Rieta Van Bree

Pharmacokinetics of chemotherapeutic agents in pregnancy: a preclinical and clinical study

Objective. To determine the impact of physiologic changes of pregnancy on pharmacokinetics of chemotherapeutic agents. Design. A preclinical and a clinical case–control trial. Setting. Institute of Primate Research Nairobi and collaborating hospitals in Belgium, the Netherlands and Czech Republic. Population. Pregnant and nonpregnant women and baboons receiving chemotherapy. Methods. Chemotherapy pharmacokinetics was compared between the pregnant and nonpregnant state. Standard‐dosed chemotherapy regimens were administered in pregnant and nonpregnant baboons/women, followed by serial blood samplings. Drug plasma levels were determined using high performance liquid chromatography and atomic absorption spectrometry. Main outcome measures. Area under the curve (AUC), maximal plasma concentration, terminal elimination half‐life, clearance and distribution volume of each drug in pregnant and nonpregnant state. Results. Intraindividual comparative pharmacokinetic data were obtained for doxorubicin and paclitaxel/platinum in three and two baboons, respectively. In the clinical trial, two patients were exposed to doxorubicin and one patient was exposed to paclitaxel/platinum during and after pregnancy. Furthermore, a pooled analysis was performed based on 16 cycles of pregnant and 11 cycles of nonpregnant women. Numbers of pregnant/nonpregnant patients were 5/2, 7/5, 4/4 and 2/2 for paclitaxel, doxorubicin, epirubicin and platinum, respectively. For all drugs tested in the preclinical and clinical study, a decreased AUC and maximal plasma concentration and an increased distribution volume and clearance were observed in pregnancy. Conclusions. Although numbers were too small for statistical significance, pregnancy‐associated physiologic alterations appear to lead to a decrease in plasma exposure of chemotherapeutic drugs. The importance of long‐term follow‐up of women treated with chemotherapy during pregnancy is underscored.

Substantial variation in transplacental transfer of chemotherapeutic agents in a mouse model.

Objective: Data on the transplacental transfer of chemotherapeutic agents are lacking. We aimed to measure the maternofetal transfer of cytotoxic drugs in a mouse model. Study design: The transplacental transfer of doxorubicin (9 mg/kg), epirubicin (11 mg/kg), vinblastine (6 mg/kg), carboplatin (50 mg/kg), paclitaxel (10 mg/kg), and cytarabine (100 mg/kg) was tested in a C57/Bl6J mouse model. Ninety minutes after intravenous (IV) drug injection on gestational day 18.5, maternal and fetal blood were collected simultaneously. Plasma drug levels were determined using high performance liquid chromatography or atomic absorption spectrometry. Results: Fetal plasma concentrations of doxorubicin, epirubicin, vinblastine, and cytarabine were 5.1% ± 0.6% (n = 8), 4.8% ± 3.8% (n = 8), 13.8% ± 5.8% (n = 6), and 56.7% ± 22.6% (n = 6) of the maternal concentrations, respectively. Total platinum passed the mouse placenta easily (117.0% ± 38.9%, n = 6). Paclitaxel could not be detected in fetal plasma samples (n = 6). Conclusions: Substantial variations in transplacental transfer were noted among the tested drugs. Current findings contribute to the understanding of reported pregnancy outcomes in humans.

Activation of TRPM3 by a potent synthetic ligand reveals a role in peptide release

Significance The cation channel TRPM3 is highly expressed in the sensory system, where it plays a key role in the detection of noxious heat and the development of inflammatory heat hypersensitivity. Our understanding of the physiological role of TRPM3 in the sensory system and other tissues is hampered by the lack of potent pharmacologic tools, however. This study describes CIM0216, a small-molecule TRPM3 agonist. Our results indicate that CIM0216 is much more potent than established TRPM3 agonists, particularly owing to its ability to open two distinct cation-permeable pores in TRPM3. Using CIM0216 as a pharmacologic tool, we reveal that activation of TRPM3 evokes the release of calcitonin gene-related peptide from sensory nerve terminals and of insulin from pancreatic islets. Transient receptor potential (TRP) cation channel subfamily M member 3 (TRPM3), a member of the TRP channel superfamily, was recently identified as a nociceptor channel in the somatosensory system, where it is involved in the detection of noxious heat; however, owing to the lack of potent and selective agonists, little is known about other potential physiological consequences of the opening of TRPM3. Here we identify and characterize a synthetic TRPM3 activator, CIM0216, whose potency and apparent affinity greatly exceeds that of the canonical TRPM3 agonist, pregnenolone sulfate (PS). In particular, a single application of CIM0216 causes opening of both the central calcium-conducting pore and the alternative cation permeation pathway in a membrane-delimited manner. CIM0216 evoked robust calcium influx in TRPM3-expressing somatosensory neurons, and intradermal injection of the compound induced a TRPM3-dependent nocifensive behavior. Moreover, CIM0216 elicited the release of the peptides calcitonin gene-related peptide (CGRP) from sensory nerve terminals and insulin from isolated pancreatic islets in a TRPM3-dependent manner. These experiments identify CIM0216 as a powerful tool for use in investigating the physiological roles of TRPM3, and indicate that TRPM3 activation in sensory nerve endings can contribute to neurogenic inflammation.

