RESUMO
The blood-brain barrier (BBB) protects the brain from toxins but hinders the penetration of neurotherapeutic drugs. Therefore, the blood-to-brain permeability of chemotherapeutics must be carefully evaluated. Here, we aimed to establish a workflow to generate primary cultures of human brain microvascular endothelial cells (BMVECs) to study drug brain permeability and bioavailability. Furthermore, we characterized and validated this BBB model in terms of quantitative expression of junction and drug-transport proteins, and drug permeability. We isolated brain microvessels (MVs) and cultured BMVECs from glioma patient biopsies. Then, we employed targeted LC-MS proteomics for absolute protein quantification and immunostaining to characterize protein localization and radiolabeled drugs to predict drug behavior at the Human BBB. The abundance levels of ABC transporters, junction proteins, and cell markers in the cultured BMVECs were similar to the MVs and correctly localized to the cell membrane. Permeability values (entrance and exit) and efflux ratios tested in vitro using the primary BMVECs were within the expected in vivo values. They correctly reflected the transport mechanism for 20 drugs (carbamazepine, diazepam, imipramine, ketoprofen, paracetamol, propranolol, sulfasalazine, terbutaline, warfarin, cimetidine, ciprofloxacin, digoxin, indinavir, methotrexate, ofloxacin, azidothymidine (AZT), indomethacin, verapamil, quinidine, and prazosin). We established a human primary in vitro model suitable for studying blood-to-brain drug permeability with a characterized quantitative abundance of transport and junction proteins, and drug permeability profiles, mimicking the human BBB. Our results indicate that this approach could be employed to generate patient-specific BMVEC cultures to evaluate BBB drug permeability and develop personalized therapeutic strategies.
Assuntos
Barreira Hematoencefálica , Células Endoteliais , Humanos , Barreira Hematoencefálica/metabolismo , Células Endoteliais/metabolismo , Proteômica , Transportadores de Cassetes de Ligação de ATP/metabolismo , PermeabilidadeRESUMO
The effect of cannabidiol (CBD), a high-affinity agonist of the transient receptor potential vanilloid-2 (TRPV2) channel, has been poorly investigated in human brain microvessel endothelial cells (BMEC) forming the blood-brain barrier (BBB). TRPV2 expression and its role on Ca2+ cellular dynamics, trans-endothelial electrical resistance (TEER), cell viability and growth, migration, and tubulogenesis were evaluated in human primary cultures of BMEC (hPBMEC) or in the human cerebral microvessel endothelial hCMEC/D3 cell line. Abundant TRPV2 expression was measured in hCMEC/D3 and hPBMEC by qRT-PCR, Western blotting, nontargeted proteomics, and cellular immunofluorescence studies. Intracellular Ca2+ levels were increased by heat and CBD and blocked by the nonspecific TRP antagonist ruthenium red (RR) and the selective TRPV2 inhibitor tranilast (TNL) or by silencing cells with TRPV2 siRNA. CBD dose-dependently induced the hCMEC/D3 cell number (EC50 0.3 ± 0.1 µM), and this effect was fully abolished by TNL or TRPV2 siRNA. A wound healing assay showed that CBD induced cell migration, which was also inhibited by TNL or TRPV2 siRNA. Tubulogenesis of hCMEC/D3 cells in 3D matrigel cultures was significantly increased by 41 and 73% after a 7 or 24 h CBD treatment, respectively, and abolished by TNL. CBD also increased the TEER of hPBMEC monolayers cultured in transwell, and this was blocked by TNL. Our results show that CBD, at extracellular concentrations close to those observed in plasma of patients treated by CBD, induces proliferation, migration, tubulogenesis, and TEER increase in human brain endothelial cells, suggesting CBD might be a potent target for modulating the human BBB.
