimentation and hydroxyurea chemotherapy in hepatoma-bearing rats. J. Surg. Res., 23: 279, 1977. 8. Copeland, E. M., Daly, J. M., and Dudrick, S. J. Nutrition as an adjunct to cancer treatment in the adult. Cancer Res. 37: 2451, 1977. 9. Copeland, E. M., MacFadyen, B. V., and Dudrick, S. J. Effect of intravenous hyperalimentation on established delayed hypersensitivity in the cancer patient. Ann. Surg. 184: 60, 1976. 10. Daly, J. M., Copeland, E. M., and Dudrick, S. J. Effects of intravenous nutrition on tumor growth and host immunocompetence in malnourished animals. Surgery 84: 655, 1978. 11. Daly, J. M., Reynolds, H. M., Dudrick, S. J., and Copeland, E. M. Effects of nutritional repletion on host and tumor response to chemotherapy. Curr. Surg. 2: 138, 1979. 12. Daly, J. M., Reynolds, H. M., Rowlands, B. J., Dudrick, S. J., and Copeland, E. M. Tumor growth in experimental animals-Nutritional manipulation and chemotherapeutic response in the rat. Ann. Surg. 191: 316, 1980. 13. Dudrick, S. J. Artificial Feeding Techniques. Presented at the Workshop on Diet and Nutrition in the Therapy and Rehabilitation of the Cancer Patient, March 26, 1975. Bethesda, Md: National Institutes of Health. 14. Harris, P. A., and Gross, J. F. Preliminary pharmacokinetic model for Adriamycin. Cancer Chemother. Rep. 59: 819, 1975. 15. Rosso, R., Esposito, M., Sala, R., and Santi, L. Distribution of Daunomycin and Adriamycin in mice-A comparative study. Biomedicine 19: 304, 1973. 16. Steiger, E., Oram-Smith, J., Miller, E., Kuo, L., and Vars, H. M. Effects of nutrition on tumor growth and tolerance to chemotherapy. J. Surg. Res. 18: 455, 1975. 17. Yesair, D. W., Schwartzbach, E., Shuck, D., Denine, E. P., and Asbell, M. A. Comparative pharmacokinetics of Daunomycin and Adriamycin in several animal species. Cancer Res. 32: 1177, 1972.