Objective
Drugs of abuse affect pregnancy outcomes, however, the mechanisms in which cannabis exerts its effects are not well understood. The aim of this study was to examine the influence of short-term (1-2 hours) exposure to cannabidiol, a major phytocannabinoid, on human placental breast cancer resistance protein function.
Study Design
The in vitro effect of short-term exposure to cannabidoil on breast cancer resistance protein in BeWo and Jar cells (MCF7/P-gp cells were used for comparison) was tested with mitoxantrone uptake, and nicardipine was used as positive control. The ex vivo perfused cotyledon system was used for testing the effect of cannabidoil on glyburide transport across the placenta. Glyburide (200 ng/mL) was introduced to maternal and fetal compartments through a recirculating 2 hour perfusion, and its transplacental transport was tested with (n = 8) or without (n = 8) cannabidoil.
Results
(1) Cannabidoil inhibition of breast cancer resistance protein-dependent mitoxantrone efflux was concentration dependent and of a noncell type specific nature ( P < .0001); (2) In the cotyledon perfusion assay, the administration of cannabidoil to the maternal perfusion media increased the female/male ratio of glyburide concentrations (1.3 ± 0.1 vs 0.8 ± 0.1 at 120 minutes of perfusion, P < .001).
Conclusion
(1) Placental breast cancer resistance protein function is inhibited following even a short-term exposure to cannabidoil; (2) the ex vivo perfusion assay emphasize this effect by increased placental penetration of glyburide to the fetal compartment; and (3) these findings suggest that marijuana consumption enhances placental barrier permeability to xenobiotics and could endanger the developing fetus. Thus, the safety of drugs that are breast cancer resistance protein substrates is questionable during cannabis consumption by pregnant women.
The potential consequences of the increasing use of psychoactive substances during pregnancy are a major concern of public health worldwide. Marijuana is the most commonly used illicit drug, before and during pregnancy. The estimated cannabis use by young women of childbearing age and pregnant women varies between 10-20%. Cannabis use preconception, during the first trimester or throughout pregnancy is associated with low birthweight, preterm labor, small for gestational age newborn, and higher rates of admission to the neonatal intensive care unit. However, assessment of the in utero effects of marijuana exposure is complex and often cannot be isolated from possible confounders (such as tobacco, alcohol, and other drugs).
Tetrahydrocannabinol (THC), cannabidiol (CBD), and cannabinol (CBN) are the most prevalent natural cannabinoids. CBD was reported to be well tolerated in high doses in humans, with minimal side effects. Moreover, because of its remarkable lack of psychoactivity and its numerous pharmacologic actions, CBD holds a promising therapeutic potential.
Pharmacokinetics of CBD is complicated and similar to that of THC. Because of their high lipophylicity (the octanol/water partition coefficients of these 2 molecules are very similar), these cannabinoids rapidly accumulate in fat tissue (and are then slowly released to the brain, blood, and other organs). The bioavailability of this cannabinoid depends on the rout of administration, and ranges between 13−19% after oral intake, although inhaled bioavailability is approximately 31%. CBD elimination half-life is about 9 hours, although a half-life of 24−31 hours was also reported, and is it excreted preferentially in the urine.
In vitro evidence suggests that CBD affects ABCG2/Breast cancer resistance protein (BCRP) and other multidrug resistance proteins (MDRs). BCRP is localized to the apical side of syncytiotrophoblasts, and possesses an ATP-dependent efflux activity, removing a broad variety of structurally diverse compounds out of cells. Hence, BCRP is regarded as a part of the placental barrier against xenobiotics. This transporter is associated with syncytial and placental survival, thought to have a protective role by defending cells from proapoptotic injuries (caused by hypoxia, for instance) and preventing differentiation in stem cells (thus maintain pluripotency).
The aim of the present study was to determine the implications of cannabis use during pregnancy (and exposure to CBD) on BCRP functionality in the human placental barrier, using choriocarcinoma cell lines as in vitro and perfused placentas as ex vivo models.