The influence of maternal cortisol and emotional state during pregnancy on fetal intrauterine growth

Background:This exploratory study investigates the influence of maternal cortisol and emotional state during pregnancy on fetal intrauterine growth (IUG). We expected higher basal cortisol levels, or more depressive and anxious complaints during pregnancy, to be associated with slower IUG and lower birth weight.Methods:A total of 91 pregnant women were recruited from the antenatal clinic and were seen once each trimester. In addition to psychological assessments, a diurnal cortisol profile was derived from saliva samples. IUG was evaluated using ultrasound.Results:In mid-pregnancy (trimester (T)2), basal cortisol levels significantly predicted the variance of weight (proportion of variance in growth variable explained (PVE) = 11.6%) and body mass index (BMI) at birth (PVE = 6.8%). In late pregnancy (T3) emotional state, particularly depressive symptoms (BMI at birth: PVE = 6.9%; ponderal index (PI) at birth: PVE = 8.2%; head circumference at T3: PVE = 10.3%; head circumference at birth PVE = 9.1%) and attachment (BMI at birth: PVE = 6.9%; PI at birth: PVE = 7.2%) had an influence on growth. Analysis of growth between T2 and T3 showed that attachment and cortisol in T3 had an influence on the variation in increase in estimated fetal weight (PVE = 12.5–8.6%).Conclusion:These data indicate basal cortisol levels were more important in T2 whereas emotional state was more important in T3.

Pancreatic islet transplantation in diabetic pregnant rats prevents acquired malformation of the ventromedial hypothalamic nucleus in their offspring

Exposure to a diabetic intrauterine environment leads to diabetogenic disturbances throughout later life in rats. This is accompanied by a fetally acquired dysplasia of the ventromedial hypothalamic nucleus (VMN) which is decisively involved in the regulation of metabolism. We investigated whether malformation of the VMN is preventable by normalization of gestational hyperglycaemia. Correction of hyperglycaemia in pregnant streptozotocin-diabetic rats was achieved by pancreatic islet transplantation. The number of neurons in the VMN was significantly reduced in adult offspring of non-treated, sham-transplanted mother rats (P<0.05), but did not differ between offspring of islet-transplanted mother rats and offspring of control mothers. In conclusion, prevention of VMN malformation in offspring of islet-transplanted diabetic mothers might be co-responsible for normalization of their glucose homeostasis during life.

Prevention by maternal pancreatic islet transplantation of hypothalamic malformation in offspring of diabetic mother rats is already detectable at weaning

Exposure to gestational diabetes (GD) in rats leads to dysplasia of the ventromedial hypothalamic nucleus (VMN), decisively involved into the regulation of body weight and metabolism. Recently, we have shown here that VMN malformation is absent in adult offspring of GD mothers treated by pancreatic islet transplantation during gestation. We therefore now investigated whether VMN malformation and its prevention are already present at the early postnatal end of the critical hypothalamic differentiation period. Already at weaning, the total number of VMN neurons, the volume of the VMN relative to total brain volume, and the numerical density of neurons in the anterior subnucleus of the VMN were reduced in offspring of sham-transplanted mothers (all P<0.05), but did not differ between offspring of islet-transplanted mothers and controls. No morphometric alterations occurred in the paraventricular hypothalamic nucleus. In conclusion, prevention of VMN malformation in offspring of islet-transplanted diabetic mothers is a direct consequence of normalized maternal metabolism during critical perinatal development.

