Daunorubicin and adriamycin metabolism in the golden Syrian hamster

I~lOCHEhIICAL

8, 352461

MEDIC10

Daunorubicin

(197.3)

and

Adriamycin

in the Golden NICHOLAS AND

Biochemistry National

Metabolism

Syrian

Hamster

R. BACHUR, MERRILL J. EGORIN, ROBERT C. HILDEBRAND

Section, lnstitutes

Baltimore Cancer of Health, 3100 Baltimore, Maryland

Received

November

Besea Wyman 21311 20,

Center, NCI, Park Driz;e,

1972

Several mammalian species are reported to metabolize the cancer chemotherapeutic antibiotics daunorubicin ( l-6) and adriamycin (3, 7, 8, 14) in uivo. Since the toxic effects that accompany clinical usage of these anthracycline glycosides may be attributable to drug metabolites, the pharmacodynamics of these compounds need to be clarified. Reports on the disposition of these drugs in mammals differ. Yesair et al. (4) found only a single metabolite of daunorubicin and no adriamycin metabolites in tissues of drug-treated rats, mice, and dogs, but found aglycone metabolites in the livers of daunorubicin-treated hamsters. Herman and co-workers (3, 14) reported that hamsters or monkeys treated with either daunorubicin or adriamycin had high tissue levels of aglycone metabolites. In our earlier work on rats (6, 8), we detected significant levels of metabolites of both daunorubicin and adriamycin. However, the aglycone levels that we observed in the rat tissues were much lower than reported for hamsters ( 3, 14). Because of these differences and because of the implication that aglycone production may be responsible for cardiotoxicity (9)) we felt that the in uivo metabolism of the anthracycline glycosides by hamsters should be studied further. METHODS

Daunorubicin hydrochloride and adriamycin hydrochloride, in clinical injection form, were obtained from the Drug Development Branch, Drug Research and Development, National Cancer Institute. Bulk adriamycin HCI and daunorubicin HCI provided by Farmitalia, Milan, Italy were purified as previously described (10) for use as analytical standards. All other chemicals were analytical grade, and water was deionized and Copyright All rights

@ 1973 by Academic Press, of reproduction in any form

352 Inc. reserved

DAUNORUBICIN

AND

ADRIAMYCIN

METABOLISM

353

distilled, Male Golden Syrian hamsters (100-150 g) were housed under laboratory conditions for 1 wk or longer prior to experimentation and provided with a Purina chow diet and water ad lib. Animal

Preparation

Animals were lightly anesthetized with diethyl ether, weighed, and the left femoral vein isolated. Drug, dissolved in 0.9% NaCl (5 or 10 mg/ml) in a volume not exceeding 0.6 ml, was injected iv over a period of 30 see via a 32-gauge needle, Following injection, the incision was closed with Michel wound clips, and the animal was allowed to recover from anesthesia in an individual cage until time of sacrifice. Paired animals were used for each sampling. The dosage of daunorubicin HCl was 25 mg/kg and that of adriamycin HCl was 5 mg/ kg (see Results).

At selected times postinjection, animals were sacrificed by a blow to the neck. Heart, lung, liver, and kidney were rapidly excised, and up to 1 g of each tissue was immediately homogenized with 20 volumes of ice cold chloroform : methanol ( 2 : I ) in a Sorvall Omni-Mixer for 2 min. Tissue homogenates were then filtered through glass wool, and the clear filtrates were evaporated to dryness under a N, stream and used for drug analysis. Hepaiinized blood samples were obtained from the sacrificed animals, and plasma was separated by centrifugation. The total drug analysis of the plasma was performed as previously described (1). In the postmortem studies, drug-treated hamsters were sacrificed 15 min postinjection. This time interval was reported to produce peak drug levels in the tissues (3). Tissues were removed as described above but were held at room temperature up to 60 min prior to extraction, For isolation and quantification of the drugs and their metabolites, the dried tissue extracts were resuspended in 0.5 ml of absolute methanol or a chloroform: methanol ( 2: 1) mixture and were analyzed by ascending thin-layer chromatography and spectrophotofluorometry as previously described ( 6 ) . The volumetric proportions of the chromatographic solvent systems used for separation and characterization of the drugs and their metabolites were as follows: System I, chloroform, methanol, water (80,20,3); System II, chloroform, methanol, glacial acetic acid, water (80,20,14,6); System III, chloroform, methanol, glacial acetic acid (IOO,2,5); System IV, chloroform, methanol, glacial acetic acid (100,2,2.5); System V. benzene, ethyl acetate, pyridine (75,25,2),

