Optimizing a therapy for opiate use disorders: Characterizing ondansetron pharmacokinetics in blood and brain

Abstract

Administration of a widely used 5‐hydroxytryptamine receptor (5HT_3AR) antagonist (ondansetron) potently inhibited the development of experimentally induced opioid dependence and withdrawal responses in mice and humans. However, in several studies examining withdrawal symptoms in subjects with chronic opioid use disorders (OUDs), ondansetron exhibited reduced or absent efficacy. Because attenuation of opioid withdrawal symptomatology is mediated within the brain, this study examined single‐dose ondansetron pharmacokinetics in the blood and brain of mice. We demonstrate that ondansetron concentrations in the brain (C _brain ng/mg) are 1000‐fold lower than the blood concentrations (C _blood ng/ml) and decrease rapidly after ondansetron administration; and that a large percentage of brain ondansetron remains in the ventricular fluid. These results indicate that the ondansetron dose, and the time window between ondansetron and opioid administration, and when withdrawal is assessed are critical considerations for clinical studies involving subjects with chronic OUD. The pharmacokinetic results and the dosing considerations discussed here can be used to improve the design of subsequent clinical trials, which will test whether a more prolonged period of ondansetron administration can provide a desperately needed therapy that can prevent the development of neonatal opioid withdrawal syndrome in babies born to mothers with chronic OUD.

Abbreviations

5HT_3AR

5‐hydroxytryptamine receptor

CNS

central nervous system

CSF

cerebrospinal fluid

DESI‐MSI

desorption electrospray ionization‐mass spectrometry imaging

SEM

standard error of the mean

Study Highlights.

• WHAT IS THE CURRENT KNOWLEDGE ON THE TOPIC?

We have previously demonstrated that ondansetron can potently inhibit opioid‐induced dependence and withdrawal symptoms through an effect on 5HT_3A receptors in brain. However, very little is known about ondansetron pharmacokinetics in brain.

• WHAT QUESTION DID THIS STUDY ADDRESS?

We examined single‐dose ondansetron pharmacokinetics in the blood and brain of mice.

• WHAT DOES THIS STUDY ADD TO OUR KNOWLEDGE?

We found that: ondansetron concentrations in brain are 1000‐fold lower than the blood concentrations; brain ondansetron concentrations decrease rapidly after ondansetron administration; and a large percentage of brain ondansetron remains within the ventricular fluid, which means that it does not have access to neuronal 5HT_3A receptors.

• HOW MIGHT THIS CHANGE CLINICAL PHARMACOLOGY OR TRANSLATIONAL SCIENCE?

These results indicate that the ondansetron dose, and the time window between when ondansetron and opioids are administered, and when opioid withdrawal is assessed are critical considerations for clinical studies involving subjects with chronic opioid exposure. The dosing considerations discussed here can be used to improve the design of subsequent clinical trials, which will test whether a more prolonged period of ondansetron administration can provide a desperately needed therapy that can prevent the development of the Neonatal Opioid Withdrawal Syndrome (NOWS) in babies born to mothers with an opioid use disorder.

INTRODUCTION

Because of the widespread (>9 million people in the United States) misuse of opioid medications, opioid use disorders (OUDs) are a substantial public health problem. ¹ Because pregnant women with OUD are typically treated with long‐acting opioids, this public health problem has impacted their babies. ² The neonatal opioid withdrawal syndrome (NOWS) is a set of symptoms that develops in infants born to mothers with OUD. Upon birth, approximately half of these infants require pharmacologic treatment of withdrawal symptoms with opioid medications, which necessitates a prolonged hospitalization (range: 7–49 days, mean 24 days). ³ Despite recent signs of a plateau in NOWS incidence, the rate remains stubbornly high at 8.8 per 1000 live births, which generates >31,000 cases a year in the United States. ⁴ Because the annual cost of caring for NOWS neonates in the United States is ~$0.5 billion, ⁵ an intervention that decreased the number of NOWS infants that require a prolonged period of inpatient treatment with opioids would reduce healthcare costs. Furthermore, as discussed in a JAMA perspective, ⁶ administration of long‐acting opioids to neonates at a critical time for brain development is not without potential (and possibly significant) downstream complications.