Functional expression of transient receptor potential channels in human endometrial stromal cells during the luteal phase of the menstrual cycle

STUDY QUESTION Are members of the transient receptor potential (TRP) channel superfamily functionally expressed in the human endometrial stroma? SUMMARY ANSWER The Ca(2+)-permeable ion channels TRPV2, TRPV4, TRPC6 and TRPM7 are functionally expressed in primary endometrial stromal cells. WHAT IS KNOWN ALREADY Intercellular communication between epithelial and stromal endometrial cells is required to initiate decidualization, a prerequisite for successful implantation. TRP channels are possible candidates as signal transducers involved in cell-cell communication, but no fingerprint is available of the functional distribution of TRP channels in the human endometrium during the luteal phase of the menstrual cycle. STUDY DESIGN, SIZE, DURATION Endometrial biopsy samples (previously frozen) from patients of reproductive age with regular menstrual cycles, who were undergoing diagnostic laparoscopic surgery for pain and/or infertility, were analysed. Samples were obtained from the menstrual (Days 1-5, n = 3), follicular (Days 6-14, n = 6), early luteal (Days 15-20, n = 5) and late luteal (Days 21-28, n = 5) phases. In addition, a total of 13 patient samples taken during the luteal phase were used to set up primary cell cultures for further experiments. PARTICIPANTS/MATERIALS, SETTING, METHODS Quantitative real-time PCR (qRT-PCR), immunocytochemistry, Fura2-based Ca(2+)-microfluorimetry and whole-cell patch clamp experiments were performed to study the functional expression pattern of TRP channels. Specific pharmacological agents, such as Δ(9)-tetrahydrocannabinol, GSK1016790A and 1-oleoyl-2-acetyl-glycerol, were used to functionally assess the expression of TRPV2, TRPV4 and TRPC6, respectively. MAIN RESULTS AND THE ROLE OF CHANCE Expression of TRPV2, TRPV4, TRPC1, TRPC4, TRPC6, TRPM4 and TRPM7 was detected at the mRNA level in endometrial biopsies (n = 19) and in primary endometrial stromal cell cultures obtained from patients during the luteal phase (n = 5) of the menstrual cycle. Messenger RNA levels of TRPV2, TRPC4 and TRPC6 were significantly increased (P < 0.01) in the late luteal phase compared with the early luteal phase. Immunocytochemistry experiments showed a positive staining for TRPV2, TRPV4, TRPC6 and TRPM7 in the plasma membrane and in the cytoplasm of primary endometrial stromal cells. Ca(2+)-microfluorimetry revealed significant increases (P < 0.001) in intracellular Ca(2+) levels when stromal cells were incubated with specific activators of TRPV2, TRPV4 and TRPC6. Further functional characterization was performed using whole-cell patch clamp experiments. Taken together, these data provide evidence for the functional activity of TRPV2, TRPV4, TRPC6 and TRPM7 channels in primary stromal cell cultures. LIMITATIONS, REASONS FOR CAUTION Although mRNA levels are detected for TRPV6, TRPC1, TRPC4 and TRPM4, the limited supply of specific antibodies and lack of selective pharmacological agents restricted any additional analysis of these ion channels. WIDER IMPLICATIONS OF THE FINDINGS Embryo implantation is a dynamic developmental process that integrates many signalling molecules into a precisely orchestrated programme. Our findings identified certain members of the TRP superfamily as candidate sensors in the epithelial-stromal crosstalk. These results are very helpful to unravel the signalling cascade required for successful embryo implantation. In addition, this knowledge could lead to new strategies to correct implantation failure and facilitate the development of novel non-hormonal contraceptives. STUDY FUNDING/ COMPETING INTERESTS This work was supported by grants from the Research Foundation-Flanders (G.0856.13N to J.V.), the Research Council of the KU Leuven (OT/13/113 to J.V. and T.D. and PF-TRPLe to T.V.) and by the Planckaert-De Waele fund (to J.V.). K.D.C. and K.H. are funded by the FWO Belgium. None of the authors have a conflict of interest.

