Renal irradiation and the pharmacology and toxicity of methotrexate and cisplatinum

0X0-3016/86 Copyright 0 1986 Pergamon

Int. 1. Raduztion Onco&~~ Biol. Phys., Vol. 12, pp. 1415-1418 Printed in the U.S.A. All rights reserved.

$3.00 + .oO Journals Ltd.

??Session VI

RENAL IRRADIATION AND THE PHARMACOLOGY AND TOXICITY OF METHOTREXATE AND CISPLATINUM JOHN E. MOULDER,

PH.D.,’ JOHN S. HOLCENBERG, M.D.,* BARTON A. KAMEN, M.D., MARY CHENG, PH.D.~ AND BRIAN L. FISH, B.S.’

PH.D.,~

‘Radiation Oncology, Medical College of Wisconsin, Milwaukee, WI; ‘Pediatrics, 4Patbology, Los Angeles Children’s Hospital, Los Angeles, CA; ‘Pediatrics, University of Texas Health Science Center, Dallas, TX We used a rat model to study

the effects of renal irradiation on the pharmacology of methotrexate (MTX) and cisplatinum (cis-Pt). Unanesthetized rats were given bilateral kidney irradiation (20 Gy in 9 fractions). At 9 months after inrrdiation, 3% of the animals had died and survivors showed moderately impaired renal function. At 15 months, 30% of the animals had died and survivors showed severely impaired renal function. Some animals were given i.v. MTX 1 week to 15 months after irradiation. In irradiated rats, the area under the MTX plasma clearance curve equaled that of controls through 6 months, and was significantly above controls from 9 months on. Other animals were given i.p. cis-Pt 1 week to 9 months after irradiation. The acute toxicity of cis-Pt was the same in control and irradiated rats when cis-Pt was given immediitely before or after irradiation. Beginning 3 months after irradiation there was a progressive increase in cis-Pt toxicity and a simultaneous decrease in urinary platinum excretion. Irradiated animals that survived cis-Pt treatment showed increased radiation nephritis; the greatest effect occurred when cis-Pt was given 3 months or more after irradiation. MTX and cis-Pt clearance decreased when renal dysfunction was first observed and changes in renal function preceded changes in drug clearance and toxicity. Methotrexate,

Cis-platinum,

Kidney, X-irradiation.

INTRODUCTION

“moderate security banier.“7 The animals were 2 months old at the start of the experiment. Rats were irradiated without anesthesia, using 250 kV X rays (HVL of 0.5 mm Cu) and Plexiglas jigs adapted from those used by Sheldon et al. I4 Parallel-opposed lateral fields were used that encompassed both kidneys with a 5 mm margin. Animals were treated with 20 Gy in nine fractions over 11 days at a dose rate of 1.3 Gy/min. Urine and serum creatinine, blood urea nitrogen (BUN), and mine protein were determined by commercial kits. N-acetyl-glucosamine (NAG) was assayed according to published methods.2 Methotrexate (MTX) was injected i.v. at 10 mg/kg (the LDzo in these rats is greater than 60 mg/kg). The animals were placed in metabolic cages; serum and urine were collected over a 48 hour period. MTX was assayed in urine and serum using a radioligand assay.g Cis-diamminedichloroplatinum (cis-Pt) was supplied by the manufacturer* and was used in the standard clinical formulation. Animals were hydrated prior to cis-Pt injection with an i.p. injection of 0.9% saline (3 ml per 100

Combined radiotherapy and chemotherapy are known to have late as well as early consequences for the host.13 When chemotherapy follows radiotherapy, toxicity may be caused by combined effects within the radiation field, or by changes in drug clearance and distribution in irradiated organs.’ Rubin13 suggested that liver irradiation in Wilm’s tumor patients leads to altered metabolism of actinomycin-D and vincristine although Holcenberg et al5 were unable to find direct evidence for this effect. Others have suggested that brain irradiation increases the permeability of the blood-brain barrier to methotrexate,4 although there is evidence to the contrary.’ This study was designed to determine whether renal irradiation effects the pharmacology of subsequently administered methotrexate or cisplatinum. METHODS

AND MATERIALS

All studies were performed with “defined microbiologically associated” female WAG/Rij rats housed in a

Presented at the Chemical Modifiers of Cancer Treatment Conference, Clearwater, Florida, 20-24 October 1985. This work was supported by National Cancer Institute Grants CA24652 (JEM) and CA34840 (JSH). Reprint requests to: John E. Moulder, Ph.D., Radiation On-

cology, MCMC Box 165, Medical College of Wisconsin, 8700 W Wisconsin Ave., Milwaukee, WI 53226. Accepted for publication 25 February 1986. * Bristol Laboratories, Syracuse, NY 13201. 1415

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August 1986, Volume 12, Number 8

grams). Cis-Pt was injected i.v. at a fixed time during the day to avoid diurnal variations in toxicity.6 The animals were then placed in metabolic cages and urine collected over a 24 hour period. Total platinum in the urine was assayed by flameless atomic absorption spectroscopy. ’’ Pharmacokinetic data was analyzed by non-compartmental analysis. I2 Fifty percent lethal doses (LD& were calculated and compared by probit analysis.3 Physiologic and pharmacokinetic data were compared by 2-tailed Mann-Whitney U-tests. All values shown are means with 95% confidence intervals except as noted for urine protein levels and all significance testing was done at the 0.05 level.

