Physiologically-based pharmacokinetic models to predict drug exposure during pregnancy.

As pregnant women are constantly exposed to drugs during pregnancy, either to treat long-term conditions or acute illnesses, drug safety is a major concern for the fetus and the mother. Clinical trials are rarely made in this population due to strict regulation and ethical reasons. However, drug pharmacokinetic (PK) parameters vary during pregnancy with an increase in distribution volume, renal clearance and more. In addition, the fetal distribution should be evaluated with the importance of placental diffusion, both active and passive. Therefore, there is a recent interest in the use of physiologically-based pharmacokinetic (PBPK) modeling to characterize these changes and complete the sparse data available on drug PK during pregnancy. Indeed, PBPK models integrate drug physicochemical and physiological parameters corresponding to each compartment of the body to estimate drug concentrations. This review establishes an overview on the current use of PBPK models in drug dosage determination for the pregnant woman, fetal exposure and drug interactions in the fetal compartment.

Brain tumours: Non-invasive techniques to treat invasive pathologies.

Brain and other central nervous system tumours are cancers of poor prognosis, for which current therapeutic possibilities do not match the expectations regarding a curative objective. If the treatment of central nervous system tumours is so difficult, it is partly due to the blood-brain barrier and the blood-tumour barrier, which need to be crossed to access the tumour. Driven by these insufficient results, more and more techniques and technologies are being explored and are evolving: the progress of surgery and radiotherapy, the growing place of immunotherapies, or the apparition of new non-invasive techniques. The latter are those which interest us here, where promising advances are taking the leap to clinical trials. Nose-to-brain delivery, receptor-mediated transcytosis and micro-bubbles-associated focused ultrasounds are three therapeutic propositions with encouraging results regarding the improvement of drug access to the brain. Even though they might have their share of limits and adverse effects, benefit-risk balance looks promising, and they may appear as new options to treat patients in the future.

Blue marine therapy: Sea as a trove of natural anticancer drugs.

The great variability of marine habitats and the species that live there allows the development of organisms with unique characteristics. These represent an excellent source of natural compounds and are therefore interesting in the search for new bioactive molecules. In recent years, many marine-based drugs have been commercialized or are currently under investigation, mainly in the treatment of cancer. This mini-review summarizes the marine-based drugs currently marketed and presents a non-exhaustive list of molecules currently in clinical trials, as monotherapy but also in combination with classical anticancer treatments.

Fine Tuning of Calcium Constitutive Entry by Optogenetically-Controlled Membrane Polarization: Impact on Cell Migration.

Anomalies in constitutive calcium entry (CCE) have been commonly attributed to cell dysfunction in pathological conditions such as cancer. Calcium influxes of this type rely on channels, such as transient receptor potential (TRP) channels, to be constitutively opened and strongly depend on membrane potential and a calcium driving force. We developed an optogenetic approach based on the expression of the halorhodopsin chloride pump to study CCE in non-excitable cells. Using C2C12 cells, we found that halorhodopsin can be used to achieve a finely tuned control of membrane polarization. Escalating the membrane polarization by incremental changes in light led to a concomitant increase in CCE through transient receptor potential vanilloid 2 (TRPV2) channels. Moreover, light-induced calcium entry through TRPV2 channels promoted cell migration. Our study shows for the first time that by modulating CCE and related physiological responses, such as cell motility, halorhodopsin serves as a potentially powerful tool that could open new avenues for the study of CCE and associated cellular behaviors.

Molecular analysis of exon 7 of the fibroblast growth factor receptor 2 (FGFR2) gene in an Indonesian patient with Apert syndrome: a case report.