Materials and Methods
Materials
In the current study, we used 2 methods to assess the effect of CBD on human placental BCRP function. Choriocarcinoma BeWo and Jar cells were used as a human placental barrier model for in vitro experiments. They provide an appropriate model in which to study some aspects of human trophoblast physiology without the aspect of interpatient variability. For ex vivo experiments, we used the technique of isolated, perfused human cotyledon, a method of choice for studying placental transport and pharmacokinetics of drugs.
BeWo and Jar cells were obtained from Dr B. Ugele, Ludwig-Maximilians University, Munich, Germany. The MCF7/P-gp cells (BCRP expressing and P-gp induced cells), were kindly provided by Prof Esther Priel (Ben Gurion University of the Negev, Beer Sheva, Israel) and were used for the assessment of the cell specificity of CBD impact. All cell culture reagents were purchased from Biological Industries (Bet Haemek, Israel). Perfusion medium M199, glucose, aminopyrine, antipyrine, ketoconazole, mitoxantrone (MX), MTT, neutral red, DMSO, and glyburide were purchased from Sigma. CBD was a kind gift from Prof Raphael Mechoulam (The Hebrew University, Jerusalem, Israel).
Methods
CBD cytotoxicity determination by MTT or NR
Cellular viability following short and long-term exposures to CBD was determined by MTT (3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide) and Neutral Red (NR) assays, respectively. Long-term exposure to CBD was carried out to properly determine the IC 50 of CBD.
MTT assay: MCF7/P-gp, BeWo and Jar cells were seeded into 96-well plates 72 hours before the experiment and grown to 80% subconfluency. On the day of the experiment culture medium was replaced with CBD containing medium (0-100 μM) for 1 hour. Cells were then washed once with phosphate buffered saline (PBS), MTT reagent (0.2 mL) was added to each well and incubated for 3 hours in the dark. Cells were washed again with PBS, 0.2 mL DMSO was added to each well for solubilization and incubated for 10 minutes at 37°C in the dark. The plate was read by ELISA reader (Bio-Rad) at a wavelength of 570 nm (with reference at 655 nm). Cellular viability was calculated as % of “control” (vehicle treated) cells.
NR assay
Chronic CBD cytotoxicity was determined after 72-hour cell incubation with various CBD concentrations. Cells were seeded in 12-well plates (0.1 × 10 6 cells/well) 96 hours before the experiments. CBD containing medium was added to each plate 24 hours after seeding and replaced every day for 72 hours. Before the NR assay cells were photographed in a light microscope (Nikon Eclipse TS100; Nikon, Melville, NY), then, washed once with PBS. Two milliliters of NR solution (NR in ethanol + appropriate culture medium, 1:100 respectively) were added to each well, and cells were incubated for 3 hours in incubator, in the dark. The cells were washed once with PBS, and 2 mL of NR-extraction solution (ethanol 25% with 1% glacial acetic acid) was added. After 15 minutes of incubation in room temperature and 30 minutes on a shaker, 300 μL from each well was transferred to 96-well plate and read by ELISA reader (Bio-Rad) at a wavelength of 540 nm. Cellular viability was determined as % of “control” (vehicle-treated) cells and IC 50 was calculated ( Appendix ; Supplementary Table 1 and Supplementary Figures 1 and 2 ). CBD concentrations were considered as nontoxic if cell viability remained above 80% compared with vehicle-treated cells.
Cell culture
MCF7/P-gp, BeWo and Jar cells were cultured as previously described. Briefly, for short-term exposure (cellular uptake experiments), the cells were seeded in 6-well plates (Corning, Amsterdam, The Netherlands) (15 × 10 4 –20 × 10 4 cells/well) and grown to confluency. Twenty-four hours before the experiments, the growth media was refreshed.
