Effects of Morphine on the Disposition of Ampicillin in Mice

ANTMCROBAL AGENTS AND CHEMOTHERAPY, Oct. 1985, p. 489-493 Vol. 28, No. 4 0066-4804/85/100489-05$02.00/0 Copyright X) 1985, American Society for Microbiology Effects of Morphine on the Disposition of Ampicillin in Mice MOSHE GARTYt AND ARYEH HURWTZ* Clinical Pharmacology Division, Department of Medicine, University of Kansas Medical Center, Kansas City, Kansas 66103 Received 11 March 1985/Accepted 16 July 1985 Morphine raised the levels of intravenously administered ampicillin in the plasma of mice. Despite higher ampicillin levels in plasma after administration of morphine, levels of this antibiotic in bile and urine were not elevated. After ligation of the common bile duct, ampicillin levels in plasma were elevated. Morphine caused a further rise in drug levels in plasma of duct-ligated mice. Ampicillin levels in plasma were higher in mice made anephric by prolonged ligation of their external urethras. n such animals, morphine also caused ampicillin levels in plasma to be even higher. These experiments suggest that morphine impairs both renal and hepatobiliary elimination of ampicillin. These effects of morphine were completely reversed by naloxone. n contrast to effects on intravenously administered ampicillin, morphine markedly reduced drug levels in plasma when ampicillin was given by gastric intubation. This resulted from delayed absorption because of retardation of gastric emptying by morphine. Opioids are commonly used clinically with other drugs. Until recently, there has been little information on interactions of opioids with concomitantly administered drugs. We have shown that morphine raises levels of the dye phenol red in plasma by reducing renal blood flow and glomerular filtration (5). Morphine also elevates levels in plasma of intravenously (i.v.) administered anionic dyes which are secreted into bile, including sulfobromophthalein and indocyanine green (6, 7). We therefore chose to evaluate the effects of morphine on the disposition of ampicillin, an antibiotic which is eliminated in both bile and urine. MATERALS AND METHODS Male Swiss Webster mice weighing 25 to 35 g were used in all studies. n each cage, 10 mice were housed over corncob bedding and kept on a 12-h light-dark cycle. Mice were fasted overnight in mesh-bottomed cages before each experiment. Morphine sulfate, naloxone, or saline was injected subcutaneously (s.c.) 30 min before administration of ampicillin sodium. The interval after s.c. injection of morphine was chosen because many narcotic effects are maximal at that time (13). Blood samples were collected in heparinized microhematocrit tubes by orbital sinus puncture. n some experiments, the animals were sacrificed by cervical dislocation after blood collection, and stomach, liver, gallbladder, or urinary bladder contents were removed. Surgical procedures, including bile duct ligation, sham abdominal surgery (laparotomy and intestinal manipulation), and external urethral ligation, were performed under ether anesthesia. Morphine sulfate was obtained from Eli Lilly & Co., ndianapolis, nd.; naloxone hydrochloride was from Endo Laboratories, nc., Garden City, N.Y.; ampicillin sodium was from Wyeth Laboratories, Philadelphia, Pa.; and polyethylene glycol (PEG; Carbowax 4000) was from Union Carbide Corp., New York. All drugs were dissolved in saline (for i.v. or s.c. administration) or in water (for gastric administration) and given at a volume of 5 ml/kg of body weight. Ampicillin was assayed fluorometrically (9) in an * Corresponding author. t Present address: Clinical Pharmacology Unit, Department Medicine "C," Beilinson Medical Center, 49 151 Petah Tiqva, srael. 