11360-3016190 $3.00 t .OO Press plc C,rpynght IC1990 Pergamon

Inr J Rodrurm Oncob~,~~tlwi Phu Vol. 19.pp. 1389-1396 Printed I” the U.S.A. All nghts resewed.

??Original Contribution

HEPATIC

JOHN

FUNCTION AND DRUG PHARMACOKINETICS AFTER TOTAL IRRADIATION PLUS BONE MARROW TRANSPLANT E. MOULDER,

PH.D.,’

BRIAN

L. FISH, B.S.,’

AND GUI-XIANG ‘Department

of Radiation Biochemistry.

SUN,

JOHN S. HOLCENBERG,

BODY

M.D.2

M.D.2

Oncology, Medical College of Wisconsin. University

of Southern

California

Milwaukee, WI; and *Departments of Pediatrics School of Medicine, San Francisco. CA. U.S.A.

and

Radiation nephritis is the principle late toxicity seen after total body irradiation in barrier-maintained rats when hematologic toxicity is prevented by bone marrow transplantation. Renal dysfunction is observed for single doses as low as 7.5 Gy. Hepatic blood flow, as measured by indocyanine green clearance, is decreased after 8.5-9.5 Gy single-dose total body irradiation. Serum albumin levels are decreased after 9.5 Gy single-dose total body irradiation. Hypoalbuminemia is a symptom of hepatic damage, but can also be caused by renal damage or edema. No decrease in total serum protein is observed, indicating that proteinuria resulting from renal damage is not the cause of hypoalbuminemia. No edema and some dehydration are observed. These data indicate that hepatic damage as well as renal damage may be occurring after total body irradiation plus bone marrow transplantation. Animals given total body irradiation plus bone marrow transplantation show decreased tolerance to a wide variety of immunosuppressive and cytotoxic drugs, even when these drugs are given months after total body irradiation. Altered drug clearance after total body irradiation plus bone marrow transplantation is observed for cis-platinum, vincristine, and adriamycin. The increase in cis-platinum toxicity after total body irradiation plus bone marrow transplantation is caused by decreased renal drug clearance. The decrease in vincristine tolerance and the alterations in adriamycin and vincristine pharmacokinetics are caused by altered drug distribution after total body irradiation plus bone marrow transplantation. These results indicate that bone marrow transplant survivors may show altered clearance of, and decreased tolerance to, a wide variety of drugs that are used after bone marrow transplantation. Total body irradiation, Bone marrow transplantation, Adriamycin.

INTRODUCTION Bone marrow transplantation has an established role in the treatment of both malignant and nonmalignant diseases (1, 7, 38). Prior to bone marrow transplant (BMT), the patient’s marrow is ablated, typically with total body irradiation (TBI) in combination with cytotoxic drugs. With the hematopoietic toxicity of TBI circumvented by BMT. the dose of TBI is usually limited by acute gastrointestinal and lung tolerance ( 14). However, TBI, both alone and in combination with drugs, has also been associated with hepatic ( 16. 20, 3 1, 32) and renal (6, 12, 3 1. 48, 5 1) damage. It is common for patients to receive drugs after TBI and bone marrow transplantation. These drugs may be given to treat graft versus host disease, bacterial, viral. or fungal infections, or tumor recurrences. The existence of

Presented at the 3 1st Annual Meeting of ASTRO. San Francisco, CA. 1-6 October 1989. Reprint requests to: Dr. John E. Moulder. Radiation Oncology, MCMC Box 165, Medical College of Wisconsin. 8700 W Wisconsin Ave., Milwaukee, WI 53226, U.S.A.

Renal function, Hepatic function, Vincristine. Cis-platinum.

multiple organ system damage after TBI and bone marrow transplantation raises the possibility that some of these drugs might show altered toxicity. Altered toxicity could be caused by additive organ toxicity (e.g., increased renal toxicity from the combination of renal radiation damage and nephrotoxic drugs such as cis-platinum (33, 47)), by altered drug clearance caused by renal or hepatic damage (e.g., alterations in renal clearance of cis-platinum (37)), or by altered drug distribution (e.g., alteration of the volume of distribution of methotrexate (26)). In previous studies we have shown that in defined-flora, barrier-maintained rats. radiation nephritis is the principle late toxicity seen after TBI when hematologic toxicity is prevented by BMT (34, 35). We also observed decreased cis-platinum clearance and increased cis-platinum, methotrexate, and cyclosporine toxicity after TBI plus BMT in the rat model (34. 36). This report presents evidence

