Preferential Delivery of an Opioid Antagonist to the Fetal Brain in Pregnant Mice

Abstract

Prolonged fetal exposure to opioids results in neonatal abstinence syndrome (NAS), a major medical problem requiring intensive care and increased hospitalization times for newborns with NAS. Multiple strategies are currently available to alleviate withdrawal in infants with NAS. To prevent NAS caused by opioid maintenance programs in pregnant women, blocking fetal dependence without compromising the mother's opiate therapy is desirable. Here we tested in pregnant mice whether a peripherally selective opioid antagonist can preferentially enter the fetal brain and, thereby, in principle, selectively protect the fetus. We show using mass spectrometry that 6β-naltrexol, a neutral opioid antagonist with very limited ability to cross the blood-brain barrier (BBB), readily crosses the placental barrier and enters the fetal brain at high levels, although it is relatively excluded from the maternal brain. Furthermore, owing to the late development of the BBB in postnatal mice, we show that 6β-naltrexol can readily enter the juvenile mouse brain until at least postnatal day 14. Taking advantage of this observation, we show that long-term exposure to morphine starting in the second postnatal week causes robust and quantifiable dependence behaviors that are suppressed by concomitant administration of 6β-naltrexol with much greater potency (ID50 0.022-0.044 mg/kg, or 1/500 the applied dose of morphine) than previously demonstrated for either the suppression of central nervous system opioid effects or the induction of withdrawal in adults. These results indicate that peripherally selective opioid antagonists capable of penetrating the placenta may be beneficial for preventing or reducing neonatal dependence and NAS in a dose range that should not interfere with maternal opioid maintenance.

Fig. 1.

6β-Naltrexol levels in embryonic and adult brain and liver at different survival times. (A) 6β-naltrexol levels in embryonic and adult brain at four different survival times after a single drug injection. (B) 6β-Naltrexol levels in embryonic and adult liver at four different survival times after drug injection. In (A) and (B), the curve for adult plasma is superimposed for comparison with solid tissues (note: units for plasma = ng/ml). The difference between embryonic and adult levels of drug was evaluated using a noncompartmental model (AUC; see Results) over all four survival time points (CIs were determined by the bootstrapping method; see Materials and Methods and Results). Secondarily, we note that the differences in brain were mathematically significant at the individual 20-, 45-, and 120-minute survival times using t tests (20 minutes, P < 0.05; 45 minutes, P < 0.01; 120 minutes, P < 0.01). n = 2 embryo brain pools (each independent embryonic sample consists of pooled brains from one litter; see Materials and Methods) and 3 adult brains at 20-minute survival, 3 embryo brain pools and 7 adult brains at 45 minutes, 2 embryo brain pools and 4 adult brains at 120 minutes, and 2 embryo brain pools and 4 adult brains at 240 minutes. n = the same for liver. Thus, in sum the AUC analysis incorporates 9 independent samples of fetal brains and 18 independent samples of adult brain (adult samples = 9 dams plus 9 additional nonpregnant females of matching age), and an equal number of liver samples.

Fig. 2.

6β-Naltrexol levels across tissues and development. (A) Levels of drug in plasma, brain and liver after a single injection at P7, P14, P20, P32, and P50. E17 and adult (>2 months old) data from Fig. 1 were added for comparison purposes. There is a 45-minute survival time for all data. A break was introduced in the plot to accommodate the extremely high level of drug in P7 liver and to better illustrate the broad range of drug levels across all tissues. Drug levels in most tissues at E17, P7, and P14 are significantly higher than in the corresponding tissues at P20, P35, and P50 (P < 0.05 by t test; not indicated in the figure). Similarly, drug levels in all adult tissues are significantly higher than corresponding tissues at P32 (P < 0.05 by t test; not indicated in the figure). (B) A comparison of drug levels in plasma, brain, and liver at P20 and P32 with two survival times, 20 and 45 minutes. Data for embryonic and adult tissues from Fig. 1 have been added for comparison. The top number on the x-axis is age (days) and the bottom number is survival time (minutes). Asterisks in (A) and (B) indicate plasma vs. brain differences at a particular age; *P < 0.05; **P < 0.01. N = 2 for all samples at P7–P50 (except for plasma at P7, n = 1); for E17 and adults, n is indicated in the Fig. 1 legend.

Fig. 3.

Suppression of withdrawal behavior by 6β-naltrexol in juvenile mice. (A) 6β-Naltrexol prevents a dependence behavior, withdrawal jumping, when delivered in combination with morphine. Total jumps were counted over a period of 15 minutes starting immediately after the injection of naloxone to induce withdrawal. We used a two-concentration ramping procedure for the morphine injections with commensurate ramping of 6β-naltrexol (see Materials and Methods). Data are plotted using the higher of the two drug concentrations. Asterisks indicate a significant difference compared with morphine-treated animals with no 6β-naltrexol; *P < 0.05; **P < 0.01. Numbers below the data-points indicate n values. (B) Kaplan-Meier plots indicating a progressive delay in time to first jump with increasing 6β-naltrexol. Note that all concentrations of 6β-naltrexol result in a significant delay relative to morphine alone (P < 0.05 by log-ratio test). (C) Inhibition of weight gain by morphine is alleviated by 6β-naltrexol. The mass of each mouse was determined before and after the 6-day morphine dosing schedule and the percentage weight change was determined. Asterisk indicates significant difference from animals that received morphine but no (“0”) 6β-naltrexol (P < 0.05). n = the same as indicated in (A). In (C), drug concentration is reported as a ratio of 6β-naltrexol to morphine to emphasize the combination treatment.