489 Aminco Bowman fluoromicrophotometer with Corning 7-51 excitation and 3-72 emission filters (Corning Glass Works, Corning, N.Y.). Samples of blood and urine and of homogenates of liver and gallbladder were assayed after appropriate dilution in water. PEG was assayed turbidimetrically (12). Mean ampicillin concentrations in plasma were fit biexponentially by the NONLN program (14). Areas under the plasma concentration-time curves from 0 to 120 min (AUCO_120) were calculated by the trapezoidal rule with AUC120 set at C120/P. (C120 is the mean concentration in plasma at 120 min, and 3 is the terminal exponential constant.) Clearance was calculated as dose/auc0, which is model independent. Data were analyzed for significant differences by analysis of variance and Duncan's test. When only two groups were compared, Student's t test was used. Data expressed in percentages were analyzed nonparametrically for significance by the Mann-Whitney test. RESULTS At 30 min after i.v. administration of ampicillin at a dose of 12.5 to 100 mg/kg of body weight, drug levels in plasma correlated well (r = 0.9997) with ampicillin dose. n all subsequent experiments, ampicillin was given within this dosage range. Morphine (12.5 mg/kg s.c.) raised the levels of ampicillin in plasma (Fig. 1). The entire plasma disappearance curve could not be derived from samples from a single mouse because an excessive volume of blood would have to have been taken. Figure 1, which represents data from 9 to 12 different animals at each time point, was therefore not amenable to statistical analysis of derived pharmacokinetic parameters. Nevertheless, the data show a 33% reduction in the clearance of ampicillin from plasma, from 17.2 ml/min after saline injection to 11.4 m/min after morphine administration. At 30 min after i.v. administration of 100 mg of ampicillin sodium per kg of body weight, levels of the antibiotic in plasma and liver were much higher in morphinetreated mice (Table 1). Despite the elevated concentrations in plasma, antibiotic levels were no higher in bile or urine after morphine treatment. Naloxone, which had no effect on ampicillin levels in plasma, completely reversed the effects of morphine (Fig. 2). Drug levels in plasma after i.v. administration of ampicillin

490 GARTY AND HURWTZ ANTMCROB. AGENTS CHEMOTHER. z 3 1-. 20 40 60 60 oo 20 FG. 1. Effect of morphine on ampicillin disappearance from plasma. Mice were injected with saline or morphine sulfate (12.5 mg/kg s.c.). After 30 min, ampicillin sodium (80 mg/kg) was given i.v. Blood samples were obtained at the times shown. Data are means ± standard error of the mean (n = 9 to 12 at each time point). Morphine-treated mice had higher ampicillin levels in plasma after 20 min (P < 0.05). were elevated at 5 h after bile duct ligation (Fig. 3). Morphine caused a further increase in ampicillin levels in plasma of duct-ligated mice and control animals which had undergone no surgery or laparotomy and visceral manipulation without ligation (sham surgery). Mice rendered functionally anephric after 5 h of urethral ligation had higher levels of ampicillin in plasma (Fig. 4). n such anephric mice, morphine caused further marked elevation of ampicillin levels. n contrast to its effects on the disposition of i.v. administered ampicillin, increasing doses of morphine progressively lowered ampicillin levels in plasma measured 30 min after gastric administration of the antibiotic (Fig. 5). By 60 min after gastric administration of ampicillin, the differences due to morphine were less pronounced, and by 2 h after administration, ampicillin levels in plasma were actually elevated after the higher morphine doses. The reduction in ampicillin levels in plasma at 30 min after gastric administration was associated with a marked increase in gastric retention of the antibiotic and of the nonabsorbable marker PEG (Table 2). The trapping of drug in the stomach resulted in lower levels of ampicillin in the gallbladder and urine. DSCUSSON The present study explored the complex interactions between morphine and the widely used antibiotic