Placental transfer of tofacitinib in the ex vivo dual-side human placenta perfusion model.

Tofacitinib is a small molecule Janus kinase (JAK) inhibitor, introduced to the European market in 2017, for the treatment of rheumatoid arthritis, psoriatic arthritis and ulcerative colitis. In the treatment of women with autoimmune diseases, pregnancy is a relevant issue, as such diseases typically affect women in their reproductive years. Currently, there is limited data on the use of tofacitinib during pregnancy. To estimate the extent of placental transfer in the absence of clinical data, we conducted ex vivo dual-side perfused human placental cotyledon perfusions. Term placentas were perfused for 180 min with tofacitinib (100 nM, added to the maternal circuit) in a closed-closed configuration. At the end of the perfusions, drug concentrations in the maternal and fetal reservoirs were near equilibrium, at 35.6 ± 5.5 and 24.8 ± 4.7 nM, respectively. Transfer of tofacitinib was similar to that observed for the passive diffusion marker antipyrine (100 µg/mL, added to the maternal reservoir). Final antipyrine maternal and fetal concentrations amounted to 36.9 ± 3.0 and 36.7 ± 1.3 µg/mL, respectively. In conclusion, in the ex vivo perfused placenta tofacitinib traverses the placental barrier rapidly and extensively. This suggests that substantial fetal tofacitinib exposure will take place after maternal drug dosing.

Does REACH provide sufficient information to regulate substances toxic to reproduction?

Hazard classification and risk assessment of substances, is essential to protect workers and consumers from hazardous substances including reproductive toxicants. The ability to classify substances for reproductive toxicity under the current REACH information requirements has been assessed. For low tonnage substances (<10 ton per annum (tpa)) information for classification is insufficient. When only a reproductive screening study is available (10-100 tpa), substances are mostly not classified in Category 1B as developmental and non-potent fertility effects may be missed. The information requirements could be improved by automatic triggering of follow-up studies in case of a Category 2 classification based on a screening study. Additionally, a study could be added to the information requirements for substances produced at 1-10 tpa. Performing a risk assessment is often problematic due to the limited study requirements at low tonnage levels. Only for substances produced at more than 100 tpa, there is a high likelihood to detect reproductive effects and perform accurate risk assessment provided that the extended-one-generation-reproductive-toxicity-study and/or extra cohorts are triggered where required. Regardless of the tonnage level, no specific studies on lactation are required. With this paper we intend to contribute to the discussion on the information requirements for reproductive toxicity in view of the REACH revision.

Predicting fetal exposure of crizotinib during pregnancy: Combining human ex vivo placenta perfusion data with physiologically-based pharmacokinetic modeling.

Commercially available physiologically-based pharmacokinetic (PBPK) modeling platforms increasingly allow estimations of fetal exposure to xenobiotics. We aimed to explore a physiology-based approach in which literature data from ex vivo placenta perfusion studies are used to parameterize Simcyp's pregnancy-PBPK (p-PBPK) model, taking crizotinib as an example. First, a physiologically-based semi-mechanistic placenta (PBMP) model was developed in MATLAB to analyze placenta perfusion data of crizotinib. Mixed-effects modeling was performed to derive intrinsic unbound clearance values across the maternal-placental barrier and fetal-placental barrier. Values were then used for parameterization of the p-PBPK model. The PBMP model adequately described the perfusion data. Clearance was estimated to be 71 mL/min and 535 mL/min for the maternal placental uptake and efflux, and 8 mL/min and 163 mL/min for fetal placental uptake and efflux, respectively. For oral dosing of 250 mg twice daily, p-PBPK modeling predicted a Cmax and AUC0-τ of 0.08 mg/L and 0.78 mg/L*h in the umbilical vein at steady-state, respectively. In placental tissue, a Cmax of 5.04 mg/L was predicted. In conclusion, PBMP model-based data analysis and the associated p-PBPK modeling approach illustrate how ex vivo placenta perfusion data may be used for fetal exposure predictions.