Cellular uptake assay
Incubation of all cell types was carried out according to protocol detailed in a previous study by our group. Cells were preincubated for 30 minutes with CBD 10 or 25 μM (working concentration previously published, initially dissolved in DMSO) or nicardipine 20 μM (positive control), dissolved in transport buffer (TB) (pH = 7.4), although “control cells” were preincubated in TB with the correlating concentration of DMSO. After preincubation MX (BCRP substrate) was added and cells were further incubated for 30 minutes. At the end of incubation plates were treated as previously detailed, and samples were stored (at −20°C) for further analysis. Intracellular MX fluorescence was quantified by Infinite M200 microplate reader (Tecan, Männedorf, Switzerland) and normalized to protein amount in the sample (determined by Lowry method). Supplementary Figures 3 and 4 provide a summary diagram of uptake experiments.
Placental perfusion assay
The placental perfusions section of our study was approved by the institutional review board of the Soroka University Medical Center. All placentas were collected after obtaining a signed informed consent, immediately after normal vaginal deliveries, of noncomplicated term pregnancies. The perfusion assays were performed using the method previously described. Placentas were examined for visible tissue damage. Within 30 minutes of delivery, maternal and fetal independent circulations were established. In the fetal side vein/artery pairs with minimal branching, supplying the same intact peripheral cotyledon, were cannulated.
Preperfusion period
To remove fetal and maternal residual blood, all the placentas were preperfused for 30 minutes with enriched M199 medium (glucose [1 g/L] and heparin [2000 U/mL]). Subsequently, the perfusion medium in the fetal and maternal compartments was exchanged with the experimental fresh enriched medium and a closed 2 hours recirculating perfusion started. Placentas were perfused with or without CBD (15 μM). The fetal and maternal perfusates were maintained at 37°C and consisted of M199 medium, with heparin (2000 U/L), gentamicin (40 mg/L), and glucose (1.0 g/L). Antipyrine (50mg/L) was added to the maternal perfusate and served as a tissue viability marker (to verify passive diffusion, and thus validate the transport capacity of the placenta during the perfusion). The method for antipyrine quantification in the maternal and fetal compartments is detailed in the Supplementary Materials and Methods . Glyburide, a BCRP substrate, was added at a therapeutic concentration of 200 ng/mL to the maternal and fetal reservoirs. Perfusates were equilibrated with a prehumidified gas mixture of 95% O 2 and 5% CO 2 in the maternal reservoir and 95% N 2 and 5% CO 2 in the fetal reservoir. Fetal artery pressure of 20-60 mm Hg, giving a flow rate of 8 mL/min in the fetal circulation and 12 mL/min in the maternal circulation, was established using an internal pressure monitor (series 50 IP-2; Philips Medizinsystems, Ontario, Canada). Perfusates were kept at physiological pH by the addition of small volumes of hydrochloric acid. During the 2-hour experimental period, 2 mL samples were taken at 0, 5, 15, 30, 45, 60, 90, and 120 minutes from maternal and fetal compartments. Placental tissue viability during the perfusion was assessed by monitoring glucose consumption, lactate, and hCG production, antipyrine diffusion, fetal arterial pressure, and fetal reservoir volume.
Perfusions were excluded if: (1) there was a loss of fetal perfusate volume greater than 5% during the experiment; (2) fetal pressure exceeded the normal range; and (3) there was no antipyrine diffusion during the experiment. Supplementary Figures 3 and 4 present a perfusion system diagram.
Glyburide quantification by high pressure liquid chromatography
Glyburide was quantified using high pressure liquid chromatography (HPLC) method according to previously published method. Chromatographic system composition: solvent delivery intelligent pump (model L-6200), equipped with a 50 μL injection loop, and a fluorescence detector (model F-1050) set to 235 ex nm and 355 em nm (Merck-Hitachi, Tokyo, Japan). A 5 μm, 150 mm x 4.6 mm Zorbax SC-C8 analytical column (Agilent, Santa Clara, CA) was used for separation. The mobile phase consisted of 45% acetonitrile, 40% buffer solution (0.05 M NH 4 H 2 PO 4 ) and 15% methanol, adjusted to pH 5.7 by diluted ammonia solution, and pumped at 1.2 mL/min flow rate, isocratically. The mobile phase was prepared daily and filtered through 0.2 μm membrane filter (Whatman, UK). The chromatograms were acquired and analyzed using D-2500 chromato-integrator (Merck-Hitachi). All work was carried out at room temperature.