This work was supported by Grant CA24652 tional Cancer Institute of the United States. Accepted for publication 2 1 June 1990.

from the Na-

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I. J. Radiation Oncology 0 Biology 0 Physics

of chronic hepatic damage in the rat syngeneic bone marrow transplant model and of altered pharmacokinetics and toxicity of drugs that are not renally excreted. METHODS

AND MATERIALS

Animals The study was done with defined microbiologically-associated WAG/RijMCW rats bred and housed in a moderate security barrier. Of particular importance to TBI studies, the barrier-maintained rats are free of Mycoplasma pulmonis, Pseudomonas, and common rat viruses. Male rats were used in single-dose TBI studies and females were used for multi-fraction TBI studies; all animals used in these studies were 6 to 8 weeks old at the time of TBI. No antibiotics, immunosuppressive drugs, or any other known nephrotoxic agents (other than those specifically mentioned in certain experiments) were used in these studies. Irradiation and bone marrow transplantation TBI was performed without anesthesia. Rats were treated with a posterior to anterior beam of 250 kVp (1 mm Cu HVL) X rays. Animals in the single-fraction TBI experiments were irradiated at a dose rate of 64 cGy/min. Animals in the fractionated TBI experiments were irradiated in 6 or 9 fractions in 60 hours at a dose rate of 20 cGy/min. Dosimetry was done with an ionization chamber in an acrylic plastic phantom. The TBI dose was defined at the midline. The dose to the lung was 5-10% higher than the midplane dose, the dose to the remainder of the body was within 5% of the midline dose. Rats were given 5 X lo7 fresh syngeneic bone marrow cells per kg, i.v., 1 to 2 hr after TBI, using techniques previously published (34). To prepare the marrow, syngeneic donor rats were killed by CO* asphyxiation, and the femora were stripped of soft tissue and removed. The marrow plug was expelled into sterile tissue culture medium containing 10% fetal bovine serum. When animals were given 10.5 Gy TBI, the acute (21-day) lethality was 100% (121/121) without BMT and 5% (4/73) with BMT (34). Because animals were barrier-maintained and the donors were syngeneic, neither infection nor graft versus host disease is an issue in this model system. Cytotoxic and immunosuppressive drugs Cis-diamminedichloroplatinum (cisPt)*, adriamycin (ADR),+ and vincristine (VCR) were used in their standard clinical formulations. Animals were hydrated prior to i.p. cisPt injection with an i.p. injection of 0.9% saline (3 ml per 100 grams). Drugs were injected at a fixed time during the day to avoid possible diurnal variations in toxicity. To assess cisPt clearance, animals were placed in metabolic cages immediately after cisPt injection, and urine

* Supplied

by Bristol-Myers

Company,

Syracuse,

NY.

December 1990. Volume 19. Number 6

was collected over a 24-hour period. CisPt was assayed by flameless atomic absorption spectroscopy (4 1). To assess VCR clearance, animals were given a 1.O mg/kg i.v. injection of tritium-labeled VCR (10.9 Ci/mole), and blood samples were taken at intervals after injection. VCR concentrations were determined by liquid scintillation counting. To assess ADR clearance, animals were given a 5 mg/kg i.p. injection of ADR, and blood samples were taken at intervals after ADR injection. ADR concentrations were determined on aliquots of plasma as total fluorescence of acid alcohol extracts (3. 25). Functional assays Blood and urine were collected at intervals after TBI, and urine creatinine, urine protein, serum albumin, total serum protein, and blood urea nitrogen (BUN) were determined by commercial assay kits. Hepatic blood flow was assessed by indocyanine green (ICG) clearance, using a modification of the method described by Paumgartner et al. (39). Rats received i.v. injection of 1.0 mg/kg ICG and blood samples were taken over a I?-minute period. ICC levels were assayed spectrophotometrically at 805 nM; the initial half-life of ICG clearance was determined from serum samples taken 1.2 and 3.2 minutes after ICG injection. The half-life for ICG clearance under these conditions is an index of hepatic blood flow (10, 39). Statistical methods Fifty percent lethal doses (LDsO’s)were calculated and compared by probit analysis (19). Physiologic data were compared by two-tailed Mann-Whitney U-tests. All values shown are means with 95% confidence intervals and all significance testing was done at the 0.05 level. VCR and ADR clearance were analyzed by noncompartmental methods (42). The area under the serum clearance curve (AUC) and the first moment of the serum concentration time curve (AUMC) were calculated by the trapezoidal rule. The mean residence time (MRT) was calculated as the AUMC divided by the AUC. RESULTS Toxicity of TBI and bone marrow transplant The 30-day LDso for single-fraction TBI was 13.3 (13.013.8) Gy when animals were given BMT, animals that survived the acute (gastrointestinal) lethality phase showed a late lethality phase, and the LDso decreased to 9.3 (8.99.5) Gy by 7 months (34). Over 60% of the animals given 9.5 Gy TBI died between 3 and 6 months after TBI; 15% of the animals given 8.5 Gy TBI died about 7 months after TBI (Fig. 1). These late deaths were accompanied by an increase in BUN and a decrease in urine creatinine levels, both symptoms of radiation nephritis (34). The 30day LDso for 6-fraction TBI was 17.4 (16.3-18.5) Gy when