Most previous and current NOWS research programs examine the effect of altering the dose (or type) of opioid used for treating NOWS infants. ⁷ However, we analyzed a murine genetic model of opioid dependence and discovered that administration of a selective 5‐hydroxytryptamine receptor (5HT_3AR) antagonist (ondansetron) could dramatically reduce opioid withdrawal symptoms and opioid‐induced hyperalgesia, and could prevent opioid dependence. ⁸ Although most interventions that show efficacy in animal models do not replicate when tested in humans, ⁹ two human translational studies confirmed our murine findings. Administration of a single dose of ondansetron ¹⁰ or of a structurally unrelated 5HT_3AR antagonist (palonosetron) ¹¹ blocked experimentally induced opiate withdrawal symptoms in healthy human volunteers. Ondansetron has a well‐established safety record in large populations. It is widely used to treat chemotherapy‐, pregnancy‐, or anesthetic‐induced nausea ¹² , ¹³ , ¹⁴ or pruritis ¹⁵ during labor. Ondansetron has been used extensively in pediatric patients, and the risk of adverse effects has been quite low. ¹⁶ , ¹⁷ , ¹⁸ We previously identified a dosing regimen that would produce ondansetron exposures in neonates that are equivalent to that of an effective anti‐emetic ondansetron dose (4 mg p.o. b.i.d.) in adults, ¹⁹ and demonstrated that this dosing regimen produced plasma ondansetron levels in babies that were within the range required for anti‐emetic efficacy. ²⁰ Based upon these findings, we conducted a randomized, placebo‐controlled clinical trial to determine whether a brief period of ondansetron treatment would reduce NOWS expression in at‐risk infants. Ondansetron treatment reduced NOWS symptom severity (p < 0.02), and there were no safety issues associated with its administration to neonates. ²⁰ Although there were concerns about potential cardiac abnormalities in neonates resulting from ondansetron treatment, this study demonstrated that there were no adverse cardiac events and the QTc interval was not altered by ondansetron treatment. Although ondansetron administration caused a 20% reduction in NOWS symptom severity, its efficacy in the setting of a chronic OUD was far less than was observed in our prior mouse and human studies ¹⁰ , ¹¹ where administration of 5HT_3AR receptor antagonists significantly inhibited or completely ablated all experimentally induced opioid withdrawal responses exhibited by the subjects.

It was possible that the reduced efficacy in the NOWS trial resulted from the use of an ondansetron dosing regimen that did not produce a sufficient level of ondansetron in the brain over a long enough period to inhibit opioid withdrawal. Although the ondansetron exposures were adequate for anti‐emetic efficacy, which is mediated by ondansetron's action at peripheral sites (vagal afferents in the gastrointestinal tract), ²¹ , ²² our murine studies demonstrated that ondansetron's effect on opioid withdrawal occurs within the central nervous system (CNS). ⁸ In addition, despite its favorable pharmacological properties (i.e., it has moderate levels of protein binding and ionization at plasma pH and good lipid solubility); ondansetron's rate of CNS entry in a prior human study was far below that predicted by its intrinsic properties. ²³ Therefore, to investigate the possibility that a different dosing regimen is required for inhibiting NOWS expression, we characterized single dose ondansetron pharmacokinetics in the brain and blood of mice.

MATERIALS AND METHODS

Chemicals

Ondansetron and its d3‐internal standard were purchased from Toronto Research Chemicals; LCMS grade acetonitrile and water were obtained from Honeywell Burdick and Jackson International; and formic acid was purchased from Pierce Thermo Scientific. Ondansetron and ondansetron‐d3 were dissolved in a 100% methanol solution for use in these experiments.

Mouse pharmacokinetic experiments

All animal experiments were performed according to protocols that were approved by the Stanford Institutional Animal Care and Use Committee. The results of these experiments are reported according to the ARRIVE guidelines. ²⁴ C57BL/6 mice were used at 8–12 weeks of age. Ondansetron (5, 10, or 25 mg/kg) was administered to the mice by intraperitoneal injection. For blood analysis, 8 μl of blood obtained by pinprick was placed in an 8 μl EDTA‐coated glass capillary tube (Vitrex Medical A/S). Blood samples were obtained just before ondansetron administration; and at 15, 30, 60, and 120 min after dosing. The capillary tube was placed within a centrifuge tube with 72‐μl of a solution that consisted of 25% isopropanol in water. The mixture was vortexed thoroughly, and subsequent dilutions were prepared from the 1/10 diluted sample. An internal standard (D3‐ondansetron) was added to the diluted samples to a final concentration of 50 ng/ml. A calibration curve with concentrations ranging from 647 to 2.5 ng/ml was made. The lower limit of quantification was estimated at 5.1 ng/ml. Known concentrations of ondansetron at low and high levels were spiked in the same matrix as the quality controls to validate the method. Three volumes of ice‐cold acetonitrile were added, proteins were precipitated, and the mixture was centrifuged. The supernatant was evaporated to dryness, and then re‐suspended in 5% acetonitrile, and 0.1% formic acid in water. The samples were analyzed on an Agilent QTOF 6545 equipped with an Infinity 1290 UPLC system. Drug concentrations were calculated relative to those of a standard curve using Agilent QTOF Quantitative Analysis software. We also demonstrated that the ondansetron levels measured in the whole blood samples were completely concordant with measurements made using simultaneously obtained mouse plasma (100 μl per sample; Figure S1). At the terminal timepoint, mice were anesthetized with isoflurane; the brains were quickly extracted; and were flash‐frozen using liquid nitrogen and then stored at −80°C. For desorption electrospray ionization‐mass spectrometry imaging (DESI‐MSI) scans, the brains were sectioned with a cryostat to produce 20 μm sections, which were placed on glass slides and stored at −80°C.