354 Metabolite

BACHUH

Isolation

ET

AL.

and Characterization

Identification of the fluorescent metabolites was achieved by comparison with known enzymatically prepared metabolites (11, 12). When sufficient quantities of metabolites were available, attempts were made to isolate and purify them for chemical analysis. Tissue extracts were pooled and chromatographed on 1 mm silica gel H thick layer plates in System I. The fluorescent bands were scraped from the plate and eluted with methanol. Eluates were evaporated to dryness under N,, resuspended in methanol, and rechromatographed with final purification achieved on 250 Frn silica gel G plates in System 1. Mass spectrograms were obtained on an LKB Model 9000 mass spectrometer. The ion source was held at 270°C while the ionizing current was 20 ,uA and 70 V. RESULTS

After the intravenous injection of daunorubicin, peak total drug fluorescence occurred at 10 or 15 min in the tissues (Table 1). The kidney had both the highest peak drug level and the most rapid decrease in total drug level over the observation period of 60 min. Total drug fluorescence did not change substantially between 30 and 60 min in the tissues examined. Plasma levels of daunorubicin were highest at 5 min (3.9 pg/ml) and decreased to 1.5 pg/mI at 30 and 60 min. TABLE TISSUE

LEVELS

OF

1

DAUNORUBICIN

Time Tissue

Drug”

5 min

10 min

AND

after

ADRIAMYCIN

injection ____

15 min

30 min

60 min

(rsldb Heart

A D

10.0 18.3

6.3 28.4

11.2 20.7

7.7 16.3

8.0 14.9

Lung

A D

7.8 18.4

5.2 34.0

7.8 29.2

4.7 21.0

5.5 14.4

Liver

A D

15.9 48.0

11.1 71.0

15.3 73.0

14.5 37.8

16.0 47.6

Kidney

A D

23.6 64.1

17.7 120.5

15.1 93.4

6.8 58.0

7.0 40.4

a Adriamycin (A) venously to hamsters b Values represent value is the average

5 mg/kg or daunorubicin (D) 25 mg/kg were administ.ered and total drug fluorescence determined in the tissues. micrograms of drug equivalents per gram tissue wet, weight,. value from paired hamst,er experiment,s.

intraEach

DAUNORUBICIN

AND

ADRIAMYCIN

355

METABOLISM

Initially we attempted to administer clinical or purified adriamycin at the same dosage as daunorubicin ( 25 mg/ kg,). However, all animals immediately exhibited acute respiratory distress and died within 30 sec. Dose reduction to 10 mg/kg produced similar results with either preparaConsequently, adriamycin dosage was 5 mg/kg in tion of adriamycin. and no all subsequent studies. At this dosage, all animals survived respiratory difficulties were observed. Tissue levels of adriamycin fluorescence reached a maximum 5-15 min postinjection and remained essentially unchanged between 30 and 60 min (Table 1). Since the dosage of adriamycin was 20% of the daunorubicin dosage, tissue levels of adriamycin fluorescence were accordingly lower than tissue levels of daunorubicin fluorescence and ranged from 13 to 55% of the daunorubicin levels. Chromatographic analysis of the tissue extracts from daunorubicintreated animals revealed four fluorescent metabolites of daunorubicin. These metabolites were daunorubicinol and three aglycones designated as DHl, DH2, and DH3 (Table 2). No metabolites more polar than daunorubicinol were observed. The aglycone metabolites of daunorubicin were most significant in the liver (Fig. 1) . At 5 min, aglycones represented 60% of total drug fluorescence in liver. By 60 min, the aglycone level had fallen and the level of daunorubicinol had increased so that daunorubicin, daunorubicinol, and ; glycones were present at approximateIy equal Auorescence levels. Of the aglycone metabolites in liver, DH2 was predominant. Aglycones DHl and DH3 accounted for less than 5% of the total drug fluorescence in liver at any one time.