T Supplied

by Adria Laboratory,

Dublin,

OH.

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Bone marrow transplant and drug pharmacokinetics 0 J. E. MOULDER el ul.

imals suggested that the older TBI survivors might also have a lower body fat content than unirradiated, agematched controls. Similarly, diminished stature compared to age cohort has been observed in pediatric bone marrow transplant survivors (4, 14).

Renal.fimction after TBI and bone marrow transplantation

0

1

2

3

4

5

6

7

6

Months after TBI+BMT Fig. 1. Animal survival after single-dose TBI and bone marrow transplant. The data shown in Figures l-5 and 9- 10 are for the same animals. In the 9.5 Gy group, 25 animals were at risk at 2 months and 14 at 4 months; in the 8.5 Gy group, 35 animals were at risk at 2 months, 28 at 4 months, and 20 at 7 months; and in the 7.5 Gy group, 28 animals were at risk at 2 months, 21 at 4 months, and 14 at 7 months.

animals were given BMT, animals that survived the acute (gastrointestinal) lethality phase showed a late lethality phase, and the LDso decreased to less than 13 Gy by 7 months (34). The 30-day LDso values for 9-fraction TBI were slightly higher (34). One of the most obvious chronic effects of TBI in the rat syngeneic bone marrow transplant model was the failure of animals to gain weight normally after TBI. Figure 2 shows weight gain after single-fraction TBI doses of 6.59.5 Gy. Significant decreases in weight gain were seen for all doses and at all time intervals. Observation of the an-

.

Altered renal function after TBI and BMT was observed at doses below the 7-month LDso. Increases in BUN levels could be seen as early as 2 months after TBI at doses of 8.5 and 9.5 Gy; a dose of 7.5 Gy produced barely detectable changes in BUN at 4 and 7 months (Fig. 3). Increased urine volume, increased urine protein levels, and decreased urine creatinine were observed 4 months after single-fraction TBI at doses as low as 5.9 Gy (34, 36). Similar renal toxicity has been observed after TBI in mice ( 13, 15, 23. 49), and dogs (43), and multiple lines of evidence indicate that the delayed lethality observed in the rat model is because of radiation nephritis (34).

H~~patic.,firrzctionqfier TBI and hone marrofl’ transplantation Indocyanine green (ICG) binds to lipoproteins in serum and is cleared by the liver (10, 39). The initial rate of clearance of ICG from serum after a bolus injection is a measure of hepatic function. At low ICG concentrations such as the 1 mg/kg dose used in this study, liver blood flow is the rate-limiting step in ICG clearance (10, 39). Figure 4 shows initial ICG clearance half-lives at intervals after single fraction TBI. Increased ICG clearance halflives (i.e., decreased hepatic blood flow) were seen 2 to 7 months after TBI in animals given 9.5 Gy and 4 to 7 months after TBI in animals given 8.5 Gy. No ICG clearance abnormalities were seen after 6.5 or 7.5 Gy TBI (Fig. 4).