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RESULTS The radiation schedule used produced a chronic nephritis with symptoms indicating progressive tubular damage. No deaths occurred in irradiated rats until 6 month after irradiation (Fig. 1A). Between 6 and 11 months, 6% of the irradiated animals died and between 11 and 16 months an additional 27% died (Fig. 1A). One animal in the control group died at 12 months; the cause of death could not be determined. BUN and urine creatinine levels did not change in control animals during the course of the experiment (Fig. 1) nor did urine volumes, urine protein levels, and NAG to creatinine ratios (Fig. 2). In irradiated animals, urine creatinine concentration decreased progressively beginning at 3 months (Fig.

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Fig. 2. Effects of renal irradiation on renal function. A: Urine protein levels in control (0) and irradiated (0) animals. Values shown are medians with 10 to 90% ranges. B: Urine volumes in control (0), irradiated (o), hydrated (0) and irradiated plus hydrated (+) animals. C: NAG to creatinine ratios in control (0) and irradiated (0) rats.

1B); this reflects an increase in urine volume (Fig. 2B), as total creatinine excreted did not change. In irradiated rats, BUN increased between 1 and 6 months and then stabilized (Fig. 1C). Urine protein levels (Fig. 2A), NAG to creatinine ratios (Fig. 2C), and blood pressure (not shown) increased 3 months after irradiation and then stabilized. The effects of renal irradiation of the MTX excretion appear in Figure 3. The area under the plasma clearance curve (AUC) is a measure of the potential for systemic toxicity. The AUC for MTX in unirradiated rats increased with age until 6 months, and then stabilized (Fig. 3A). In irradiated rats, AUC was the same as in controls through

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Fig. 1. Effects of renal irradiation on survival and renal function. A: Survival in irradiated (solid line) and control (dashed line) groups. The number of animals at risk is shown in parentheses. B: Urine creatinine levels in control (0) and irradiated (a) animals. C: BUN levels in control (0) and irradiated (0) animals.

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Fig. 3. Effect of renal irradiation on MTX excretion. A: Area under the MTX plasma clearance curve for control (0) and irradiated (0) groups. Values are relative to that measured at the start of the experiment. B: Percent of MTX injected that was cleared into the urine in the first 24 hr after injection in control (0) and irradiated (0) animals.

Renal irradiation and pharmacology 0 J. E. MOULDER

6 months and 50% greater from 9 months on (Fig. 3A). The volume of distribution decreased in control animals between 3 and 6 months and then stabilized. The volume of distribution in irradiated rats was the same as in control animals through 6 months and was significantly below controls from 9 months on. The mean residence time of MTX was unchanged by age or irradiation, as was the amount of MTX excreted in the urine during the first 4, 24, or 48 hr (Fig. 3B). The lack of a detectable effect on renal excretion is expected because 99% of the MTX is cleared from the plasma within 2 hr, making even a 4 hr urine collection too long. The effects of renal irradiation on the excretion and toxicity of cis-Pt appear in Figure 4. The fraction of injected platinum excreted in the urine within the first 24 hr after cis-Pt injection is shown in Figure 4A. Platinum excretion in control animals rose with age, whereas platinum excretion in irradiated animals dropped slightly with age. Three months following irradiation, the difference was not significant; but at 6 and 9 months the irradiated animals were excreting half as much platinum as the control animals (Fig. 4A). The decrease in cis-Pt clearance paralleled an increase in acute cis-Pt toxicity (Fig. 4B). Animals given cis-Pt alone died 3-8 days later or lived for at least 12 months. The acute toxicity of cis-Pt (assessed 10 days after drug injection) did not change significantly with age, in the unirradiated animals, although there was a trend towards lower tolerance (Fig. 4B). In irradiated animals cis-Pt tolerance dropped steadily after irradiation. The difference was not significant at 3 months, but was significant at 6 and 9 months. Some late toxicity was seen in animals given cis-Pt alone; a few animals given cis-Pt at the start of the experiment died at 12- 15 months. Irradiated animals that survived the acute effects of cis-Pt showed more severe radiation nephritis than animals that received irradiation alone. As a result of this late toxicity, LDSO’s assessed 12 months after irradiation (regardless of when cis-Pt was given) are much lower than acute LDsO’s (Fig. 4B). When cis-Pt was given immediately before or after irradiation the effect was barely detectable; but when cisPt was given 3, 6, or 9 months after irradiation the combined late toxicity was dramatic (Fig. 4B). DISCUSSION It has long been speculated that radiation damage to organs might effect the pharmacokinetics of subsequently