CHROMATOGRAPHIC

Rf's

TABLE OF ANTHR~CYCLINE

2 ANTIBIOTICS

SND

System

METABOLITESO System _____--

Component

I

IV

V

Component,

II

Daunorubicin Daunorubicinol Daunorubicinol aglycone Daunorubicin aglycone I)111 DH2 DH3 Deoxydaunorubicinol aglycone Deoxydaunorubicin algycone

22 .t4 .67 .78 .x0 .71 .56 .71 ,230

.oo .oo .25 .45 .57 .37 .14 .36 .57

00 .oo .05 .I6 .29 .13 .03 .I3 .29

Adriamycin AHPl AHP2 AH1 AH2 AH3 A4H4

.73 .57 .43 1.0 1.0 1.0 1.0

a The compounds dried, and developed

were dissolved in methanol, in an ascending fashion for

applied 15 cm.

to 250 pm silica

III .OO .oo .oo .47 .3R .26 .19

gel G plates,

356

BACHIJR

ET AL.

* )_,.. . . . . -..* I -?-:- r--q

FIG. 1. Drug and metabolite levels in hamster tissues after iv daunorubicin (25 or adriamycin (5 mg/kg) treatment. Each point represents the average value from paired animal experiments. a-0, daunorubicin; l - - - - -0, daunoruaglycones; O-O adriamycin; 0- - - - -0, bicinol; l . . . . . 0, total daunorubicin adriaadriamycin aglycone AH4; 0 . . . . . 0, adriamycin aglycone AH2; &-A, mycin polar metabolite AHP2. mg/kg)

In heart, lung, and kidney, daunorubicin was the major drug form during the entire experiment ( Fig. 1) , and the total aglycone fluorescence never exceeded 4% of the total drug fluorescence extracted. The major biotransformation was the conversion of parent drug to daunorubicinol. At 60 min, levels of daunorubicinol reached 30 to 35% of the total drug fluorescence extracted in heart and kidney and about 12% of the drug extracted from lung. Adriamycin was converted to four aglycone metabolites (AH& AH2, AH3, and AH4) and two more polar metabolites (AHPl and AHPZ) (Table 2). AH2, AH4 and AHP2 were the major metabolites in the tissues (Fig. 1). Although several metabolites of adriamycin were found, the degree of biotransformation of adriamycin was less than that of daunorubicin. In all tissues examined, parent drug remained the predominant fluorescent specie throughout the 60 min (Fig. 1). Adriamycin aglycone levels in heart, lung, and kidney were low, never exceeding 7% of the total drug extracted. Only liver contained appreciable levels of the aglycones, but these levels never approached those of parent drug. Postmortem Studies Since we were unable to demonstrate the high levels of aglycones in heart and lung previously reported (3, 14), we carded out post-

hamster

DAUNORUBICIN

AKD

ADFUAMYCIN

METABOLIS~f

357

FIG. 2. Postmortem changes in tissue daunorubicin after iv daunorubicin (25 mg/kg). Animals were sacrificed 15 min after drug administration, and the tissues were analyzed from time of death (0 min) to 60 min later. Each point represents the average value from paired animal experiments. 0-Q total daunorubicin fluorescence; o-0, daunorubicin; @---a, daunorubicinol; l . . . * . 0, total daunorubicin aglycones.