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Months after TBI + BMT Fig. 2. Body weight gain for male rats after TBI and bone marrow transplant. Body weights are shown at intervals after TBI for males given 6.5 (0), 7.5 (Cl), 8.5 (A), or 9.5 (0) Gy single-fraction TBI, and for age-matched unirradiated males (a). Body weights are shown with 95% confidence intervals; body weights at all time intervals and doses were significantly lower in TBI animals than in age-matched controls. At 1,2 and 4 months there were 1 I - 14 animals in each control group and 5-7 in each irradiated group; at 7 months there were 32 animals in the control group, 14-20 animals in the 6.5, 7.5, and 8.5 Gy groups, and just 3 survivors at 9.5 Gy.

20

0

1.

“1.

1

2

Months after TBI + BMT

Fig. 3. Blood urea nitrogen (BUN), an index of renal function, after TBI and bone marrow transplant. BUN levels were determined at intervals after TBI for animals given 6.5 (0), 7.5 (El), 8.5 (A). or 9.5 (0) Gy single-fraction TBI. BUN levels in agematched unirradiated animals (0) did not differ significantly from those of animals given 6.5 Gy TBI. BUN levels are shown with 95% confidence intervals. The number of animals per group is the same as in Figure 2.

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1. J. Radiation Oncology 0 Biology 0 Physics

December 1990. Volume 19. Number 6

dence of dehydration. However, in view of the severe renal dysfunction observed after 9.5 Gy TBI (Fig. 3), this hypoalbuminemia cannot be unambiguously attributed to hepatic damage.

Cis-phtinwn

1.or

.

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,

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Months after TBI + BMT Fig. 4. Indocyanine green (KG) clearance, an index of hepatic blood flow, after TBI and bone marrow transplant. Initial ICG clearance half-lives were determined at intervals after TBI for animals given 7.5 (0). 8.5 (A), or 9.5 (,?) Gy single-fraction TBI. Clearance half-lives are also shown for age-matched unirradiated animals (0). ICG half-lives are shown with 95% confidence intervals. Values which were significantly different than those of age-matched control animals are indicated (*). The number of animals per group is the same as in Figure 2.

Moderately minemia

severe

liver

(low serum

damage

albumin

can cause

levels),

hypoalbu-

but hypoalbumin-

emia is not specific for liver damage.

It can also be caused by edema, ascites. malnutrition, or renal disease. Figure 5 shows total serum protein and serum albumin levels after single-fraction TBI. No significant changes in serum protein levels were observed after TBI, but a significant decrease in serum albumin levels was seen 4 and 7 months after 9.5 Gy TBI. The animals showing hypoalbuminemia showed no evidence of ascites or edema and some evi-

tosicity und excretion in TBI survivors

All male animals that survived single-dose TBI with BMT in early experiments (34) were given a single i.p. dose of cisPt (5-10 mg/kg) 9 months after TBI. If TBI/ BMT survivors had increased drug sensitivity because of decreased renal function, the effect should be most dramatic for a drug like cisPt which was both nephrotoxic and renally excreted. Survivors consisted of 26 animals that had received 4.9-6.7 Gy, 19 animals that had received 7.6-8.5 Gy, and 47 age-matched controls (36). The acute (14-day) cisPt tolerance of the TBI/BMT survivors was significantly lower than that of age-matched animals that had not received TBI (36). or of younger weight-matched animals that had not received TBI. The rate of urinary excretion of Platinum is an index of the systemic exposure to cisPt. with decreased excretion generally associated with increased toxicity (33). TBI/ BMT survivors show a dose-related decrease in Platinum excretion (Fig. 6), with a significant reduction in excretion for doses of 7.6 Gy and above.

Vincristinr toxicity und cleurunce in TBI swvivors All female animals that survived 6- and 9-fraction TBI in early experiments (34) were given a single i.p. dose of VCR’( 1.O-2.5 mg/kg) 9 months after TBI. VCR is neither nephrotoxic nor renally excreted. It is excreted in the bile and has major neurological toxicity and minor hepatic

21”“.‘.‘.““‘J 0

1

2

3

4

5

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Months after TBI + BMT Fig. 5. Total serum protein and serum albumin after TBI and bone marrow transplant. Total serum protein (0. A, R) and serum albumin (0, A, 0) are shown for animals that received 6.5-7.5 (0, ?? ) or 8.5-9.5 (A, A) Gy single-fraction TBI, and for age-matched unirradiated controls (0,O). Data are shown with 95% confidence intervals, and values which were significantly different than those of age-matched control animals are indicated (*). At 1 to 2 months there were 20 animals in the control group, 12 in the 6.5-7.5 Gy group, and 20 in the 8.5-9.5 Gy group; at 4 months there were lo-14 animals in each group: and at 7 months there were 31 animals in the control group, 30 in the 6.5-7.5 Gy group, and 15 in the 8.5-9.5 Gy group.