ampicillin. TABLE 1. Effect of morphine and naloxone on disposition of ampicillin administered i.v. Ampicillin level in: Treatment' Plasma Urine Gallbladder Liver (>Lg/ml) (mg) (Lg) (ttg/g) Saline 53.5 ± 5.0 1.86 ± 0.13 30.1 ± 4.5 117.6 ± 9.3 Morphine 73.0 ± 3.8b 1.67 ± 0.11 24.3 ± 4.5 212.3 ± 17.lb Morphine 55.6 ± 3.5 2.09 ± 0.77 30.1 ± 4.1 113.7 ± 7.2 + naloxone asaline or morphine sulfate (10 mg/kg s.c.) or morphine (10 mg/kg) plus naloxone (lmg/kg s.c.) was given to mice 30 min before the administration of ampicillin sodium (100 mg/kg i.v.). Urethral ligation was done under ether anesthesia just before narcotic administration. Mice were bled and sacrificed 30 min after administration of ampicillin. n = 8 in each group. b P < 0.01 compared with saline control. Ampicillin is administered orally and parenterally. After distribution, it is eliminated rapidly by active secretion into bile and urine. Morphine caused levels of ampicillin in plasma to rise and reduced ampicillin clearance by one-third after i.v. administration. Since only mean concentration data could be obtained in mice, only the model-independent clearance calculation based on area under the plasma concentration-time curve was considered to be a meaningful parameter. Volumes of distribution and elimination rates based on mean data have been shown to be inappropriate (3). Furthermore, for drugs with multicompartmental characteristics, such as ampicillin, biologic half-life and volume of distribution are hybrid parameters, and "neither is useful for quantitating true changes in distribution space or elimination rates" (10, 15). Evidence for a renal effect of morphine is provided by the # 50 3 3o z 20-6 o -,.. TME (nmn) FG. 2. Effects of morphine and naloxone on ampicillin levels in plasma. Mice were injected s.c. with saline or morphine sulfate (12.5 mg/kg), naloxone hydrochloride (1 mg/kg), or both morphine and naloxone. After 30 min, ampicillin sodium (40 mg/kg) was given i.v., and after another 30 min blood samples were obtained. Data are means ± standard error of the mean (n = 8 in each group). *, P < 0.01 compared with saline-treated mice. *, Morphine; [, saline, A, naloxone plus morphine; O,naloxone plus saline. 120

VOL. 28, 1985 EFFECTS OF MORPHNE ON DSPOSTON OF AMPCLLN 491 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ NO SURGERY SHAM UGATED 60 20 30 60 0 30 60 12 TME (minutes) FG. 3. Effects of bile duct ligation and morphine on levels of ampicillin in plasma. Common bile ducts were ligated under ether anesthesia in one group of mice. Five hours later, saline or morphine sulfate (12.5 mg/kg s.c.) was injected into these mice and into nonligated mice. At 30 min later, ampicillin sodium (100 mg/kg) was given i.v., and after another 30 min the first blood samples were taken. Data are means ± standard error of the mean (n = 6 to 9 at each point)., Saline-treated mice; -----, morphine-treated mice. *, P < 0.05 compared with corresponding saline-treated mice; t, P < 0.005 compared with nonligated mice. absence of higher urinary ampicillin levels, despite transport of a higher concentration of antibiotic to the kidneys in plasma of morphine-treated mice (Table 1). After bile duct ligation, ampicillin levels in plasma were higher, as expected when a major site of elimination is obstructed. Morphine caused further elevation, indicating interference with the one remaining site of elimination, the kidneys (Fig. 3). The renal effect of morphine is in agreement with the report of its impairment of renal blood flow and glomerular filtration (5). Prolonged obstruction of the urinary tract, such as that caused by external urethral ligation, renders an animal functionally anephric (2). n such animals, which are unable to excrete ampicillin in the urine, antibiotic levels were elevated in plasma. Morphine caused a further rise in ampicillin levels by interfering with its