Effects of tumor necrosis factor on undifferentiated and syncytialized placental choriocarcinoma BeWo cells.

Tumor necrosis factor (TNF) regulates trophoblast turnover during the formation of the placental syncytium and can be a potentially relevant target for adverse effects of xenobiotics. We mimicked syncytialization in vitro by stimulating BeWo cells with 50 µM forskolin. Undifferentiated and syncytialized BeWo cells were exposed to TNF (10 pg/mL-10 ng/mL) for 48 h after which cell viability, progesterone release and gene expression of a selected set of markers representative for placental function were assessed. In undifferentiated BeWo cells, high TNF levels (1-10 ng/mL) increased gene expression of TNF, NF-κB, and TNFRSF1B to maximally 99 ± 17, 2.2 ± 0.2, and 3.0 ± 0.4 of control values, respectively (p < 0.001). These effects were also found in syncytialized BeWo cells but less pronounced. Additionally, TNF may induce syncytialization in BeWo cells as it upregulated ERVW-1 expression by 1.55 ± 0.14-fold (p < 0.05). On the contrary, TNF levels of 10 and 100 pg/mL did not affect gene expression in both undifferentiated and syncytialized BeWo cells, but did enhance cell viability in syncytialised BeWo cells (p < 0.001). In conclusion, we found that high TNF levels (1-10 ng/mL) increased gene expression of TNF, NF-κB, and TNFRSF1B especially in undifferentiated BeWo cells, while physiological TNF concentrations positively affected cell viability and while there was no effect on any of the investigated functional markers.

Placental disposition of eculizumab, C5 and C5-eculizumab in two pregnancies of a woman with paroxysmal nocturnal haemoglobinuria.

Eculizumab is known to cross the placenta to a limited degree, but recently therapeutic drug levels in cord blood were found in a single case. We report maternal, cord and placental levels of unbound eculizumab, C5 and C5-eculizumab in two pregnancies of a paroxysmal nocturnal haemoglobinuria patient who received 900 mg eculizumab every 2 weeks. In both pregnancies, cord blood concentrations of unbound eculizumab were below 4 µg/mL, while C5-eculizumab levels were 22 and 26 µg/mL, suggesting that a considerable fraction of C5 was blocked in the newborn. Concentrations in each placenta of unbound eculizumab were 41 ± 3 and 45 ± 4 µg/g tissue, of C5-eculizumab 19 ± 2 and 32 ± 3 µg/g, and of C5 20 ± 3 and 30 ± 2 µg/g (mean ± SD, in three tissue samples per placenta). Placental levels of unbound eculizumab were higher than those of C5-eculizumab complexes, while maternal concentrations were approximately equal, suggesting selective transport of unbound eculizumab across the placenta.

Toxicity of anticancer drugs in human placental tissue explants and trophoblast cell lines.

The application of anticancer drugs during pregnancy is associated with placenta-related adverse pregnancy outcomes. Therefore, it is important to study placental toxicity of anticancer drugs. The aim of this study was to compare effects on viability and steroidogenesis in placental tissue explants and trophoblast cell lines. Third trimester placental tissue explants were exposed for 72 h (culture day 4-7) to a concentration range of doxorubicin, paclitaxel, cisplatin, carboplatin, crizotinib, gefitinib, imatinib, or sunitinib. JEG-3, undifferentiated BeWo, and syncytialised BeWo cells were exposed for 48 h to the same drugs and concentrations. After exposure, tissue and cell viability were assessed and progesterone and estrone levels were quantified in culture medium. Apart from paclitaxel, all compounds affected both cell and tissue viability at clinically relevant concentrations. Paclitaxel affected explant viability moderately, while it reduced cell viability by 50% or more in all cell lines, at 3-10 nM. Doxorubicin (1 µM) reduced viability in explants to 83 ± 7% of control values, whereas it fully inhibited viability in all cell types. Interference with steroid release in explants was difficult to study due to large variability in measurements, but syncytialised BeWo cells proved suitable for this purpose. We found that 1 µM sunitinib reduced progesterone release to 76 ± 6% of control values, without affecting cell viability. While we observed differences between the models for paclitaxel and doxorubicin, most anticancer drugs affected viability significantly in both placental explants and trophoblast cell lines. Taken together, the placenta should be recognized as a potential target organ for toxicity of anticancer drugs.