Mouse brain tissues of known weight were homogenized in methanol using a Precelly 24 (Bertin Technologies) homogenizer. Fifty microliters of homogenate were aliquoted, and ondansetron‐d3 was added as internal standard to a final concentration of 50 ng/ml. After the mixture was incubated with three volumes of cold acetonitrile at −20°C, the solution was centrifuged at 14,000 x g for 10 min. The supernatant was dried in a speed vacuum and re‐suspended in the original volume. A standard curve was prepared using brain homogenate from an untreated mouse with a linear range of 0.5–250 ng/ml. Statistical analysis of the ondansetron measurements was performed using the formulas in Excel (Microsoft).

Desorption electrospray ionization‐mass spectrometry imaging

The DESI‐MSI of the mouse brains was performed using a Waters DESI‐XS source coupled with a SELECT SERIES Cyclic Ion‐Mobility mass spectrometer. The data were collected in positive ion mode. The spatial resolution for the imaging was set at 125 μm, and the rate was set at 125 μm/s. A solution (90% methanol, 5% acetonitrile, 5% water, 0.01% formic acid, and 100 pg/μl of leucine enkephalin [Leu/Enk]) was delivered at 5 μl/min by a Hamilton syringe pump. The collected data were processed to produce the two‐dimensional images using Waters High‐Definition Imaging software. The mass accuracy of ondansetron (m/z 294.1601) was corrected by using Lue/Enk (556.2771) as the reference mass.

RESULTS

Ondansetron pharmacokinetics in blood and brain

A single dose of ondansetron (5, 10, or 25 mg/kg i.p.) was administered to C57BL/6 mice, and ondansetron concentration levels in blood (C _blood in ng/ml) and brain (C _brain in ng/mg brain tissue) were measured at various times after dosing (Figure 1, Figure S1, Table 1). The correlation between plasma ondansetron levels and anti‐emetic efficacy are not absolute, but trough plasma levels measured in patients treated with ondansetron doses that exhibited anti‐emetic efficacy were between 10 and 200 ng/ml. ²⁵ Blood ondansetron concentrations in the mice were above that required for anti‐emetic efficacy for over 60 min at all doses examined. In contrast, the C _brain levels were quite low and decreased more rapidly than the C _blood levels (Figure 1). The C _blood/C _brain ratio was >1000 at all doses and timepoints where brain and blood samples were simultaneously obtained.

FIGURE 1.

Ondansetron pharmacokinetics in blood and brain. Ondansetron (5, 10, or 25 mg/kg IP) was administered to C57BL/6 mice, and the ondansetron concentrations (ng/ml) in blood (a) or brain (ng/mg tissue) (b) samples were measured at the indicated time after dosing. Each datapoint is the average ± SEM of the measurements obtained from three mice per group. Note that the scales used for the brain sample data (ng/mg tissue) are over 1000‐fold lower than those used for the blood samples (ng/ml). In addition, the blood ondansetron concentration remained above that required for anti‐emetic efficacy (10–200 ng/ml) for over 60 min at all doses tested.

TABLE 1.

Ondansetron levels in blood (ng/ml, C_blood) and brain (ng/mg tissue, C_brain) were measured at the indicated times after a single i.p. dose of ondansetron (10 mg/kg); or after 5 or 25 mg/kg doses were administered to C57BL/6 mice

Minutes	Brain		Blood		Cblood/Cbrain
	ng/mg	SEM	ng/ml	SEM
15	1.6	0.1	2013	141	1241
30	1.3	0.1	1427	145	1122
60	0.4	0.1	477	59	1212
120	0.1	0.0	206	37	2010

Dose (mg/kg)	Min	5	5	Min	25	25
		#1	#2		#3	#4
Blood ng/ml	30	234.6	724.2	30	4895	5745
Brain ng/kg	40	0.22	0.54	35	3.7	4.1
Cblood/Cbrain		1063	1340		1310	1411

Note: The ratios of the ondansetron concentration in blood and brain (C_blood/C_brain) were >1000 at all doses and times assessed.