hepatobiliary elimi- nation (Fig. 4). This is consistent with the data in Table 1, which failed to show morphine-induced elevation of ampicillin content in the gallbladder, despite higher levels of the antibiotic in plasma and liver. n earlier studies, opioids were found to elevate levels of anionic dyes in plasma and liver (6, 7). Morphine did not reduce renal or biliary excretion of ampicillin by competing for secretory mechanisms. n the presence of morphine, naloxone completely reversed its effects on ampicillin disposition (Fig. 2), indicating involvement of opioid receptors, not of transport sites. Prior studies have shown that morphine reduces clearance of iothalamate and indocyanine green, suggesting opioid-mediated reduction of blood flow in the kidneys and liver (5, 7). Synthetic narcotics with chemical structures different from that of 0- E40 2 30 C) w 20 z -3 la -.~. 1.4t 3 6 30S 60=120 FG. 4. Morphine effects on ampicillin levels in plasma of functionally anephric mice. External urethras were ligated under ether anesthesia in one group of mice. After 5 h, saline or morphine sulfate (12.5 mg/kg s.c.) was injected into these mice and into nonligated mice; 30 min later, ampicillin sodium (20 mg/kg) was given i.v., and after another 30 min, blood samples were taken. Data are means + standard error of the mean (n = 6 to 8 at each point). *, P < 0.05 compared with nonligated mice; t, P < 0.05 compared with corresponding saline-treated mice. *, Ligation plus morphine; 0, ligation plus saline; *, nonligation plus morphine; O, nonligation plus saline. t

492 GARTY AND HURWTZ ANTMCROB. AGENTS CHEMOTHER. TABLE 2. Effect of morphine on distribution of ampicillin given by gastric intubation Ampicillin level of PEG in Treatmenta Plasma Liver Urine Gallbladder Stomach stomach (% of dose) (,ug/ml) (,ug/g) (p.g) (,ug) p.g % of dose Treatment' Stomach ~~~~~~~~~~~~~~~~~~~~~~~Level Saline 17.8 ± 1.3 46.2 ± 4.5 209.7 ± 11.1 3.66 ± 0.38 198 ± 67 4.4 2.8 Morphine' 8.0 ± 1.2 25.3 ± 3.6 64.8 ± 7.0 1.69 ± 0.12 1,960 ± 184 43.6 42.6 a Saline or morphine sulfate (12.5 mg/kg) was given s.c. 30 min before gastric ampicillin (100 mg/kg) in 3% PEG. Mice were bled and sacrificed 30 min after administration of ampicillin. n = 8 in each group. b p < 0.01. morphine also reduced renal and hepatic clearance of compounds with blood flow-limited elimination. These earlier findings support the view that opioid receptors mediate changes in ampicillin clearance by affecting hepatic and renal blood flow. The sites of these receptors and their subtypes remain to be elucidated. The effects of morphine on gastric emptying account for the delayed absorption of ampicillin after its gastric administration. This was proven by increased retention of the antibiotic in the stomach, with reduced levels at all other sites (Table 2). After the initial delay in gastric emptying, C) 4 '0r 0.5)_ 30 60 120 FG. 5. Effects of morphine on levels of ampicillin given by gastric intubation in plasma. Fasted mice were given saline or morphine sulfate s.c. at the doses indicated. After 30 min, ampicillin sodium (100 mg/kg) was given by gastric intubation. Blood samples were obtained at 30, 60, and 120 min after the administration of ampicillin. Data are means ± standard error of the mean (n = 7 in each group). *, P < 0.05 compared with mice which received no morphine (saline controls). Doses of morphine were as follows: 0, 0 mg/kg; *, 2 mg/kg; 0, 5 mg/kg; A, 12.5 mg/kg. \ ampicillin did reach the absorptive sites in the proximal small intestine. Drug levels at later times were therefore elevated in plasma of morphine-treated mice, probably from delayed absorption and impaired elimination, as demonstrated in experiments with i.v. administered antibiotic. The dose of morphine which reduced ampicillin clearance in mice in the present study was 10 to 12.5 mg/kg. This may seem too high for human relevance. When