BACKGROUND: Apert syndrome, Online Mendelian Inheritance in Man number 101200, is a rare genetic condition, with autosomal dominant inheritance, characterized by craniosynostosis, midfacial malformation, and severe symmetrical syndactyly. Apert syndrome is associated with other systemic malformations, including intellectual disability. At least seven mutations in fibroblast growth factor receptor 2 (FGFR2) gene have been found to cause Apert syndrome. Most cases of Apert syndrome are caused by one of the two most frequent mutations located in exon 7 (Ser252Trp or Pro253Arg). CASE PRESENTATION: A 27-year-old Javanese man presented borderline intellectual functioning and striking dysmorphisms. A clinical diagnosis of Apert syndrome was previously made based on these clinical features. Furthermore, POSSUM software was used before molecular analysis and the result showed suspected Apert syndrome with a cut-off point of 14. Molecular genetic analysis of FGFR2, targeting exon 7, was performed by direct sequencing. In this patient, a missense mutation c.755C>G was detected, changing a serine into a tryptophan (p.Ser252Trp). CONCLUSION: We report the case of an Indonesian man with Apert syndrome with a c.755C>G (p.Ser252Trp) mutation in the FGFR2 gene. Our patient showed similar dysmorphism to previously reported cases, although cleft palate as a typical feature for p.Ser252Trp mutation was not present. In spite of the accessibility of molecular genetic testing in a few parts of the world, the acknowledgement of clinically well-defined syndromes will remain exceptionally imperative in developing countries with a lack of diagnostic facilities.

A functional assay to study the pathogenicity of CHD7 protein variants encountered in CHARGE syndrome patients.

CHARGE syndrome is a rare genetic disease characterized by numerous congenital abnormalities, mainly caused by de novo alterations of the CHD7 gene. It encodes a chromodomain protein, involved in the ATP-dependent remodeling of chromatin. The vast majority of CHD7 alterations consists in null alleles like deletions, nonsense substitutions or frameshift-causing variations. The aim of this study was to develop a biological test of CHD7 protein, to study the impact upon protein functionality of rare allelic variants in the CHD7 gene that elicits changes in the amino acid sequence. Using an expression vector encoding CHD7, three amino acid substitutions and one five-amino acid insertion were generated via site-directed mutagenesis. Then CHD7 proteins, either wild-type (WT) or variants, were overexpressed in HeLa cell line. Protein expression was highlighted by western blot and immunofluorescence. We then used real-time RT-PCR to study CHD7 functionality by evaluating the transcript amounts of five genes whose expression is regulated by CHD7 according to the literature. These reporter genes are 45S rDNA, SOX4, SOX10, ID2, and MYRF. We observed that, upon WT-CHD7 expression, the reporter gene transcriptions were downregulated, whereas the four variant alleles of CHD7 had no impact. This suggests that these alleles are not polymorphisms because the variant proteins appeared nonfunctional. Therefore, these variations can be considered as disease-causing of CHARGE syndrome.

CFTR-France, a national relational patient database for sharing genetic and phenotypic data associated with rare CFTR variants.

Most of the 2,000 variants identified in the CFTR (cystic fibrosis transmembrane regulator) gene are rare or private. Their interpretation is hampered by the lack of available data and resources, making patient care and genetic counseling challenging. We developed a patient-based database dedicated to the annotations of rare CFTR variants in the context of their cis- and trans-allelic combinations. Based on almost 30 years of experience of CFTR testing, CFTR-France (https://cftr.iurc.montp.inserm.fr/cftr) currently compiles 16,819 variant records from 4,615 individuals with cystic fibrosis (CF) or CFTR-RD (related disorders), fetuses with ultrasound bowel anomalies, newborns awaiting clinical diagnosis, and asymptomatic compound heterozygotes. For each of the 736 different variants reported in the database, patient characteristics and genetic information (other variations in cis or in trans) have been thoroughly checked by a dedicated curator. Combining updated clinical, epidemiological, in silico, or in vitro functional data helps to the interpretation of unclassified and the reassessment of misclassified variants. This comprehensive CFTR database is now an invaluable tool for diagnostic laboratories gathering information on rare variants, especially in the context of genetic counseling, prenatal and preimplantation genetic diagnosis. CFTR-France is thus highly complementary to the international database CFTR2 focused so far on the most common CF-causing alleles.

Functional and splicing defect analysis of 23 ACVRL1 mutations in a cohort of patients affected by Hereditary Hemorrhagic Telangiectasia.