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51’ Renal CIS-PT Clearance 4. (% Cleared in 24 hrs)

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Fig. 4. Effect of renal irradiation on cis-Pt excretion and toxicity. A: Percent of cis-Pt injected that was cleared into the urine in the first 24 hr after injection in control (0) and irradiated (0) animals. B: LDso for cis-I? in control (0, 0) and irradiated (0, +) animals. Toxicity was assessedeither 10 days after cis-F’t injection (0, 0) or at 12 months after the irradiation (0, 4).

administered drugs.‘,‘3 We have demonstrated that renal irradiation can affect the clearance of two common anticancer agents, MTX (Fig. 3A) and cis-Pt (Fig. 4A), and that for cis-Pt, the decrease in excretion correlates with an increase in drug toxicity (Fig. 4B). This finding is of particular importance because the kidneys are irradiated to doses near their tolerance in a number of treatment protocols that include the use of chemotherapy: combined therapy for Wilm’s tumor, lower hemi-body irradiation, and the total body irradiation used with bone marrow transplantation. lo For the 20 Gy in 9 fractions renal irradiation schedule, effects on drug clearance were first shown 6 months after irradiation. The effect on drug clearance was apparent only after the first evidence of renal dysfunction, as changes in renal function were seen at 3 months. The changes in drug clearance, however, occur well before lifethreatening radiation nephritis develops, as no significant mortality from renal irradiation appears until 11 months (Fig. 1A). When rats are treated with lower doses of renal radiation, renal function abnormalities occur later as do changes in MTX clearance (J. E. Moulder and J. S. Holcenberg, unpublished observation, May, 1985).

REFERENCES 1. Brown, J.M.: Drug or radiation changes to the host which could affect the outcome of combined modality therapy. Znt. J. Radiat. Oncol. Biol. Phys. 5: 1151-l 163, 1979. 2. Dance, N., Price, R.G., Robings, D., Stirling, J.L.: Betagalactosidase, beta-glucosidase, and N-acetyl-betaglucosamine in human kidney. Clin. Chem. Acta 24: 189-197,1969.

Finney, D.J.: Probit Analysis, 3rd edition. Oxford, Cambridge Univ. Press, 197 1. Griffin, T.W., Rasey, J.S., Bleyer, W.A.: The effect of photon irradiation on blood-brain barrier permeability to methotrexate in mice. Cancer 40: 1109- 1111, 1977. Holcenberg, J.S., Kun, L.E., Ring, B.J ., Evans, W.E.: Effect

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of hepatic irradiation on the toxicity and pharmacokinetics of adriamycin in children. Int. J. Radiat. Oncol. Biol. Phys. 7: 953-956, 1981. Hrushesky, W.J.M., Levi, F.A., Halberg, F., Kennedy, B.J.: Circadian stage dependence of cis-diamminedichloroplatinum lethal toxicity in rats. Cancer Rex 42: 945-949, 1982. Jonas, A.M., Besch, E.L., Brennan, P.C., Foster, H.L., Knapka, J.J., Kruckenberg, S.M., Serrano, L.J.: Long-term holding of laboratory rodents. Inst. Lab. Animal Resources News 29: l-26, 1976. Kamen, B.A., Moulder, J.E., Kun, L.E., Ring, B.J., Adams, S.M., Fish, B.L., Holcenberg, J.S.: Effects of single-dose and fractionated cranial irradiation on rat brain accumulation of methotrexate. Cancer Rex 44: 5092-5094, 1984. Kamen, B.A., Takach, P.L., Vatev, R., Caston, J.D.: A rapid, radiochemical-ligand binding assay for methotrexate. Anal. Biochem. 70: 54-63, 1976.

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10. Moulder, J.E., Fish, B.L., Abrams, R.A.: Renal toxicity following total body irradiation and syngeneic bone marrow transplantation. Transplantation (In press). 11. Pera, M.F., Harder, H.C.: Analysis for platinum in biological

material by flameless atomic absorption spectroscopy. Clin. Chem. 23: 1245-1249, 1977. 12. Perrier, D., Mayerson, M.: Non-compartmental determination of steady-state volumes-of-distributions for any mode of administration. J. Pharm. Sci. 71: 372-373, 1982. 13. Rubin, P.: Late effects of chemotherapy and radiation therapy: a new hypothesis. Int. J. Radiat. Oncol. Biol. Phys. 10: 5-34, 1984. 14. Sheldon, P.W., Hill, S.A., Moulder, J.E.: Radioprotection by pentobarbitone sodium of a murine tumor in vivo. Int. J. Radiat. Biol. 32: 571-575, 1976.