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Zebrafish as a Screening Model for Testing the Permeability of Blood–Brain Barrier to Small Molecules
Abstract
The objective of this study was to evaluate the permeability of small molecules into the brain via the blood-brain barrier in zebrafish and to investigate the possibility of using this animal model as a screening tool during the early stages of drug discovery. Fifteen compounds were used to understand the permeation into the brain in zebrafish and mice. The ratio of brain-to-plasma concentration was compared between the two animal models. The partition coefficient (Kp,brain), estimated using the concentration ratio at designated times (0.167, 0.25, 0.5, or 2 h) after oral administrations (per os, p.o), ranged from 0.099 to 5.68 in zebrafish and from 0.080 to 11.8 in mice. A correlation was observed between the Kp,brain values obtained from the zebrafish and mice, suggesting that zebrafish can be used to estimate Kp,brain to predict drug penetration in humans. Furthermore, in vivo transport experiments to understand the permeability glycoprotein (P-gp) transporter-mediated behavior of loperamide (LPM) in zebrafish were performed. The zebrafish, Kp,brain,30min of LPM was determined to be 0.099 ± 0.069 after dosing with LPM alone, which increased to 0.180 ± 0.115 after dosing with LPM and tariquidar (TRQ, an inhibitor of P-gp). In mouse, the Kp,brain,30min of LPM was determined to be 0.080 ± 0.004 after dosing with LPM alone and 0.237 ± 0.013 after dosing with LPM and TRQ. These findings indicate that the zebrafish could be used as an effective screening tool during the discovery stages of new drugs to estimate their distribution in the brain.
... It displays neurochemistry profiles and hormones comparable to those of mammals. It is also an attractive model because of its high fecundity, easy husbandry, and transparency, allowing high-content screening [15][16][17]. ...
... PQ was orally administered to adult male zebrafish (wild type, 10 months old, N = 5) at a dose of 50 mg/kg using Silastic Laboratory Tubing according to the oral gavage method previously reported [16]. After 30 min, the fish blood and brain tissues were collected PQ was extracted by adding methanol to the blood and brain samples, which were then homogenized using the sonicator. ...
... In this study, our data demonstrated for the first time that internal PQ was highly accumulated in brain tissue by entering the BBB in adult zebrafish model. We previously demonstrated that BBB permeability study using adult zebrafish model could be used as an effective screening tool for predicting the penetration of CNS drug in rodents and humans BBB [16]. Therefore, this finding suggests that PQ could be have a neurotoxic effect in the zebrafish model, brain accumulation of PQ resulted in abnormalities in behaviors, neurochemistry, and EEG profiles (Figs. 2, 3, 4). ...
Paraquat is a polar herbicide protecting plant products against invasive species, it requires careful manipulation and restricted usage because of its harmful potentials. Exposure to paraquat triggers oxidative damage in dopaminergic neurons and subsequently causes a behavioral defect in vivo. Thereby, persistent exposure to paraquat is known to increase Parkinson’s disease risk by dysregulating dopaminergic systems in humans. Therefore, most studies have focused on the dopaminergic systems to elucidate the neurotoxicological mechanism of paraquat poisoning, and more comprehensive neurochemistry including histaminergic, serotonergic, cholinergic, and GABAergic systems has remained unclear. Therefore, in this study, we investigated the toxicological potential of paraquat poisoning using a variety of approaches such as toxicokinetic profiles, behavioral effects, neural activity, and broad-spectrum neurochemistry in zebrafish larvae after short-term exposure to paraquat and we performed the molecular modeling approach. Our results showed that paraquat was slowly absorbed in the brain of zebrafish after oral administration of paraquat. In addition, paraquat toxicity resulted in behavioral impairments, namely, reduced motor activity and led to abnormal neural activities in zebrafish larvae. This locomotor deficit came with a dysregulation of dopamine synthesis induced by the inhibition of tyrosine hydroxylase activity, which was also indirectly confirmed by molecular modeling studies. Furthermore, short-term exposure to paraquat also caused simultaneous dysregulation of other neurochemistry including cholinergic and serotonergic systems in zebrafish larvae. The present study suggests that this neurotoxicological profiling could be a useful tool for understanding the brain neurochemistry of neurotoxic agents that might be a potential risk to human and environmental health.
... While mouse models have provided valuable information regarding the role of P-gp and ABCG2 at the BBB, they are expensive to maintain and are not amenable to high-throughput screening or direct imaging of the CNS. The zebrafish (Danio rerio, Dr) has emerged as a potential model for studying the role of transporters at the BBB [9][10][11] . Like higher vertebrate organisms, zebrafish have a BBB comprised of endothelial cells that form tight junctions characterized by expression of claudin-5 and zona occludens-1 as well as decreased transcytosis [12][13][14] . ...
... However, which of the two proteins (Abcb4 or Abcb5) was expressed at the BBB was unknown. Preliminary studies demonstrated that transporters expressed at the zebrafish BBB can efflux known P-gp substrates such as rhodamine 123, loperamide and some tyrosine kinase inhibitors 11,17 . Both Abcb4 and Abcb5 have been shown to transport some substrates of human P-gp 15,18,19 ; however, detailed substrate specificity testing of the two transporters has not been performed. ...