Assuntos
Neoplasias Encefálicas/irrigação sanguínea , Canabidiol/farmacologia , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Endoteliais/metabolismo , Microvasos/patologia , Canais de Cátion TRPV/metabolismo , Barreira Hematoencefálica/metabolismo , Cálcio/metabolismo , Cannabis/química , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Impedância Elétrica , Temperatura Alta , Humanos , Extratos Vegetais/farmacologia , Rutênio Vermelho/farmacologia , Canais de Cátion TRPV/antagonistas & inibidores , ortoaminobenzoatos/farmacologiaRESUMO
Treatment of late-stage sleeping sickness requires drugs that can cross the blood-brain barrier (BBB) to reach the parasites located in the brain. We report here the synthesis and evaluation of four new N-hydroxy and 12 new N-alkoxy derivatives of bisimidazoline leads as potential agents for the treatment of late-stage sleeping sickness. These compounds, which have reduced basicity compared to the parent leads (i.e., are less ionized at physiological pH), were evaluated in vitro against Trypanosoma brucei rhodesiense and in vivo in murine models of first- and second-stage sleeping sickness. Resistance profile, physicochemical parameters, in vitro BBB permeability, and microsomal stability also were determined. The N-hydroxy imidazoline analogues were the most effective in vivo, with 4-((1-hydroxy-4,5-dihydro-1H-imidazol-2-yl)amino)-N-(4-((1-hydroxy-4,5-dihydro-1H-imidazol-2-yl)amino)phenyl)benzamide (14d) showing 100% cures in the first-stage disease, while 15d, 16d, and 17d appeared to slightly improve survival. In addition, 14d showed weak activity in the chronic model of central nervous system infection in mice. No evidence of reduction of this compound with hepatic microsomes and mitochondria was found in vitro, suggesting that N-hydroxy imidazolines are metabolically stable and have intrinsic activity against T. brucei. In contrast to its unsubstituted parent compound, the uptake of 14d in T. brucei was independent of known drug transporters (i.e., T. brucei AT1/P2 and HAPT), indicating a lower predisposition to cross-resistance with other diamidines and arsenical drugs. Hence, the N-hydroxy bisimidazolines (14d in particular) represent a new class of promising antitrypanosomal agents.
Assuntos
Tripanossomicidas/uso terapêutico , Trypanosoma brucei brucei/patogenicidade , Trypanosoma brucei rhodesiense/patogenicidade , Tripanossomíase Africana/tratamento farmacológico , Animais , Modelos Animais de Doenças , Feminino , Imidazolinas/uso terapêutico , Camundongos , Trypanosoma brucei brucei/efeitos dos fármacos , Trypanosoma brucei rhodesiense/efeitos dos fármacosRESUMO
Antibodies normally do not cross the blood-brain barrier (BBB) and cannot bind an intracellular cerebral antigen. We demonstrate here for the first time that a new class of antibodies can cross the BBB without treatment. Camelids produce native homodimeric heavy-chain antibodies, the paratope being composed of a single-variable domain called VHH. Here, we used recombinant VHH directed against human glial fibrillary acidic protein (GFAP), a specific marker of astrocytes. Only basic VHHs (e.g., pI=9.4) were able to cross the BBB in vitro (7.8 vs. 0% for VHH with pI=7.7). By intracarotid and intravenous injections into live mice, we showed that these basic VHHs are able to cross the BBB in vivo, diffuse into the brain tissue, penetrate into astrocytes, and specifically label GFAP. To analyze their ability to be used as a specific transporter, we then expressed a recombinant fusion protein VHH-green fluorescent protein (GFP). These "fluobodies" specifically labeled GFAP on murine brain sections, and a basic variant (pI=9.3) of the fusion protein VHH-GFP was able to cross the BBB and to label astrocytes in vivo. The potential of VHHs as diagnostic or therapeutic agents in the central nervous system now deserves attention.