mortem studies to determine the effects of delayed extraction on drug metabolism. After death, total daunorubicin fluorescence decreased in the tissues examined 2543% ( Fig. 2). The relative proportions of aglycones changed little in heart or lung but increased in liver and kidney. By 10 min postmortem, these aglycones, primarily DM2, represented the major fluorescent species in liver. Concomitant with this rise of the aglycone level, there was a fall of the level of parent drug in liver and to a lesser degree in kidney. The parent drug continued as the predominant fluorescent form in heart, lung, and kidney. Daunorubicinol levels remained essentially stable except for a decrease in the kidney. Total adriamycin fluorescence did not decrease to the same degree as daunorubicin in the postmortem tissues (Table 3A). No decrease was observed in lung, but the other tissues did lose fluorescence with the largest loss of 16% in heart. Adriamycin is converted primarily to aglycones in the postmortem liver and kidney, but little, if any, biotransformation of adriamycin to aglycones is seen in the heart and lung (Table 3B). Questionable production of the more polar metabolites occurs in both heart and lung. Identification

of Metabolites

After the daunorubicin metabolites were isolated, they were compared to known daunorubicin derivatives that were prepared chemically or en-

358

BACHUR

ET

AL.

A

Time (min) 0 60

I_--.--~ Heart 11.2

9.4

-..-~~. ~__Heart Component Adriamycin AHPl AHP2 AH1 AH2 AH3 .4H4

0 min 60min _-.. 87.6 91.4 0.8 5.3 1.3 1.6 3.3 NDb 0.4 0.5 1.7 0.7 5. 1 0. 6

__-

pg jg tissllr .~~ --~ Lung Liver

Kidney

7.8

1.5 3

15.1

8.2

1:3.4

14.0

B y0 of total tissue fluorescencea Lung -I____ 0 min 60 min -___ 94.4 81.2 10 3.5 3 0 1.2 ND 4.1 3.5 ND 4.4 ND 26 1.1

__ Omin

Liver

73.4 I 3 13 1.1 6.2 0.6 16.0

60 min 63.6 2.6 2. 0 0. 6 2.8 0. :3 28.0

Kidney 0 min 60 min 87.8 06 1.6 0.6 3 6 0. 6 5.3

72.3 1.9 1.7 ND 8.0 ND 16.1

a Fifteen minutes aft,er hamsters were dosed with 5 mg/kg adriamycin HCl they were sacrificed. Tissues were analyzed immediately (0 min) or allowed to stand at room temperat,ure for 60 min before analysis. b ND, not detectable.

zymatically. Hamster tissue daunorubicinol was chromatographically identical to the daunorubicinol obtained from enzymatic preparation. The most prominent aglycone metabolite, DH2, had chromatographic mobilities and fluorescent spectra identical to those of enzymatically prepared deoxydaunorubicinol aglycone (Table 2). In addition, mass spectroscopy assigned a mass of 384 to metabolite DH2. Although the daunorubicin metabolite DHl had chromatographic mobilities in three systems which corresponded to deoxydaunorubicin aglycone, mass spectral data were inconclusive. DH3 and the adriamycin metabolites were not obtained in sufficient quantity for characterization, DISCUSSION

Although the anthracycline antibiotics daunorubicin and adriamycin are metabolized in vitro by rat and hamster tissue preparations (g-13)) the degree, extent, and nature of in viva metabolism is under question. It was reported that the metabolic production of anthracycline aglycones was related to increased coronary flow pressures in dog heart-