expressed as dose per unit of body surface area, however, the extrapolation from 12.5 mg/kg calculates to less than 1.5 mg/kg in humans (11). This is well below the lethal dose in nontolerant humans. Tolerant humans survive morphine doses reported to be up to 20-fold higher (4). Since tolerance to all the pharmacological effects of narcotics does not develop (8), gastric, renal, and hepatobiliary effects in addicts taking high doses may cause changes in ampicillin disposition. The present studies thus suggest effects of possible clinical significance. Although the delay in absorption of orally administered ampicillin may have been transient, morphine did reduce peak levels of antibiotic. f intermittent high peaks are needed to drive ampicillin into infected sites (1), flattening of the concentration-time curve by morphine may reduce the efficacy of ampicillin. Concomitant administration of opioids may interfere with the desired localization of adequate concentrations of this antibiotic. Despite higher levels of i.v. administered ampicillin in plasma after morphine administration, the present studies suggest that more antibiotic may have to be given to achieve effective concentrations in bile and urine. ACKNOWLEDGMENT We thank S. K. Melethil for extremely valuable assistance in analyzing and interpreting the pharmacokinetic data. LTERATURE CTED 1. Barza, M., J. Brusch, M. G. Bergeron, and L. Weinstein. 1974. Penetration of antibiotics into fibrin loci in vitro.. ntermittent vs. continuous infusion and the effect of probenecid. J. nfect. Dis. 129:73-78. 2. Becker, B. A., and J. E. Gibson. 1967. A simple method for the production of anuria in mice. Proc. Soc. Exp. Biol. Med. 124:296-298. 3. Cocchetto, D. M., W. A. Wargin, and J. W. Crow. 1980. Pitfalls and valid approaches to pharmacokinetic analysis of mean concentration data following intravenous administration. J. Pharmacokinet. Biopharm. 8:539-552. 4. Cox, T. C., M. R. Jacobs, A. E. Leblanc, and J. A. Marshman. 1983. Drugs and drug abuse: a reference text. Addiction Research Foundation, Toronto. 5. Hurwitz, A. 1981. Narcotic effects on phenol red disposition in mice. J. Pharmacol. Exp. Ther. 216:90-94. 6. Hurwitz, A., and H. R. Fisher. 1983. Narcotic effects on hepatic disposition of sulfobromophthalein in rats. J. Pharmacol. Exp. Ther. 227:68-72.

VOL. 28, 1985 EFFECTS OF MORPHNE ON DSPOSTON OF AMPCLLN 493 7. Hurwitz, A., H. R. Fischer, J. D. nnis, S. Ronsse, and Z. Ben-Zvi. 1985. Opioid effects on hepatic disposition of dyes in mice. J. Pharmacol. Exp. Ther. 232:617-623. 8. Jaffe, J. H. 1980. Drug addiction and drug abuse, p. 535-584. n A. G. Gilman, L. S. Goodman, and A. Gilman (ed.), Goodman and Gilman's the pharmacological basis of therapeutics, 6th ed. Macmillan Publishing Co., nc., New York. 9. Jusko, W. J. 1971. Fluorometric analysis of ampicillin in biological fluids. J. Pharm. Sci. 60:728-732. 10. Jusko, W. J., and M. Gibaldi. 1972. Effects of change in elimination on various parameters of the two-compartment open model. J. Pharm. Sci. 61:1270-1273. 11. Klaassen, C. D., and J. Doull. 1980. Evaluation of safety: toxicologic evaluation, p. 21-22. n J. Doull, C. D. Klaassen, and M. 0. Amdur (ed.), Casarett and Doull's toxicology, 2nd ed. Macmillan Publishing Co., nc., New York. 12. Malawer, S. J., and D. W. Powell. 1967. An improved turbidimetric analysis of polyehtylene glycol utilizing an emulsifyer. Gastroenterology 53:250-256. 13. McGilliand, K. L., and A. E. Takemori. 1978. Antagonism by naloxone of narcotic-induced respiratory depression and analgesia. J. Pharmacol. Exp. Ther. 207:495-503. 14. Metzler, C. M., G. L. Elfring, and A. J. McEwen. 1974. A user's manual for NONLN and associated programs. The Upjohn Co., Kalamazoo, Mich. 15. Perrier, D., and M. Gibaldi. 1974. Clearance and biologic half-life as indices of intrinsic hepatic metabolism. J. Pharmacol. Exp. Ther. 191:17-24.