0

1

2

3

4

5

6

7

6

9

TBI Dose (Gy) Fig. 6. Urinary excretion of cisPt after TBI and bone marrow transplant. The percent of injected Platinum that was cleared into the urine within 24 hrs of cisPt injection (5-10 mg/kg) is shown for animals which survived for 9 months after singlefraction TBI (0) and for age-matched unirradiated animals (0). Excretion values are shown with 95% confidence intervals; values which were significantly different than those in age-matched control animals are indicated (*). There were 35 animals in the control group, and 6- 10 animals in each irradiated group. Data from 32 control animals and 3 1 irradiated animals from a previous report [Moulder et al. (36)] are included in this figure.

Bone marrow transplant and drug pharmacokinetics 0 J. E. MOULDER

(1 1. 17, 27). VCR was used to assess whether survivors had increased drug sensitivity for TBI/BMT reasons other than renal damage. Survivors consisted of 31 animals that had received 6.8-10.0 Gy, 44 animals that had received 10.5- 12.0 Gy, and 34 age-matched controls. The acute (21-day) LDso for VCR was 1.50 (1.201.80) mg/kg in the high-dose TBI survivors, 1.90 (1.552.25) mg/kg in the low-dose TBI survivors, and 1.90 ( 1.602.45) in age-matched animals that had not received TBI (Fig. 7). The difference in VCR tolerance between the high-dose TBI survivors and the age-matched controls was statistically significant. Interpretation of the increase in VCR toxicity in survivors of TBI/BMT is complicated by the observation that VCR tolerance increased with age, and thus with weight, in unirradiated control animals. At 9 months, high-dose TBI survivors had an average weight of 145 g, whereas the age-matched controls weighed 185 g. When the VCR tolerance of high-dose TBI survivors was compared to younger control animals whose average weight was 150 g. there was no difference in VCR tolerance. Serum VCR clearance was assessed in 7 high-dose TBI survivors and 9 age-matched controls (Fig. 8). The mean residence time (MRT) of VCR was 33 minutes in the control animals and 32 minutes in the TBI group. The area under the serum clearance curve (AUC), on the other hand, was 48 pg - min/ml in the controls and 60 yg - min/ ml in the TBI animals. The 25% increase in AUC can account for the 25% decrease in VCR tolerance observed in the high-dose TBI group (Fig. 7). The change in AUC without a change in MRT indicates that the volume of distribution of VCR had decreased in the TBI survivors.

1393

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so

40

80

after Vincristine Injection (min)

Fig. 8. Vincristine serum clearance after TBI and bone marrow transplant. A single dose of I mg/kg VCR was given to 7 animals that had survived for 9 months after I OS- 12.0 Gy (0) fractionated TBI (6 or 9 fractions in 60 hours at 20 cGy/min) and to 9 unirradiated age-matched controls (0). Serum VCR levels are shown with 95% confidence intervals.

Since there is clinical evidence that ADR clearance can be affected by hepatic damage (8, 25, 29); further studies of drug pharmacokinetics were carried out with adriamycin. Like VCR, ADR is neither nephrotoxic nor renally excreted; it is metabolized in the liver and eliminated by biliary excretion (2, 5). Animals were given single-fraction TBI at doses of 6.5, 7.5, 8.5. and 9.5 Gy, followed by a bone marrow transplant. ADR serum clearance was assessed 1, 3, 4, and 7 months later. Figure 9 shows ADR serum clearance 7 months after TBI in unirradiated animals and in animals that had received 8.5 Gy TBI. There are abnormalities in the clearance of ADR in the irradiated animals, but the pattern is different from that seen for VCR (Fig. 8). For VCR, drug levels were higher in irradiated animals: for ADR the drug

Vincristine Dose (mg/kg)

Fig. 7. Vincristine tolerance after TBI and bone marrow transplant. A single dose of VCR was given to animals that had survived for 9 months after TBI. The morbidity dose-response curves assessed at 2 1 days are shown for animals that had received 6.8-10.0 Gy (0) or 10.5-12.0 Gy (0) fractionated TBI (6 or 9 fractions in 60 hours at 20 cGy/min). A morbidity dose-response curve is also shown for unirradiated age-matched controls (m). LDSo values are shown with 95% confidence intervals. For low (1 mg/kg) and high (2.5 mg/kg) VCR doses 4-8 animals were used per dose group: for the intermediate VCR doses 10-l 4 animals were used per dose group.