Calibration curve: glyburide was dissolved to a stock solution of 1 mg/mL in acetonitrile. Dilutions were made to give 0-500 ng/mL concentrations. The internal standard ketoconazole (IS) stock solution was prepared at 1 mg/mL, in acetonitrile, with further dilutions to a concentration of 50 μg/mL. All standards and stock solutions were prepared once weekly and stored at 4 to 8▫C. To 0.5 mL sample with 25 μL of 20 μg/mL ketoconazole, 100 μL of acetonitrile were added to precipitate proteins. Each sample was then vortex-mixed and centrifuged for 2 minutes at 12,000 g. The supernatant solution was transferred into a clean tube and 50 μL aliquot was injected into HPLC system. Glyburide and ketoconazole retention times were approximately 4 and 7.5 minutes.
Statistical analysis
Statistics and graphs were carried out using GraphPad Prism5 software (GraphPad, San Diego, CA) and Microsoft Excel (Microsoft, Redmond, WA). One-way ANOVA followed by appropriate Bonferroni corrections and Student t test were used when suitable.
Results
CBD cytotoxicity
Short-term exposure of MCF7/P-gp, Jar and BeWo cells to 1-100 μM CBD was associated with >90% viability of these cells ( Table 1 ). To determine the CBD IC 50 for each cell type, we performed long-term exposure of these cells to various concentrations of CBD. Results, including visible morphological appearance of the cells and full CBD concentration list in all 3 cell lines are presented in Supplementary Figures 1 and 2 . The highest non-lethal CBD dose during long-term exposure (72 hours) for all 3 cell lines was 15 μM ( Table 1 ; Supplementary Figure 1 , A-C). Exposure to more than 25 μM CBD was associated with morphologic changes in the cell appearance ( Supplementary Figure 2 , A-C). The current study concentrated on the effect of a short-term exposure to CBD. Therefore, we used 10 μM/25 μM and 15 μM concentration for the in vitro and ex vivo assays, respectively.
| Cell type | Cell viability: short-term (1 h) exposure to 1-100 μM CBD | IC 50 – μM CBD (for long-term [72 h] exposure) | Highest nonlethal dose a (for 72 h exposure) – μM CBD |
|---|---|---|---|
| MCF7/P-gp | >90% | 35.1 | 15 |
| Jar | >92% | 23.5 | 15 |
| BeWo | >95% | 38.7 | 15 |
a Cellular viability >80% compared with vehicle-treated cells. Viability data presented as percentage of viable cell population compared to vehicle-treated (control) cells, from at least 3 independent experiments in each cell line.
Acute exposure to CBD: BCRP inhibition in vitro
Cells were subjected to MX uptake experiments. MX intracellular concentrations were significantly elevated in the presence of CBD 10 and 25 μM, in BeWo, Jar, and MCF7/P-gp cells. CBD 25 μM yielded higher intracellular MX accumulation compared with CBD 10 μM ( P < .0001) ( Figure 1 ). Nicardipine strongest and weakest effects were seen in Jar and MCF7/P-gp cells, respectively ( Figure 1 ). These results correlate with cellular BCRP expression: the highest levels of BCRP expression was observed in Jar cells and the lowest in MCF7/P-gp cells (data not shown).