Assuntos
Astrócitos/metabolismo , Barreira Hematoencefálica/metabolismo , Encéfalo/metabolismo , Proteína Glial Fibrilar Ácida/metabolismo , Anticorpos de Domínio Único/metabolismo , Animais , Astrocitoma/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , Ensaio de Imunoadsorção Enzimática , Feminino , Imunofluorescência , Proteína Glial Fibrilar Ácida/genética , Proteína Glial Fibrilar Ácida/imunologia , Humanos , Imuno-Histoquímica , Camundongos , Camundongos Endogâmicos C57BL , Mutagênese Sítio-Dirigida , Plasmodium berghei/patogenicidade , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Anticorpos de Domínio Único/genética , Anticorpos de Domínio Único/imunologiaRESUMO
Human cerebral microvascular endothelial cell line hCMEC/D3 is an established model of the human blood-brain barrier (BBB). The purpose of the present study was to determine, by means of quantitative targeted absolute proteomics, the protein expression levels in hCMEC/D3 cells of multiple transporters, receptors and junction proteins for comparison with our previously reported findings in isolated human brain microvessels. Among 91 target molecules, 12 transporters, 2 receptors, 1 junction protein and 1 membrane marker were present at quantifiable levels in plasma membrane fraction of hCMEC/D3 cells. ABCA2, MDR1, MRP4, BCRP, GLUT1, 4F2hc, MCT1, ENT1, transferrin and insulin receptors and claudin-5 were detected in both hCMEC/D3 cells and human brain microvessels. After normalization based on Na(+)/K(+) ATPase expression, the differences in protein expression levels between hCMEC/D3 cells and human brain microvessels were within 4-fold for these proteins, with the exceptions of ENT1, transferrin receptor and claudin-5. ABCA8, LAT1, LRP1 and γ-GTP were below the limit of quantification in the cells, but were found in human brain microvessels. ABCA3, ABCA6, MRP1 and ATA1 were found only in hCMEC/D3 cells. Furthermore, compared with human umbilical vein endothelial cells (HUVECs) as reference nonbrain endothelial cells, MDR1 was found only in hCMEC/D3 cells, and GLUT1 expression was 15-fold higher in hCMEC/D3 cells than in HUVECs. In conclusion, this is the first study to examine the suitability and limitations of the hCMEC/D3 cell line as a BBB functional model in terms of quantitative expression levels of transporters, receptors and tight junction proteins.
Assuntos
Barreira Hematoencefálica/metabolismo , Cérebro/irrigação sanguínea , Cérebro/metabolismo , Células Endoteliais/metabolismo , Endotélio Vascular/metabolismo , Proteínas de Membrana/metabolismo , Microvasos/metabolismo , Proteoma/metabolismo , Transporte Biológico , Linhagem Celular , Membrana Celular/metabolismo , Circulação Cerebrovascular , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Proteínas de Membrana Transportadoras/metabolismo , Modelos Biológicos , Proteômica/métodosRESUMO
Transient receptor potential vanilloid 1-4 (TRPV1-4) expression and functionality were investigated in brain microvessel endothelial cells (BMEC) forming the blood-brain barrier (BBB) from rat and human origins. In rat, Trpv1-4 were detected by qRT-PCR in the brain cortex, brain microvessels, and in primary cultures of brain microvessel endothelial cells [rat brain microvessel endothelial cells (rPBMEC)]. A similar Trpv1-4 expression profile in isolated brain microvessels and rPBMEC was found with the following order: Trpv4 > Trpv2 > Trpv3 > Trpv1. In human, TRPV1-4 were detected in the BBB cell line human cerebral microvessel endothelial cells D3 cells (hCMEC/D3) and in primary cultures of BMEC isolated from human adult and children brain resections [human brain microvascular endothelial cells (hPBMEC)], showing a similar TRPV1-4 expression profile in both hCMEC/D3 cells and hPBMECs as follow: TRPV2 > > TRPV4 > TRPV1 > TRPV3. Western blotting and immunofluorescence experiments confirmed that TRPV2 and TRPV4 are the most expressed TRPV isoforms in hCMEC/D3 cells with a clear staining at the plasma membrane. A fluorescent dye Fluo-4 AM ester was applied to record intracellular Ca2+ levels. TRPV4 functional activity was demonstrated in mediating Ca2+ influx under stimulation with the specific agonist GSK1016790A (ranging from 3 to 1000 nM, EC50 of 16.2 ± 4.5 nM), which was inhibited by the specific TRPV4 antagonist, RN1734 (30 µM). In contrast, TRPV1 was slightly activated in hCMEC/D3 cells as shown by the weak Ca2+ influx induced by capsaicin at a high concentration (3 µM), a highly potent and specific TRPV1 agonist. Heat-induced Ca2+ influx was not altered by co-treatment with a selective potent TRPV1 antagonist capsazepine (20 µM), in agreement with the low expression of TRPV1 as assessed by qRT-PCR. Our present study reveals an interspecies difference between Rat and Human. Functional contributions of TRPV1-4 subtype expression were not identical in rat and human tissues reflective of BBB integrity. TRPV2 was predominant in the human whereas TRPV4 had a larger role in the rat. This interspecies difference from a gene expression point of view should be taken into consideration when modulators of TRPV2 or TRPV4 are investigated in rat models of brain disorders.