... We next compared the ability of human P-gp and zebrafish Abcb4 and Abcb5 to transport known fluorescent substrates of human P-gp using flow cytometry. We also examined the ability of some common P-gp inhibitors to abrogate transporter activity, as P-gp inhibitors are frequently used as inhibitors of zebrafish Abcb4 and Abcb5 11,15,18 , despite the fact that their efficacy with different substrates has not been carefully examined. The substrates tested included calcein AM, rhodamine 123, BODIPY-prazosin, Flutax, BODIPY-vinblastine, LDS 751, BODIPY EDA, and TMRE. ...
Capillary endothelial cells of the human blood–brain barrier (BBB) express high levels of P-glycoprotein (P-gp, encoded by ABCB1 ) and ABCG2 (encoded by ABCG2 ). However, little information is available regarding ATP-binding cassette transporters expressed at the zebrafish BBB, which has emerged as a potential model system. We report the characterization and tissue localization of two genes that are similar to ABCB1, zebrafish abcb4 and abcb5. When stably expressed in HEK293 cells, both Abcb4 and Abcb5 conferred resistance to P-gp substrates; however, Abcb5 poorly transported doxorubicin and mitoxantrone compared to zebrafish Abcb4. Additionally, Abcb5 did not transport the fluorescent P-gp probes BODIPY-ethylenediamine or LDS 751, while they were transported by Abcb4. High-throughput screening of 90 human P-gp substrates confirmed that Abcb4 has an overlapping substrate specificity profile with P-gp. In the brain vasculature, RNAscope probes for abcb4 colocalized with staining by the P-gp antibody C219, while abcb5 was not detected. The abcb4 probe also colocalized with claudin-5 in brain endothelial cells. Abcb4 and Abcb5 had different tissue localizations in multiple zebrafish tissues, potentially indicating different functions. The data suggest that zebrafish Abcb4 functionally phenocopies P-gp and that the zebrafish may serve as a model to study the role of P-gp at the BBB.
... The interest in BBB function in human health and disease is at the forefront of neuroscience, due to the key role that the BBB plays in the onset of several diseases such as Alzheimer's disease [11]. More recently, the mammalian BBB has been studied for the purpose of optimizing therapeutic drug-delivery into the CNS, where the goal is to hijack existing transport mechanisms of the BBB to allow selective entry of therapeutic molecules, while minimizing the ill effects of a leakier BBB [12][13][14]. Current research has focused mainly on pathological conditions; however, this perspective is limited. ...
... Therefore, it is essential to consider the diversity of environments inhabited by fish, and that other fish species should be examined to make broad conclusions about piscine bloodbrain barriers. Tracer studies in teleost fish demonstrate that there are compounds such as sulfo-NHS-biotin, Evan's blue, and horseradish peroxidase (HRP) which cannot pass from the endothelial cells into the brain parenchyma [3,14,180]. Unlike other fish, however, elasmobranchs and sturgeons do not have an endothelial BBB (Figure 3). ...
... In elasmobranchs, this glial barrier is joined by tight junctions, while in sturgeons the glial cells are linked by glial lamellae instead, similar to that observed in cuttlefish [20,178]. Tracer studies in teleost fish demonstrate that there are compounds such as sulfo-NHS-biotin, Evan's blue, and horseradish peroxidase (HRP) which cannot pass from the endothelial cells into the brain parenchyma [3,14,180]. Unlike other fish, however, elasmobranchs and sturgeons do not have an endothelial BBB (Figure 3). ...
The need to protect neural tissue from toxins or other substances is as old as neural tissue itself. Early recognition of this need has led to more than a century of investigation of the blood-brain barrier (BBB). Many aspects of this important neuroprotective barrier have now been well established, including its cellular architecture and barrier and transport functions. Unsurprisingly, most research has had a human orientation, using mammalian and other animal models to develop translational research findings. However, cell layers forming a barrier between vascular spaces and neural tissues are found broadly throughout the invertebrates as well as in all vertebrates. Unfortunately, previous scenarios for the evolution of the BBB typically adopt a classic, now discredited ‘scala naturae’ approach, which inaccurately describes a putative evolutionary progression of the mammalian BBB from simple invertebrates to mammals. In fact, BBB-like structures have evolved independently numerous times, complicating simplistic views of the evolution of the BBB as a linear process. Here, we review BBBs in their various forms in both invertebrates and vertebrates, with an emphasis on the function, evolution, and conditional relevance of popular animal models such as the fruit fly and the zebrafish to mammalian BBB research.
... The zebrafish (Danio rerio) has emerged as a potential model for studying the role of transporters at the BBB [9][10][11]. Similar to higher vertebrate organisms, zebrafish have a BBB comprised of endothelial cells that form tight junctions characterized by expression of claudin-5 and zona occludens-1 and decreased transcytosis [12][13][14]. ...