Acute exposure to CBD: BCRP inhibition ex vivo
Characteristics of all collected placentas are summarized in Table 2 . There were no statistical differences in placental and women characteristics between the CBD group and the “control” group of placentas. In addition, drug transport in both experimental groups was gender independent. CBD presence had no effect on antipyrine diffusion profile (data presented in Supplementary Materials and Methods ), or other viability markers. Antipyrine disappearance from the maternal circulation correlated to antipyrine appearance in the fetal circulation. Placentas perfused with CBD had a higher F/M concentration ratio or glyburide than placentas that were perfused without it, reaching the most significant difference at the end of the 2 hours perfusion ( P < .0001) ( Figure 2 ). F/M ratios of glyburide concentrations with CBD were higher by 44, 40, 60, and 62% at 45, 60, 90, and 120 minutes of perfusion, respectively (eg, F/M ratio 1.3 ± 0.1 vs 0.8 ± 0.1 at 120 minutes, P < .001).
| Characteristics | Control (n = 8) | CBD (n = 8) | P value a |
|---|---|---|---|
| Woman | NS | ||
| Age | 27.4 ± 7.2 | 27.3 ± 4.1 | NS |
| Pregnancy no. | 3.8 ± 4.2 | 3.8 ± 1.9 | NS |
| Pregnancy week | 39.1 ± 1.1 | 39.3 ± 1.3 | NS |
| Newborn | NS | ||
| Weight, g | 3051.9 ± 480 | 3319.2 ± 322 | NS |
| Sex | M-5, F-3 | M-4, F-4 | NS |
| Umbilical pH | 7.4 ± 0.08 | 7.4 ± 0.04 | NS |
| Placenta weight, g | 548.9 ± 112 | 533.7 ± 88 | NS |
| Cotyledon weight, g | 24.0 ± 12 | 24.1 ± 9 | NS |
a Statistical analysis between the “control” and the CBD groups was made using the Student t test.
Results
CBD cytotoxicity
Short-term exposure of MCF7/P-gp, Jar and BeWo cells to 1-100 μM CBD was associated with >90% viability of these cells ( Table 1 ). To determine the CBD IC 50 for each cell type, we performed long-term exposure of these cells to various concentrations of CBD. Results, including visible morphological appearance of the cells and full CBD concentration list in all 3 cell lines are presented in Supplementary Figures 1 and 2 . The highest non-lethal CBD dose during long-term exposure (72 hours) for all 3 cell lines was 15 μM ( Table 1 ; Supplementary Figure 1 , A-C). Exposure to more than 25 μM CBD was associated with morphologic changes in the cell appearance ( Supplementary Figure 2 , A-C). The current study concentrated on the effect of a short-term exposure to CBD. Therefore, we used 10 μM/25 μM and 15 μM concentration for the in vitro and ex vivo assays, respectively.
| Cell type | Cell viability: short-term (1 h) exposure to 1-100 μM CBD | IC 50 – μM CBD (for long-term [72 h] exposure) | Highest nonlethal dose a (for 72 h exposure) – μM CBD |
|---|---|---|---|
| MCF7/P-gp | >90% | 35.1 | 15 |
| Jar | >92% | 23.5 | 15 |
| BeWo | >95% | 38.7 | 15 |
a Cellular viability >80% compared with vehicle-treated cells. Viability data presented as percentage of viable cell population compared to vehicle-treated (control) cells, from at least 3 independent experiments in each cell line.
Acute exposure to CBD: BCRP inhibition in vitro
Cells were subjected to MX uptake experiments. MX intracellular concentrations were significantly elevated in the presence of CBD 10 and 25 μM, in BeWo, Jar, and MCF7/P-gp cells. CBD 25 μM yielded higher intracellular MX accumulation compared with CBD 10 μM ( P < .0001) ( Figure 1 ). Nicardipine strongest and weakest effects were seen in Jar and MCF7/P-gp cells, respectively ( Figure 1 ). These results correlate with cellular BCRP expression: the highest levels of BCRP expression was observed in Jar cells and the lowest in MCF7/P-gp cells (data not shown).