Ondansetron distribution in brain

To characterize ondansetron's distribution in the brain, DESI‐MSI ²⁶ was performed on brain tissue sections obtained from mice after administration of a single dose of ondansetron. In DESI‐MSI, a thin tissue section placed on a slide is bombarded with solvent microdroplets to dissolve hundreds of chemicals from the sample surface; the secondary microdroplets that are formed enter a mass spectrometer and their chemical content is analyzed. A movable stage enables multiple pixels (in a row by column matrix) across the entire thin section to be sampled, which generates a detailed two‐dimensional chemical map of the drug distribution within the brain section. The measured amount of ondansetron in each pixel is used to produce an image of its relative abundance throughout the brain section. We previously used DESI‐MSI to characterize a genetic mechanism underlying susceptibility to a drug‐induced CNS toxicity. ²⁷ DESI‐MSI revealed that the highest ondansetron concentrations in the brain were within the ventricular fluid 35 min after the mice received a very high dose of ondansetron (25 mg/kg i.p.; Figure 2), which is when the pharmacokinetic analysis indicated that brain ondansetron levels were rapidly decreasing. Of note, this image is shown for illustrative purposes because this ondansetron dose is six‐fold higher than the dose (4 mg/kg i.p.) we previously demonstrated was sufficient to reduce opiate responses in C57BL/6 mice. ⁸ Very low ondansetron levels were detected in brain sections obtained 15 min administration of a 10 mg/kg i.p. dose of ondansetron, and ondansetron was virtually undetectable in brain sections prepared 30 and 60 min after dosing (Figure 3a). The results of analyzing sequential DESI‐MSI obtained after administration of the highest ondansetron dose (25 mg/kg i.p.; Figure 3b) were consistent with the brain pharmacokinetic results: a very low level of ondansetron was present in brain 60 min after dosing, and ondansetron was undetectable at the 120 min timepoint.

FIGURE 2.

(a) Top: A brain sagittal section (20 μM thickness) was prepared from a mouse 35 min after administration of ondansetron (25 mg/kg i.p.), and the slide with a tissue section is shown. The location of the frontal cortex (FC) and cerebellum (CB) are indicated. Bottom: The DESI‐MSI image of this brain section is shown. Brain regions with the highest (yellow), next highest (red) and low/absent (blue) amounts of ondansetron are visualized in this brain section. (b) A diagram from http://gensat‐public.rockefeller.edu/imagenavigator.jsp?imageID=2058 is superimposed upon the DESI‐MSI obtained for this brain section. It is noteworthy that areas with highest ondansetron concentrations are within CSF‐containing lateral septum and the third and fourth ventricles. CSF, cerebrospinal fluid; DESI‐MSI, desorption electrospray ionization‐mass spectrometry imaging; NAc, nucleus accumbens; VTA, ventral tegmental area

FIGURE 3.

(a) DESI‐MSI of sagittal brain sections obtained from mice 15, 30, or 60 min after ondansetron administration (10 mg/kg i.p.). Brain regions with the highest (yellow), next highest (red), and low/absent (blue) amounts of ondansetron are visualized. It is noteworthy that ondansetron is virtually absent in brain tissue obtained 30 and 60 min after ondansetron dosing. (b) Top: Brain slide orientation is shown on the slide, and the measured amount of ondansetron (ng per mg brain tissue) in brain sections obtained from 30, 60, or 120 min after ondansetron administration (25 mg/kg i.p.). Bottom: DESI‐MSI of brain sections obtained from mice 30, 60, or 120 min after administration of a high dose of ondansetron (25 mg/kg i.p.). Consistent with the brain pharmacokinetic measurements, only a minimal amount of ondansetron is detected in the brain at 60 min after dosing, and ondansetron is undetectable at 120 min after dosing. DESI‐MSI, desorption electrospray ionization‐mass spectrometry imaging