RESUMO
Organic cation transporters (OCTs) participate in the handling of compounds in kidneys and at the synaptic cleft. Their role at the blood-brain barrier (BBB) in brain drug delivery is still unclear. The presence of OCT1,2,3 (SLC22A1-3) in mouse, rat and human isolated brain microvessels was investigated by either qRT-PCR, quantitative proteomics and/or functional studies. BBB transport of the prototypical substrate [3H]-1-methyl-4-phenylpyridinium ([3H]-MPP+) was measured by in situ brain perfusion in six mouse strains and in Sprague Dawley rats, in primary human brain microvascular endothelial cells seeded on inserts, in the presence or absence of OCTs and a MATE1 (SLC49A1) inhibitor. The results show negligible OCT1 (SLC22A1) and OCT2 (SLC22A2) expression in either mice, rat or human brain microvessels, while OCT3 expression was identified in rat microvessels by qRT-PCR. The in vitro human cellular uptake of [3H]-MPP+ was not modified by OCTs/MATE-inhibitor. Brain transport of [3H]-MPP+ remains unchanged between 2- and 6-month old mice, and no alteration was observed in mice and rats with inhibitors. In conclusion, the evidenced lack of expression and/or functional OCTs and MATE at the BBB allows the maintenance of the brain homeostasis and function as it prevents an easy access of their neurotoxicant substrates to the brain parenchyma.
RESUMO
The aryl hydrocarbon receptor (AhR) is a ligand-dependent transcription factor activated by a variety of widespread persistent environmental pollutants such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). It can transactivate the expression of several target genes. Recently AhR transcripts were detected in isolated human brain microvessels and in the hCMEC/D3 human cerebral microvascular endothelial cell line, an in vitro model of the human cerebral endothelium. To date AhR implication in the co-regulation of ABCB1, ABCG2 and CYP1B1 at human cerebral endothelium has not been addressed. Here we investigated whether AhR could co-regulate ABCB1, ABCG2 and CYP1B1 expressions in the hCMEC/D3 cell line. Exposure to TCDD induced a concentration-dependent increase in CYP1B1 expression. We demonstrated AhR involvement in the TCDD-mediated increase in CYP1B1 expression by using small interfering RNA against AhR. Western blotting analysis also revealed an increase in CYP1B1 protein expression following TCDD exposure in hCMEC/D3. Regarding ABCB1 and ABCG2, exposure to TCDD had no effect on their protein expressions and functional activities. In conclusion our data indicated a differential modulation of CYP1B1 and ABCB1/ABCG2 expressions in hCMEC/D3 cells following TCDD exposure.
Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Córtex Cerebral/irrigação sanguínea , Córtex Cerebral/efeitos dos fármacos , Citocromo P-450 CYP1B1/metabolismo , Proteínas de Neoplasias/metabolismo , Dibenzodioxinas Policloradas/toxicidade , Receptores de Hidrocarboneto Arílico/metabolismo , Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP , Linhagem Celular , Córtex Cerebral/metabolismo , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Humanos , Microvasos/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacosRESUMO
OBJECTIVE: The vascular system is adapted to specific functions in different tissues and organs. Vascular endothelial cells are important elements of this adaptation, leading to the concept of 'specialized endothelial cells'. The phenotype of these cells is highly dependent on their specific microenvironment and when isolated and cultured, they lose their specific features after few passages, making models using such cells poorly predictive and irreproducible. We propose a new source of specialized endothelial cells based on cord blood circulating endothelial progenitors (EPCs). As prototype examples, we evaluated the capacity of EPCs to acquire properties characteristic of cerebral microvascular endothelial cells (blood-brain barrier (BBB)) or of arterial endothelial cells, in specific inducing culture conditions. APPROACH AND RESULTS: First, we demonstrated that EPC-derived endothelial cells (EPDCs) co-cultured with astrocytes acquired several BBB phenotypic characteristics, such as restricted paracellular diffusion of hydrophilic solutes and the expression of tight junction proteins. Second, we observed that culture of the same EPDCs in a high concentration of VEGF resulted, through activation of Notch signaling, in an increase of expression of most arterial endothelial markers. CONCLUSIONS: We have thus demonstrated that in vitro culture of early passage human cord blood EPDCs under specific conditions can induce phenotypic changes towards BBB or arterial phenotypes, indicating that these EPDCs maintain enough plasticity to acquire characteristics of a variety of specialized phenotypes. We propose that this property of EPDCs might be exploited for producing specialized endothelial cells in culture to be used for drug testing and predictive in vitro assays.
Assuntos
Artérias/citologia , Artérias/metabolismo , Barreira Hematoencefálica/citologia , Barreira Hematoencefálica/metabolismo , Células Endoteliais/metabolismo , Fenótipo , Células-Tronco/metabolismo , Animais , Astrócitos/citologia , Astrócitos/metabolismo , Permeabilidade Capilar , Técnicas de Cultura de Células , Técnicas de Cocultura , Células Endoteliais/citologia , Sangue Fetal/citologia , Perfilação da Expressão Gênica , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Ratos , Células-Tronco/citologia , Transcriptoma , Veias/metabolismoRESUMO
Wnt morphogens released by neural precursor cells were recently reported to control blood-brain barrier (BBB) formation during development. Indeed, in mouse brain endothelial cells, activation of the Wnt/ß-catenin signaling pathway, also known as the canonical Wnt pathway, was shown to stabilize endothelial tight junctions (TJs) through transcriptional regulation of the expression of TJ proteins. Because Wnt proteins activate several distinct ß-catenin-dependent and independent signaling pathways, this study was designed to assess whether the noncanonical Wnt/Par/aPKC planar cell polarity (PCP) pathway might also control TJ integrity in brain endothelial cells. First we established, in the hCMEC/D3 human brain endothelial cell line, that the Par/aPKC PCP complex colocalizes with TJs and controls apicobasal polarization. Second, using an siRNA approach, we showed that the Par/aPKC PCP complex regulates TJ stability and reassembling after osmotic shock. Finally, we provided evidence that Wnt5a signals in hCMEC/D3 cells through activation of the Par/aPKC PCP complex, independently of the Wnt canonical ß-catenin-dependent pathway and significantly contributes to TJ integrity and endothelial apicobasal polarity. In conclusion, this study suggests that the Wnt/Par/aPKC PCP pathway, in addition to the Wnt/ß-catenin canonical pathway, is a key regulator of the BBB.
Assuntos
Barreira Hematoencefálica/fisiologia , Polaridade Celular , Células Endoteliais/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Junções Íntimas/metabolismo , Proteínas Wnt/metabolismo , Via de Sinalização Wnt/fisiologia , Proteínas Adaptadoras de Transdução de Sinal , Barreira Hematoencefálica/citologia , Barreira Hematoencefálica/metabolismo , Técnicas de Cultura de Células , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Microscopia de Fluorescência , Permeabilidade , Proteína Quinase C/genética , Proteína Quinase C/metabolismo , Proteínas Proto-Oncogênicas/genética , RNA Interferente Pequeno/genética , Reação em Cadeia da Polimerase em Tempo Real , Junções Íntimas/genética , Junções Íntimas/fisiologia , Proteínas Wnt/genética , Via de Sinalização Wnt/genética , Proteína Wnt-5aRESUMO
The Blood-brain barrier (BBB), present at the level of the endothelium of cerebral blood vessels, selectively restricts the blood-to-brain paracellular diffusion of compounds; it is mandatory for cerebral homeostasis and proper neuronal function. The barrier properties of these specialized endothelial cells notably depend on tight junctions (TJs) between adjacent cells: TJs are dynamic structures consisting of a number of transmembrane and membrane-associated cytoplasmic proteins, which are assembled in a multimolecular complex and acting as a platform for intracellular signaling. Although the structural composition of these complexes has been well described in the recent years, our knowledge about their functional regulation still remains fragmentary. Importantly, pericytes, embedded in the vascular basement membrane, and perivascular microglial cells, astrocytes and neurons contribute to the regulation of endothelial TJs and BBB function, altogether constituting the so-called neurovascular unit.The present review summarizes our current understanding of the structure and functional regulation of endothelial TJs at the BBB. Accumulating evidence points to a correlation between BBB dysfunction, alteration of TJ complexes and progression of a variety of CNS diseases, such as stroke, multiple sclerosis and brain tumors, as well as neurodegenerative diseases like Parkinson's and Alzheimer's diseases. Understanding how TJ integrity is controlled may thus help improve drug delivery across the BBB and the design of therapeutic strategies for neurological disorders.