Assessment of Placental Disposition of Infliximab and Etanercept in Women With Autoimmune Diseases and in the Ex Vivo Perfused Placenta.

Tumor necrosis factor (TNF) inhibitors are increasingly applied during pregnancy without clear knowledge of the impact on placenta and fetus. We assessed placental transfer and exposure to infliximab (n = 3) and etanercept (n = 3) in women with autoimmune diseases. Furthermore, we perfused healthy term placentas for 6 hours with 100 µg/mL infliximab (n = 4) or etanercept (n = 5). In pregnant women, infliximab transferred into cord blood but also entered the placenta (cord-to-maternal ratio of 1.6 ± 0.4, placenta-to-maternal ratio of 0.3 ± 0.1, n = 3). For etanercept, a cord-to-maternal ratio of 0.04 and placenta-to-maternal ratio of 0.03 was observed in one patient only. In ex vivo placenta perfusions, the extent of placental transfer did not differ between the drugs. Final concentrations in the fetal compartment for infliximab and etanercept were 0.3 ± 0.3 and 0.2 ± 0.2 µg/mL, respectively. However, in placental tissue, infliximab levels exceeded those of etanercept (19 ± 6 vs. 1 ± 3 µg/g, P < 0.001). In conclusion, tissue exposure to infliximab is higher than that of etanercept both in vivo as well as in ex vivo perfused placentas. However, initial placental transfer, as observed ex vivo, does not differ between infliximab and etanercept when administered in equal amounts. The difference in placental tissue exposure to infliximab and etanercept may be of clinical relevance and warrants further investigation. More specifically, we suggest that future studies should look into the occurrence of placental TNF inhibition and possible consequences thereof.

Editor's Highlight: PlacentalDisposition and Effects of Crizotinib: An Ex Vivo Study in the Isolated Dual-Side Perfused Human Cotyledon.

Tyrosine kinase inhibitors (TKIs) play an important role in cancer pharmacotherapy, yet there is limited data on their use during pregnancy. We studied placental disposition and placental toxicity of crizotinib, a TKI used to treat nonsmall cell lung cancer. Term placentas were perfused for 3 h with crizotinib (1 µM) using the ex vivo dual-side cotyledon perfusion technique. Interference of TKIs with trophoblast viability was studied using BeWo cells. Expression of P-glycoprotein (P-gp) and Breast Cancer Resistance Protein (BCRP) in placental tissue was assessed by immunohistochemistry and inhibition of these transporters was determined in vitro by transport studies with membrane vesicles overexpressing human P-gp or BCRP. We found that crizotinib rapidly and strongly accumulates in cotyledon perfusion experiments, reaching a concentration of 3.1 ± 0.4 µM in placental tissue. Final drug concentrations in the maternal and foetal reservoirs were 0.2 ± 0.05 and 0.08 ± 0.01 µM, respectively. Furthermore, crizotinib inhibited BeWo cell viability (IC50: 234 nM, 95% CI: 167-328 nM) 10 times more potently than other TKIs tested. In vitro transport studies revealed that crizotinib is a potent inhibitor of the transport activities of BCRP (IC50: 5.7 µM, 95% CI: 2.7-11.8 µM) and P-gp (IC50: 7.8 µM, 95% CI: 3.4-18.0 µM). In conclusion, crizotinib strongly accumulated in placental tissue at clinically relevant concentrations. IC50 values for transporter inhibition and trophoblast cell viability were similar to the tissue concentrations reached, suggesting that crizotinib can inhibit placental BCRP and P-gp function and possibly affect trophoblast viability.