Hereditary Hemorrhagic Telangiectasia syndrome (HHT) or Rendu-Osler-Weber (ROW) syndrome is an autosomal dominant vascular disorder. Two most common forms of HHT, HHT1 and HHT2, have been linked to mutations in the endoglin (ENG) and activin receptor-like kinase 1 (ACVRL1or ALK1) genes respectively. This work was designed to examine the pathogenicity of 23 nucleotide variations in ACVRL1 gene detected in more than 400 patients. Among them, 14 missense mutations and one intronic variant were novels, and 8 missense mutations were previously identified with questionable implication in HHT2. The functionality of missense mutations was analyzed in response to BMP9 (specific ligand of ALK1), the maturation of the protein products and their localization were analyzed by western blot and fluorescence microscopy. The splicing impairment of the intronic and of two missense mutations was examined by minigene assay. Functional analysis showed that 18 out of 22 missense mutations were defective. Splicing analysis revealed that one missense mutation (c.733A>G, p.Ile245Val) affects the splicing of the harboring exon 6. Similarly, the intronic mutation outside the consensus splicing sites (c.1048+5G>A in intron 7) was seen pathogenic by splicing study. Both mutations induce a frame shift creating a premature stop codon likely resulting in mRNA degradation by NMD surveillance mechanism. Our results confirm the haploinsufficiency model proposed for HHT2. The affected allele of ACVRL1 induces mRNA degradation or the synthesis of a protein lacking the receptor activity. Furthermore, our data demonstrate that functional and splicing analyses together, represent two robust diagnostic tools to be used by geneticists confronted with novel or conflicted ACVRL1 mutations.

Involvement of the Cdc42 pathway in CFTR post-translational turnover and in its plasma membrane stability in airway epithelial cells.

Cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel that is expressed on the apical plasma membrane (PM) of epithelial cells. The most common deleterious allele encodes a trafficking-defective mutant protein undergoing endoplasmic reticulum-associated degradation (ERAD) and presenting lower PM stability. In this study, we investigated the involvement of the Cdc42 pathway in CFTR turnover and trafficking in a human bronchiolar epithelial cell line (CFBE41o-) expressing wild-type CFTR. Cdc42 is a small GTPase of the Rho family that fulfils numerous cell functions, one of which is endocytosis and recycling process via actin cytoskeleton remodelling. When we treated cells with chemical inhibitors such as ML141 against Cdc42 and wiskostatin against the downstream effector N-WASP, we observed that CFTR channel activity was inhibited, in correlation with a decrease in CFTR amount at the cell surface and an increase in dynamin-dependent CFTR endocytosis. Anchoring of CFTR to the cortical cytoskeleton was then presumably impaired by actin disorganization. When we performed siRNA-mediated depletion of Cdc42, actin polymerization was not impacted, but we observed actin-independent consequences upon CFTR. Total and PM CFTR amounts were increased, resulting in greater activation of CFTR. Pulse-chase experiments showed that while CFTR degradation was slowed, CFTR maturation through the Golgi apparatus remained unaffected. In addition, we observed increased stability of CFTR in PM and reduction of its endocytosis. This study highlights the involvement of the Cdc42 pathway at several levels of CFTR biogenesis and trafficking: (i) Cdc42 is implicated in the first steps of CFTR biosynthesis and processing; (ii) it contributes to the stability of CFTR in PM via its anchoring to cortical actin; (iii) it promotes CFTR endocytosis and presumably its sorting toward lysosomal degradation.

Orphan missense mutations in the cystic fibrosis transmembrane conductance regulator: A three-step biological approach to establishing a correlation between genotype and phenotype.

More than 1860 mutations have been found within the human cystic fibrosis transmembrane conductance regulator (CFTR) gene sequence. These mutations can be classified according to their degree of severity in CF disease. Although the most common mutations are well characterized, few data are available for rare mutations. Thus, genetic counseling is particularly difficult when fetuses or patients with CF present these orphan variations. We describe a three-step in vitro assay that can evaluate rare missense CFTR mutation consequences to establish a correlation between genotype and phenotype. By using a green fluorescent protein-tagged CFTR construct, we expressed mutated proteins in COS-7 cells. CFTR trafficking was visualized by confocal microscopy, and the cellular localization of CFTR was determined using intracellular markers. We studied the CFTR maturation process using Western blot analysis and evaluated CFTR channel activity by automated iodide efflux assays. Of six rare mutations that we studied, five have been isolated in our laboratory. The cellular and functional impact that we observed in each case was compared with the clinical data concerning the patients in whom we encountered these mutations. In conclusion, we propose that performing this type of analysis for orphan CFTR missense mutations can improve CF genetic counseling.