RESUMO
The blood-brain barrier (BBB) selectively controls the exchanges between the blood and the brain: it is formed by tight junctions (TJs) between adjacent microvascular endothelial cells. The transmembrane protein claudin-5 is known as a key TJ protein at the BBB, although, the molecular mechanisms by which it regulates TJ tightness are poorly understood. To identify putative claudin-5 partners that contribute to TJ integrity, claudin-5-enriched membrane microdomains were prepared by cell fractionation, using the human brain endothelial cell line hCMEC/D3 and claudin-5 immunoprecipitates were submitted to tandem mass spectrometry. Because a high concentration of mannitol is known to transiently destabilize TJs, this analysis was performed in basal conditions, after mannitol treatment, and after recovery of TJ integrity. We here demonstrate that the G-protein subunit αi2 (Gαi2) interacts with claudin-5 and that association is correlated with TJ integrity in hCMEC/D3 cells; also, a selective expression of Gαi2 is observed in human brain vasculature in situ. Moreover, small interfering RNA-mediated depletion of Gαi2 or claudin-5 in hCMEC/D3 cells similarly increases their paracellular permeability and delays TJ recovery after mannitol treatment. Altogether, our results identify Gαi2 as a novel claudin-5 partner required for TJ integrity in brain endothelial cells.
Assuntos
Barreira Hematoencefálica/metabolismo , Claudinas/metabolismo , Células Endoteliais/metabolismo , Subunidade alfa Gi2 de Proteína de Ligação ao GTP/metabolismo , Junções Íntimas/metabolismo , Barreira Hematoencefálica/citologia , Linhagem Celular , Claudina-5 , Claudinas/genética , Células Endoteliais/citologia , Subunidade alfa Gi2 de Proteína de Ligação ao GTP/genética , Técnicas de Silenciamento de Genes , Humanos , Manitol/química , Microdomínios da Membrana/genética , Microdomínios da Membrana/metabolismo , Ligação Proteica , RNA Interferente Pequeno/genética , Junções Íntimas/genéticaRESUMO
To improve the blood-brain barrier permeability of the trypanocidal lead compound 4,4'-bis(imidazolinylamino)diphenylamine (1), five N-alkoxy analogues were synthesized from bis(4-isothiocyanatophenyl)amine and N-alkoxy-N-(2-aminoethyl)-2-nitrobenzenesulfonamides following successive chemical reactions in just one reactor ("one-pot procedure"). This involved: (a) formation of a thiourea intermediate, (b) removal of the amine protecting groups, and (c) intramolecular cyclization. The blood-brain barrier permeability of the compounds determined in vitro by transport assays through the hCMEC/D3 human cell line, a well-known and characterized human cellular blood-brain barrier model, showed that the N-hydroxy analogue 16 had enhanced blood-brain barrier permeability compared with the unsubstituted lead compound. Moreover, this compound displayed low micromolar IC(50) against Trypanosoma brucei rhodesiense and Plasmodium falciparum and moderate activity by intraperitoneal administration in the STIB900 murine model of acute sleeping sickness.