0023 0

4

8

12

16

20

24

28

32

Time after ADR Injection (hrs)

Fig. 9. Adriamycin serum clearance after TBI and bone marrow transplant. A single dose of 5 mg/kg ADR was given to 11 animals that had survived for 7 months after 8.5 Gy (0) TBI delivered in a single fraction at 64 cGy/min and to 17 unirradiated age-matched controls (0). Serum ADR levels are shown with 95% confidence intervals. The data shown in Figures 1-5 and 9-10 are for the same animals.

1394

1. J. Radiation Oncology 0 Biology 0 Physics

levels were lower after irradiation. Figure 10 shows a detailed pharmacokinetic analysis of the ADR clearance data. The mean residence time of ADR was 9.0 (8.6-9.4) hours in the control animals and did not vary with age. Seven months after TBI, the mean residence time for animals given 8.5-9.5 Gy TBI was 10.0 (8.6-l 1.4) hours; for all other TBI doses and intervals, mean MRT values ranged from 8.0 to 9.4 hours. None of these differences in MRT were statistically significant, although the IOhour value 7 months after high-dose TBI approached significance (p = 0.07). The area under the ADR serum clearance curve (AUC) varied with age in the control animals from a minimum of 5.0 (3.9-6.2) pg. hr/ml 1 month after sham TBI to a maximum of 7.1 (5.3-8.9) pg. hr/ml4 months after sham TBI. There was a trend towards increasing AUC with age, but the trend was not statistically significant. AUC levels were significantly decreased starting 2 to 7 months after irradiation in animals given 8.5-9.5 Gy TBI, and at 7 months after irradiation in animals given 6.5-7.5 Gy TBI. The change in AUC without a change in MRT indicates that the volume of distribution of ADR had increased by 30-50% in the TBI survivors. There was no correlation of body weight and AUC in the irradiated animals, so that the effect of TBI on ADR is unlikely to be caused by the growth stunting observed after TBI (Fig. 2).

0

1

2

3

4

5

6

7

Months after TBI + BMT Fig. 10. Adriamycin serum pharmacokinetics after TBI and bone marrow transplant. A single dose of 5 mg/kg ADR was given at intervals after 6.5-7.5 (0) or 8.5-9.5 (0) Gy single-fraction TBI. Pharmacokinetic data are also shown for age-matched unirradiated controls (0). TOP: Mean residence time (MRT) with 95% confidence intervals. BOTTOM: Area under the clearance curve (AUC) with 95% confidence intervals; values which were significantly different than those of age-matched control animals are indicated (*). At 1 month there were 6 animals in the control and irradiated groups; at 2 and 4 months there were 11-14 animals in each control and irradiated group; and at 7 months there were 30 animals in the control group, 29 in the 6.5-7.5 Gy group and 12 in the 8.5-9.5 Gy group.