Endosomal SNARE proteins regulate CFTR activity and trafficking in epithelial cells.

The Cystic Fibrosis Transmembrane conductance Regulator (CFTR) protein is a chloride channel localized at the apical plasma membrane of epithelial cells. We previously described that syntaxin 8, an endosomal SNARE (Soluble N-ethylmaleimide-sensitive factor Attachment protein REceptor) protein, interacts with CFTR and regulates its trafficking to the plasma membrane and hence its channel activity. Syntaxin 8 belongs to the endosomal SNARE complex which also contains syntaxin 7, vti1b and VAMP8. Here, we report that these four endosomal SNARE proteins physically and functionally interact with CFTR. In LLC-PK1 cells transfected with CFTR and in Caco-2 cells endogenously expressing CFTR, we demonstrated that endosomal SNARE protein overexpression inhibits CFTR activity but not swelling- or calcium-activated iodide efflux, indicating a specific effect upon CFTR activity. Moreover, co-immunoprecipitation experiments in LLC-PK1-CFTR cells showed that CFTR and SNARE proteins belong to a same complex and pull-down assays showed that VAMP8 and vti1b preferentially interact with CFTR N-terminus tail. By cell surface biotinylation and immunofluorescence experiments, we evidenced that endosomal SNARE overexpression disturbs CFTR apical targeting. Finally, we found a colocalization of CFTR and endosomal SNARE proteins in Rab11-positive recycling endosomes, suggesting a new role for endosomal SNARE proteins in CFTR trafficking in epithelial cells.

Transient receptor potential vanilloid 1 (TRPV1) channels in cultured rat Sertoli cells regulate an acid sensing chloride channel.

Sertoli cells provide a controlled microenvironment for regulation and maintenance of spermatogenesis for which an acidic milieu is crucial for male fertility. Sertoli cells also contribute to protection of spermatogenetic cells. Here, we showed that TRPV1 is expressed in rat Sertoli cells and regulates an acid sensing Cl(-) channel (ASCC). The expression of TRPV1 in rat Sertoli cells was demonstrated by RT-PCR, immunostaining and calcium measurement experiments. ASCC activity was inhibited by capsaicin (IC(50)=214.3+/-1.6 nM), olvanil (IC(50)=400+/-1.7 pM) and resiniferatoxin (IC(50)=9.3+/-1.5 nM) but potentiated by capsazepine (EC(50)=5.3+/-1.3 microM) and ruthenium red (EC(50)=2.3+/-1.5 microM). In the human airway epithelial cell line Calu-3 in which ASCC can be detected but not TRPV1, capsaicin and capsazepine were without any effect. Finally the application of the non-steroidal anti-inflammatory drug ibuprofen prevented the control of ASCC by TRPV1. Our study provides the first evidence for a regulation by TRPV1 of an acid sensing chloride channel in rat Sertoli cells. TRPV1 and ASCC may thus be considered as new potential physiological regulators of spermatogenesis and targets for pharmacological treatments of reproductive disorders as cryptorchidism, Sertoli cell tumors or torsion of the spermatic cord.

Mini-dystrophin expression down-regulates overactivation of G protein-mediated IP3 signaling pathway in dystrophin-deficient muscle cells.