Vinblastine and doxorubicin administration to pregnant mice affects brain development and behaviour in the offspring

BACKGROUND Administration of chemotherapy during the fetal phase of pregnancy may put late-developing organs like the central nervous system at risk. METHODS Transplacental transfer of doxorubicin and vinblastine was measured in C57/BJ mice by high-performance liquid chromatographic detection of the drugs in maternal and fetal plasma, 90 min after intravenous injection. Further, doxorubicin, vinblastine or saline were administered to pregnant C57/6J mouse dams on gestational day 17.5. Effects on brain morphology of the offspring were examined at 24h p.i. (immediate phase) and at 4-5 months p.i. (residual phase), using light- and electron microscopy. At the age of 3 months, offspring performed a behavioural test battery addressing neuromotor performance, exploration and anxiety, and learning and memory. RESULTS Fetal plasma levels of doxorubicin and vinblastine reached respectively 5.0+/-0.2% and 13.9+/-2.4% of the maternal plasma levels. In the immediate phase, pathological examination revealed endothelial and perivascular parenchymal damage to the neocortical subventricular zone and a less constant thickening of the leptomeninx, in some cases also cortical lamination defects were noted. Brain histology was within normal limits in the mice of the residual phase group. Behavioural testing revealed subtle differences between drug-exposed and control mice. Grip strength was reduced in drug-exposed mice, but other tests for motor performance were normal. Several exploratory measures were altered, and there were some indications of increased anxiety in the drug-exposed mice. In the passive avoidance task, step-through latency was shorter in the drug-exposed mice, but their normal performance in the Morris water maze indicated that this was probably not due to impaired memory. CONCLUSION The current preclinical data reveal subtle changes in behaviour and transiently also in brain morphology in the mice that were prenatally exposed to vinblastine or doxorubicin.

In Vitro Validation of Survivin as Target Tumor-associated Antigen for Immunotherapy in Uterine Cancer.

Survivin is an antiapoptotic protein, not expressed in terminally differentiated adult tissues, yet overexpressed in several tumors. Therefore, it is an interesting target for immunotherapeutic strategies. In addition to specific overexpression in tumors, tumor survival is mediated by survivin and hence, tumor survival can be tackled by targeting survivin. Survivin expression in uterine cancer was validated by quantitative real-time polymerase chain reaction and immunohistochemistry. In addition, we evaluated survivin immunogenicity by analyzing spontaneous B-cell and T-cell responses in patients. Survivin as a protein was expressed in only a minority of normal tissues, whereas it was being expressed in all of the currently analyzed uterine cancers, both endometrial carcinoma (n=52) and uterine sarcoma (n=52). Survivin RNA transcripts were overexpressed in more aggressive tumors and survivin protein was overexpressed in recurrent endometrial tumors compared with primary tumors. Spontaneous T-cell responses were seen in 10/39 endometrial cancer patients and 3/16 uterine sarcoma patients. In normal controls, T-cell responses were found only in 1 donor (n=21). Although increased antibody titers were found in more aggressive and far-advanced tumors, no differences in B-cell responses were seen. Overall, when compared with normal controls, a B-cell response was only measured in 1/41 uterine sarcoma patients. In conclusion, we currently validated the presence of survivin in uterine cancer. In addition, spontaneous T-cell responses were found in 23.6% of the total patient population. These data indicate that a survivin-specific immune response may be induced spontaneously in patients, further fortifying the eligibility of survivin as an immunotherapeutic target.

Lack of Evidence That Male Fetal Microchimerism is Present in Endometriosis.

Introduction: Fetal microchimerism has been implicated in the etiology of autoimmune diseases. This study was done to test the hypothesis that male fetal microchimerism is present in eutopic and ectopic endometrium (EM) obtained from women with endometriosis but not in eutopic EM from women without endometriosis. Methods: A total of 31 patients were selected, including women with endometriosis (paired eutopic and ectopic EM; n = 19) and women without endometriosis (eutopic EM; n = 12). Tricolor interphase fluorescence in situ hybridization analysis was performed by cohybridization of CEP Y SpectrumAqua and CEP X SpectrumGreen (SG)/CEP Y SpectrumOrange probes. Results: Ectopic EM from women with endometriosis had 75% XX chromosomes (double SG signals) and 25% X chromosomes (single SG signal). Y chromosomes were not observed in any of the eutopic/ectopic endometrial tissues from cases or controls. Conclusions: We were unable to confirm our hypothesis that male fetal microchimerism is present in eutopic and/or ectopic EM obtained from women with endometriosis.