December 1990, Volume 19. Number 6

DISCUSSION In contrast to the general observation that pneumonitis is the major late toxicity after TBI and bone marrow transplant (30,40,49), nephritis is the major late toxicity observed in the rat (34-36). The changes in renal function seen after TBI and bone marrow transplant in the rat match both qualitatively and quantitatively the changes seen after local renal irradiation, and there is little doubt that the late deaths seen after TBI plus BMT (Fig. 1) are caused by radiation nephritis (34). Similar radiation nephritis after TBI has been reported in mice (13, 15, 23, 49) and dogs (43) and recently there have been reports of radiation nephritis in humans after TBI plus bone marrow transplant (6, 12, 31, 48, 5 1). We now have evidence that there may also be chronic hepatic damage in rats after TBI and bone marrow transplant. The hepatic damage is less severe than the renal damage, and we have no evidence that it is a contributing cause of the late morbidity observed (Fig. 1). The alteration in ICG clearance (Fig. 4) is our best evidence for a hepatic effect, as alterations in ICG clearance are considered to be diagnostic for alterations in hepatic function ( 10, 39). ICG clearance is affected by both hepatic blood flow and by the ability of hepatocytes to take up the dye, but at the low ICG concentration used in this study, hepatic blood flow is the rate-limiting step in ICG clearance (10, 39). Further studies using other ICG concentrations are planned, as such studies would allow us to assess both hepatic blood flow and hepatocyte function (39). The other indications for hepatic damage are less definitive. The hypoalbuminemia observed after high doses of TBI (Fig. 5) is consistent with hepatic damage, but hypoalbuminemia can also be caused by renal disease. In view of the severe renal dysfunction observed after 9.5 Gy TBI (Fig. 3). this hypoalbuminemia cannot be unambiguously attributed to hepatic damage. Similarly. the alterations in area under the vincristine and adriamycin serum clearance curves are consistent with chronic hepatic damage, but are not diagnostic. If the alterations in VCR and ADR clearance had been caused by a change in the mean residence time, we would have had stronger evidence for hepatic damage (29): alterations in volume of distribution, however, might have non-hepatic causes (e.g., alteration in body fat content). To complement this pharmacokinetic data, studies of cyclophosphamide pharmacokinetics after TBI plus BMT are planned. Such studies would assess a different aspect of hepatic function, the ability of the liver to activate a drug. The hepatic damage observed in the rat syngeneic bone marrow transplant model does not resemble the venocelusive disease (VOD) of the liver which is frequently observed after TBI plus BMT in humans (16, 20, 32). VOD in humans occurs within several weeks of transplant and is characterized by jaundice, hepatomegaly, and ascites; most patients with serious VOD die within 2 months

Bone marrow transplant and drug pharmacokinetics 0 J. E. MOULDER

of transplant. The hepatic damage observed after TBI in rats occurs months after transplant, and we have not observed either jaundice, hepatomegaly, or ascites. Radiation hepatitis is not a common complication of radiotherapy: a recent review by Schacter et al. (46) found only 32 cases in the literature. In addition, there are few reports of chronic hepatic radiation injury in animal models ( 18) except when very high radiation doses were used (24) or when radiation was accompanied by partial hepatectomy (2 1). An exception is the report of altered ‘“I-rose bengal clearance one year after 20 Gy total liver irradiation in rats (22). The idea that organ damage caused by radiation could affect drug distribution and clearance is not new (9, 2.5. 28,45), and there are reasons to expect such effects after bone marrow transplant. Patients who relapse after TBI and bone marrow transplant appear to have low tolerance for Phase I agents ((44); J. S. Holcenberg, unpublished observations, September, 1987). and we have observed a decrease in cisPt and methotrexate clearance after lowdose renal irradiation in rats (26, 37). The alteration in cisPt excretion (Fig. 6) and toxicity was anticipated, and the mechanism for the effect was obvious (36). The alteration in VCR and ADR clearance (Figs. 7- 10) was less expected, since renal function is not thought to play a

1395

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significant role in the clearance of either VCR (I 1, 27) or ADR (2, 5). As discussed above, this effect cannot be unambiguously attributed to hepatic damage, since it is the distribution of the drugs rather than their clearance rate which is altered after TBI. The altered distribution could also be related to the change in the growth of the animals (Fig. 2). or it could be caused by subclinical effects on other organs. We have demonstrated in the rat syngeneic bone marrow transplant model that the TBI used in the conditioning regimen toxicity renally

can

affect

of a broad excreted

the

range

distribution,

of drugs,

or nephrotoxic.

clearance,

and

not all of which The

effects

of TBI

are on

human bone marrow transplantation by organ damage caused by nephrotoxic (e.g., cyclosporine, gentamicin. amphotericin (50)) or hepatotoxic (e.g., cyclophosphamide, busulfan, azathiopurine (52)) agents used during or after BMT. It could also be accentuated if the bone marrow transplant patient had received prior radiotherapy involving hepatic or renal irradiation or had received nephrotoxic or hepatotoxic agents in prior chemotherapy regimens. Detailed studies of drug pharmacokinetics and toxicity after TBI plus bone marrow transplant will be required to resolve these issues.

drug

clearance

and toxicity

could

be accentuated

in

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