We present here evidence for the enhancement of an inositol 1,4,5-trisphosphate (IP3) mediated calcium signaling pathway in myotubes from dystrophin-deficient cell lines (SolC1(-)) as compared to a cell line from the same origin but transfected with mini-dystrophin (SolD(+)). With confocal microscopy, we demonstrated that calcium rise, induced by the perifusion of a solution containing a high potassium concentration, was higher in SolC1(-) than in SolD(+) myotubes. The analysis of amplitude and kinetics of the calcium increase in SolC1(-) and in SolD(+) myotubes during the exposure with SR Ca2+ channel inhibitors (ryanodine and 2-APB) suggested the presence of two mechanisms of SR calcium release: (1) a fast SR calcium release that depended on ryanodine receptors and (2) a slow SR calcium release mediated by IP3 receptors. Detection analyses of mRNAs (reverse transcriptase [RT]-PCR) and proteins (Western blot and immunolocalization) demonstrated the presence of the three known isoforms of IP3 receptors in both SolC1(-) and SolD(+) myotubes. Furthermore, analysis of the kinetics of the rise in calcium revealed that the slow IP3-dependent release may be increased in the SolC1(-) as compared to the SolD(+), suggesting an inhibitory effect of mini-dystrophin in this signaling pathway. Upon incubation with pertussis toxin (PTX), an inhibitory effect similar to that of the IP3R inhibitor (2-APB) was observed on K+-evoked calcium release. This result suggests the involvement of a Gi protein upstream of the IP3 pathway in these stimulation conditions. A hypothetical model is depicted in which both Gi protein and IP3 production could be involved in K+-evoked calcium release as well as a possible interaction with mini-dystrophin. Our findings demonstrate the existence of a potential relationship between mini-dystrophin and SR calcium release as well as a regulatory role of mini-dystrophin on intracellular signaling.

The cystic fibrosis mutation G1349D within the signature motif LSHGH of NBD2 abolishes the activation of CFTR chloride channels by genistein.

Cystic fibrosis (CF) is a common lethal genetic disease caused by autosomal recessive mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel that belongs to the ATP-Binding Cassette (ABC) family of transporters. The class III CF mutations G551D and G1349D are located within the "signature" sequence LSGGQ and LSHGH of NBD1 and NBD2, respectively. We have constructed by site-directed mutagenesis vectors encoding green fluorescent protein (GFP)-tagged wild-type (wt) CFTR or CFTR containing delF508, G551D, G1349D and G551D/G1349D to study their pharmacology after transient expression in COS-7 cells. We show that IBMX and the benzo[c]quinolizinium derivative MPB-91 stimulates the activity of G1349D-, G551D- and G551D/G1349D-CFTR only in the presence of cAMP-promoting agents like forskolin or cpt-cAMP. Similar half-maximal effective concentrations (EC(50)) of MPB-91 (22-36microM) have been determined for wt-, G551D-, G1349D- and G551D/G1349D-CFTR. The isoflavone genistein stimulates wild-type (wt)- and delF508-CFTR channel activity in a non-Michaelis-Menten manner. By contrast, the response of G1349D- and G551D-CFTR to genistein is dramatically altered. First, genistein is not able to stimulate G1349D- and G551D/G1349D-CFTR. Second, genistein stimulates G551D-CFTR without any inhibition at high concentration. We conclude from these results that whereas G551 in NBD1 is an important molecular site for inhibition of CFTR by genistein, the symmetrical G1349 in NBD2 is also one major site but for the activation of CFTR by genistein. Because both mutations alter specifically the mechanism of CFTR channel activation by genistein, we believe that the signature sequences of CFTR act as molecular switches that upon interaction with genistein turn on and off the channel.

Syntaxin 8 impairs trafficking of cystic fibrosis transmembrane conductance regulator (CFTR) and inhibits its channel activity.

The cystic fibrosis transmembrane conductance regulator (CFTR) is a cyclic AMP-dependent chloride channel that mediates electrolyte transport across the luminal surface of epithelial cells. In this paper, we describe the CFTR regulation by syntaxin 8, a t-SNARE protein (target soluble N-ethylmaleimide-sensitive factor attachment protein receptor) involved in the SNARE endosomal complex. Syntaxin family members are key molecules implicated in diverse vesicle docking and membrane fusion events. We found that syntaxin 8 physically interacts with CFTR: recombinant syntaxin 8 binds CFTR in vitro and both proteins co-immunoprecipitate in HT29 cells. Syntaxin 8 regulates CFTR-mediated currents in chinese hamster ovary (CHO) cells stably expressing CFTR and syntaxin 8. Iodide efflux and whole-cell patch-clamp experiments on these cells indicate a strong inhibition of CFTR chloride current by syntaxin 8 overexpression. At the cellular level, we observed that syntaxin 8 overexpression disturbs CFTR trafficking. Confocal microscopy shows a dramatic decrease in green fluorescent protein-tagged CFTR plasma membrane staining, when syntaxin 8 is coexpressed in COS-7 cells. Using antibodies against Lamp-1, TfR or Rab11 we determined by immunofluorescence assays that both proteins are mainly accumulated in recycling endosomes. Our results evidence that syntaxin 8 contributes to the regulation of CFTR trafficking and chloride channel activity by the SNARE machinery.