DAUNORUBICIN

AND

ADRIAMYCIN

METABOLISM

359

lung preparations (9) and to disturbed electrocardiograms with cardiotoxicity in viva in hamsters injected with anthracycline antibiotics (3). Our present data are similar to what we found in the rat (6, 8). Only small ( <7%) quantities of aglycones are found in heart and lung of adriamycinor daunorubicin-treated hamsters. In the liver, however. substantial quantities of aglycones were seen with both drugs, indicating either breakdown of the compounds in the liver or the extraction b> liver of aglycone produced elsewhere. The predominant aglycone formed was deoxydaunorubicinol aglycone. This is in contrast to the reports aglycone as of Herman and co-workers (3, 14) who find daunorubicin the sole metabolite. The level of deoxydaunorubicinol aglycone in both liver and kidney was higher a few minutes after drug administration when the animal was anesthetized and probably slightly hypoxic. Concomitant with recovery of the animals from anesthesia (S-10 min) the levels of aglycones decreased in both liver and kidney. The enzyme for the production of deoxyaglycones is known to be inhibited by oxygen ( 12). Since we were unable to substantiate the findings of Herman et al. (3) and Mhatre et al. (14), we felt that a conversion of the drugs to aglycones may have been a postmortem effect aided by the hypoxia produced in the postmortem state. In our postmortem studies, however, we were unable to obtain aglycone levels greater than a few percent in either heart or lung although postmortem liver and kidney produced elevated Ievels of aglycones. We assume that these aglycones were produced enzymatically in situ by the anthracycline glycosidases (11-B). Although the glycosidases are present in both heart and lung ( ll), thcx): apparently are not as active as the enzymes in liver and kidney. Of interest was the decrease in total daunorubicin fluorescence in postmortem tissues, indicating either a binding of the daunorubicin that was more resistant to the extraction technique or a metabolism of the anthracycline ring system with a loss of fluorescence. This loss of &unorubicin fluorescence did not appear to be associated with any particular metabolic form of daunorubicin. Total adriamycin fluorescence, on the other hand, did not change significantly in the postmortem tissues, although there was a conversion of the adriamycin to other metabolic components. We have questioned why we do nut observe the aglycone metabolites in heart and lung as previously reported (3, 14). Since we have used the same animals, given the same dose, at least of daunorubicin, and sacrificed at the same time intervals, we feel that the most likely causes for the differences observed are extraction and isolation &&niques. We have utilized a rapid tissue removal with immediate tissne dell:l.

360

BACHUR

ET AL.

turation and extraction by organic solvents at low temperature. This extraction technique stopped further enzymatic degradation of the anthracyclines and liberated drug and metabolites from tissue debris ( 10). Since the anthracycline antibiotics have limited stability, especially in acidic conditions, prolonged exposure of the drugs or their metabolites to acids are minimized through separation with thin-layer chromatography. The stable extracts were chromatographed with nondestructive solvent systems on silica gel H thin layer for a short period of time (0.5-2 hr). Studies that utilized a water homogenization of the tissues with chromatography of this water homogenate on paper under acidic conditions (3, 14) may allow both enzymatic and chemical conversion of the anthracycline glycosides to aglycones. The aqueous homogenates have increased glycosidase activity (ll), probably because of decreased oxygen tension. Another unsuspected observation is the rapid acute toxicity of the adriamycin. Although the hamsters tolerate the large dose of daunorubicin ( 25 mg/kg) , they die immediately on receiving adriamycin at this dosage but are tolerant to an adriamycin dosage that is reduced by 80% ( 5 mg/kg ) . Th e c~1 mica1 dosage of daunorubicin ranges from 1 to 3 mglkg, while that of adriamycin is somewhat lower. When based on body surface area, however, the hamster dosage used here and human dosage are not very different. In the clinical usage of adriamycin, no similar acute toxicity has been reported. The observed human cardiotoxicity is a cumulative dose-dependent chronically produced cardiac decompensation or “pump failure.” The differences observed in the disposition, metabolism, and toxicity of adriamycin and daunorubicin may relate to the differences in antitumor activity seen in these structurally similar drugs. ACKNOWLEDGMENTS The authors appreciate the technical assistance of assistance of Mrs. Barbara Dressel in the preparation of tion of Dr. Henry Fales and Mr. William Comstock of Natronlcl Heart and Lung Institute in obtaining mass

Mr. Thomas Cooper and the this manuscript. The cooperathe Laboratory of Chemistry, spectra is appreciated.