DISCUSSION

This pharmacokinetic analysis reveals that ondansetron levels in the brain are quite low (relative to blood) and decrease rapidly after a single dose of ondansetron is administered, and a large percentage of brain ondansetron remains within the ventricular fluid. Our results reveal why different types of studies examining ondansetron's effect on opioid withdrawal symptoms have produced divergent results. In our prior studies involving healthy human volunteers, experimentally induced opioid withdrawal symptoms were completely ablated ¹⁰ , ¹¹ when a single dose of a 5HT_3AR antagonist was administered prior to opioid exposure. This dosing schedule enabled a sufficient level of the 5HT_3AR antagonist to be present in the brain at the time of opioid exposure. In contrast, in our NOWS study, ²⁰ the fetus of a mother with a chronic OUD is continuously exposed to opioids throughout the pregnancy, but there is only a brief period at the end of the pregnancy when a single dose of ondansetron was administered to the mother. This dosing regimen produces fetal brain ondansetron levels that could inhibit opioid responses for only a short period of time; it was not long enough to cause a sufficient reduction in withdrawal symptoms that would eliminate the need for treating the infant with a prolonged course of opioids. Similarly, another study examining individuals with chronic OUD due to chronic back pain (with a flawed experimental design ²⁸ ), administered a single dose of ondansetron just before opioid withdrawal was experimentally induced, and found that ondansetron treatment did not decrease the opioid withdrawal symptoms. ²⁹ To prevent opioid withdrawal from developing after chronic opioid use, ondansetron must be present in the brain when opioids are administered over a more prolonged period; it cannot only be administered for a short period when the opioids are withdrawn. To robustly inhibit NOWS expression in babies born to mothers with a chronic OUD, our brain pharmacokinetic data and other considerations indicate that a more prolonged period of ondansetron treatment given to the mother prior to delivery should be explored in subsequent clinical trials. Moreover, because its anti‐opioid effect requires ondansetron to be present in the brain, a higher dose of ondansetron may be required for inhibiting NOWS expression than is required for its anti‐emetic effect. These dosing alterations will ensure that a sufficient level of ondansetron is present in the fetal brain for a long enough period to inhibit NOWS expression after delivery when maternal opioids are no longer available. Of note, this study examined mice that are equivalent to human adults. Hence, it may be useful for subsequent studies to examine younger mice in order to provide a better model for neonates.

Our DESI‐MSI results indicate that a large percentage of brain ondansetron is within the ventricular fluid, which means that it is not available for inhibiting opioid responses. The passage of drugs from blood into brain is regulated by a specialized multicellular vascular structure (i.e., the blood–brain barrier [BBB]) that is essential for maintaining brain function. ³⁰ , ³¹ , ³² Small lipophilic drugs readily enter the brain by passive diffusion. However, because the CNS to blood efflux transporters exist for nearly all types of molecules, many drugs accumulate in the brain at a much lower rate than expected based upon their physiochemical properties. ³¹ Ondansetron is a known substrate for the ABCB1 transporter. ³³ The cerebrospinal fluid (CSF) is produced by the choroid plexus lining the brain ventricles; it flows from the lateral and midline third and fourth ventricles into the cisterna magna, from which it flows through the subarachnoid space and is re‐absorbed into the venous circulation. Of importance, the blood‐CSF barrier is anatomically and functionally distinct from the BBB. Therefore, drug entry into the CSF is not a measure of BBB permeability, and drugs within the CSF do not distribute to brain parenchyma but are returned to the blood. ³⁴ Overall, drugs in clinical development for CNS diseases have a >50% lower probability of reaching the marketplace (7 vs. 15%) relative to drugs for other types of diseases. ³⁵ , ³⁶ Although multiple factors contribute to this problem, the ability to achieve sufficient brain exposure levels has been a contributing factor. Because the BBB is also present in the fetus, ³⁷ the dosing considerations discussed above are directly applicable to candidate drugs for preventing NOWS expression. The time window among ondansetron administration, opioid administration, and when opioid withdrawal is assessed are critical considerations for clinical studies examining ondansetron's effect on opioid withdrawal. The pharmacokinetic results and the dosing considerations discussed here can be used to improve the design of subsequent clinical trials, which will test whether ondansetron administration can provide a desperately needed therapy that can prevent NOWS expression.

AUTHOR CONTRIBUTIONS

G.P. wrote the manuscript. G.P. designed the research. Z.C., M.W., Y.T., and T.L. performed the research. G.P., Y.T., and M.W. analyzed the data.

FUNDING INFORMATION

This work was supported by an NIH/NIDA award (5U01DA04439902) to GP. The funder had no role in the writing of this paper.

CONFLICT OF INTEREST

The authors declared no competing interests for this work.

ETHICS APPROVAL

All animal experiments were performed according to protocols that were approved by the Stanford Institutional Animal Care and Use Committee.

Supporting information

Figure S1

Wu M, Cheng Z, Le AT, Tan Y, Peltz G. Optimizing a therapy for opiate use disorders: Characterizing ondansetron pharmacokinetics in blood and brain. Clin Transl Sci. 2023;16:216‐223. doi: 10.1111/cts.13440