Regulation of the cystic fibrosis transmembrane conductance regulator channel by beta-adrenergic agonists and vasoactive intestinal peptide in rat smooth muscle cells and its role in vasorelaxation.

The signaling events that regulate vascular tone include voltage-dependent Ca(2+) influx and the activities of various ionic channels; which molecular entities are involved and their role are still a matter of debate. Here we show expression of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel in rat aortic smooth muscle cells. Immunoprecipitation and in vitro protein kinase A phosphorylation show the appearance of mature band C of CFTR. An immunohistochemistry study shows CFTR proteins in smooth muscles of aortic rings but not in skeletal muscles. Using the iodide efflux method, a combination of agonists and pharmacological agents was used to dissect the function of CFTR. Agonists of the cAMP pathway, the beta-adrenergic agonist isoproterenol, and the neuropeptide vasoactive intestinal peptide activate CFTR-dependent transport from cells maintained in a high but not low extracellular potassium-rich saline, suggesting that depolarization of smooth muscle is critical to CFTR activation. Smooth muscle CFTR possesses all of the pharmacological attributes of its epithelial homologues: stimulation by the CFTR pharmacological activators MPB-07 (EC(50) = 158 microm) and MPB-91 (EC(50) = 20 microm) and inhibition by glibenclamide and diphenylamine-2-carboxylic acid but not by 5,11,17,23-tetrasulfonato-25,26,27,28-tetramethoxy-calix[4]arene. Contraction measurements on isolated aortic rings were performed to study the contribution of CFTR to vascular tone. With aortic rings (without endothelium) preconstricted by high K(+) saline or by the alpha-adrenergic agonist norepinephrine, CFTR activators produced a concentration-dependent relaxation. These results identify for the first time the expression and function of CFTR in smooth muscle where it plays an unexpected but fundamental role in the autonomic and hormonal regulation of the vascular tone.

A Novel voltage-dependent chloride current activated by extracellular acidic pH in cultured rat Sertoli cells.

Sertoli cells from mammalian testis are key cells involved in development and maintenance of stem cell spermatogonia as well as secretion of a chloride- and potassium-rich fluid into the lumen of seminiferous tubules. Using whole-cell patch clamp experiments, a novel chloride current was identified. It is activated only in the presence of an extracellular acidic pH, with an estimated half-maximal activation at pH 5.5. The current is strongly outwardly rectifying, activated with a fast time-dependent onset of activation but a slow time-dependent kinetic at depolarization pulses. The pH-activated chloride current was not detected at physiological or basic pH and is not sensitive to intracellular or extracellular Ca2+ variation. Diphenylamine-2-carboxylic acid and 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid blocked the induced currents, and its anionic selectivity sequence was Cl- > Br- > I-> gluconate. We have performed a reverse transcription-PCR analysis to search for voltage-dependent chloride rClC channels in cultured rat Sertoli cells. Among the nine members of the family only rClC-2, rClC-3, rClC-6, and rClC-7 have been identified. The inwardly rectifying rClC-2 chloride current was activated by hyperpolarization but not by pH variation. A different depolarization-activated outwardly rectifying chloride current was activated only by hypotonic challenge and may correspond either to rClC-3 or rClC-6. Immunolocalization experiments demonstrate that rClC-7 resides in the intracellular compartment of Sertoli cells. This study provides the first functional identification of a native acid-activated chloride current. Based on our molecular analysis of rClC proteins, this new chloride current does not correspond to rClC-2, rClC-3, rClC-6, or rClC-7 channels. The potential physiological role of this native current in an epithelial cell from the reproductive system is discussed.