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D.,

BACHUR,

N. R., AND HOLTZMAN,

J., Clin.

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Ther. 12, 96

(1971). 2. DIFRONZO, G., GAMBETTA, R. A., AND LENAZ, L., Reu. Eur. Etudes C&n. Biol. 16, 572 (1971). 3. HERMAN, E., MHATRE, R., LEE, I. P., VICK, J., AND WARAVDEKAR, V. S., Phapmucobgy 6, 230 ( 1971). 4. YESAIR, D. W., SCHWARTZBACH, E., SHUCK, D., DENINE, E. P., AND ASBELL, M. A., Cancer Rm. 32, 1177 (1972).

DAUNORUBICIN

5. HUFFMAN,

AND

ADRIAMYCIN

R. S., AND

D. H., BENJAMIN,

361

METABOLISM

N. R., C&L ~ha~c~~. Ther.

BACHUR,

13, 895 (1972). 6. CRADOCK,

J. C.,

EGORIN,

M.

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202, 48 ( 1973). 7. BENJAMIN, R. S., HUFFMAN, D. H., WIERNIK, Ass. Cancer Res. 13, 115 (1972).

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J. C., EGORIN, M. J., AND BACHUR, TV. R. In preparation. R., HERMAN, E., HUIDOBRO, A., AND WARAVDEKAR, V., J. Pharmacol. Exp. Ther. 178, 216 (1971). BACHUR, N. R., AND CRADOCK, J. C., J. Pharmacol. Exp. T/w. 175, 331 (1970). BACIIUR, N. R., AND GEE, M., J. Pharmacol. Exp. Ther. 1’77, 567 (1971). BACHLJR, N. R., AND GEE, M., Fed. PTOC. 31, 835 (1972). ASBELL, M. A., SCHWARTZBACH, E., BULLOCK, F. J., AND YESAIR, 1). W., J. Pharmucol. Exp. Ther. 182, 63 ( 1972). MHATRE, R. M., HER~~AN, E. H., WARAVDEKAR, V. S., AND LEE, I. p., &&em. Med. 6, 445 ( 1972).

8. CRALXXK,

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P. H., AND BACHUR,

BIOCHEMICAL

MEDICINE

Identification

8,

and

362-370

( 1973)

Quantitative

Determination

in Biological M. W. COUCH, Veterans

Fluids

N. P. DAS, K. N. SCOTT, Administration University

Hospital

of Florida Gainesville,

of Saccharin

AND

C. M. WILLIAMS

and Department College of Medicine, Florida 32601

of Radiology,

AND

R. L. FOLTZ Battelle

Received

Columbus Columbus,

Laboraton’es, Ohio

November

20, 1972

The determination of saccharin in biological fluids has in the past been carried out by paper and thin-layer chromatography (l-5), ion exchange chromatography ( 6), infrared spectrometry ( 7)) and ultraviolet spectrometry (S), but no techniques for its determination by gas-liquid chromatography (glc) have been described to our knowledge. We have been engaged in the identification of low molecular weight constituents of urine and cerebrospinal fluid, and this paper concerns the identification of saccharin as its N-methyl derivative in urine and plasma and provides a simple method for its quantitative determination by gas chromatography ( gc). The identification procedure involved the isolation of the compounds from urine and plasma by preparative gc, followed by preliminary characterization by high and low resolution mass spectrometry. Further confirmation of the structure of the compounds was accomplished through ‘H (PMR) and ‘“C (CMR j nuclear magnetic resonance spectroscopy of the urinary compounds, which can generally be obtained in larger quantity than the compounds from the plasma. Once the structure of the compounds was established by mass and nuclear magnetic resonance (NMR) spectroscopy, final proof of the structure was obtained by comparison with the mass and NMR spectra and gc characteristics of the authentic compound. Copyright AH rights

0 1973 by Academic Press, of reproduction in any form

362 Inc. reserved.