... However, which of the two proteins (Abcb4 or Abcb5) was expressed at the BBB was unknown. Preliminary studies demonstrated that transporters expressed at the zebrafish BBB are able to efflux known P-gp substrates such as rhodamine 123, loperamide and some tyrosine kinase inhibitors [11,17]. Both Abcb4 and Abcb5 have been shown to transport some substrates of 105 and is also made available for use under a CC0 license. ...
... Consistent with these previous reports, we also found C219 reactivity in the endothelial cells of the zebrafish brain, and using RNA in situ techniques, have identified abcb4 as the gene responsible for the observed C219 staining. Thus, the zebrafish could indeed be a powerful tool to study the role of transporters in limiting the brain penetration of drugs, and initial studies have shown that the zebrafish could be comparable to mouse models to determine whether drugs can cross the BBB [11]. ...
Given its similarities with mammalian systems, the zebrafish has emerged as a potential model to study the blood-brain barrier (BBB). Capillary endothelial cells at the human BBB express high levels of P-glycoprotein (P-gp, encoded by the ABCB1 gene) and ABCG2 (encoded by the ABCG2 gene). However, little information has been available about ATP-binding cassette transporters expressed at the zebrafish BBB. In this study, we focus on the characterization and tissue localization of two genes that are similar to human ABCB1 , zebrafish abcb4 and abcb5 . Cytotoxicity assays with stably-transfected cell lines revealed that zebrafish Abcb5 cannot efficiently transport the substrates doxorubicin and mitoxantrone compared to human P-gp and zebrafish Abcb4. Additionally, zebrafish Abcb5 did not transport the fluorescent probes BODIPY-ethylenediamine or LDS 751, while they were readily transported by Abcb4 and P-gp. A high-throughput screen conducted with 90 human P-gp substrates confirmed that zebrafish Abcb4 has overlapping substrate specificity with P-gp. Basal ATPase activity of zebrafish Abcb4 and Abcb5 was comparable to that of human P-gp. In the brain vasculature, RNAscope probes to detect abcb4 colocalized with staining by the P-gp antibody C219, while abcb5 was not detected. Zebrafish abcb4 also colocalized with claudin-5 expression in brain endothelial cells. Abcb4 and Abcb5 had different tissue localizations in multiple zebrafish tissues, consistent with different functions. The data suggest that zebrafish Abcb4 most closely phenocopies P-gp and that the zebrafish may be a viable model to study the role of the multidrug transporter P-gp at the BBB.
... BPF penetration of the BBB was measured using waterborne exposure in adult zebrafish according to a previously reported method (Kim et al., 2017). Maintained in a 3-L tank, four animals in each treatment group were exposed for 168 h to BPF at one of four concentrations (0, 0.001, 0.01, and 0.1 mg/L) spanning the range observed in the environment. ...
... The BBB serves as a gatekeeper to protect the neuronal microenvironment, restricting the penetration of hazardous substances from the circulating blood into the central nervous system (CNS). The BBB in zebrafish and mammals share enough biological similarity (Jeong et al., 2008;Kim et al., 2017) to support the testing of BBB penetration in the adult zebrafish for chemicals with neurotoxicity potential in humans (Kim et al., 2020;Zheng, 2001). Therefore, our data of BPF bioaccumulation in the zebrafish CNS under naturally occurring levels of BPF exposure strongly suggest that chronic BPF exposure poses a potential risk to CNS functions, including cognition and behavior, in humans. ...
Although bisphenol F (BPF), the main replacement for bisphenol A, has been commonly used in polycarbonate production, its neurotoxicity and the underlying mechanisms remain poorly understood. To address this knowledge gap, this study aimed to assess the neurotoxicity caused by chronic exposure to BPF and to identify its underlying mechanisms. We exposed adult zebrafish chronically to BPF at environmentally relevant concentrations (0.001, 0.01, and 0.1 mg/L) for 4 weeks. The results revealed that with BPF crossing the blood–brain barrier and bioaccumulating in brain tissues, chronic exposure to BPF resulted in anxiety-like behaviors and disruptions in learning and memory function in adult zebrafish. Furthermore, BPF toxicity in the zebrafish brain involved the dysregulation of metabolic pathways for choline and kynurenine in neurotransmitter systems and for 17β-estradiol, cortisol, pregnenolone-sulfate, and Dehydroepiandrosterone (DHEA)-sulfate in neurosteroid systems. RNA-seq analysis revealed that BPF exposure affected metabolic pathways, calcium signaling pathways, neuroactive ligand–receptor interactions, tight junctions, gap junctions, and the gonadotropin-releasing hormone signaling pathway. Our results indicate that chronic exposure to BPF alters the neurochemical profile of the brain and causes neurobehavioral effects, such as anxiety and cognitive decline. Overall, the multimodal approach, including behavioral and neurochemical profiling technologies, has great potential for the comprehensive assessment of potential risks posed by environmental pollutants to human and ecosystem health.
... Notably, the structure, function and anatomical morphology of the BBB in zebrafish is highly comparable to that of mammals [43][44][45]. Furthermore, the permeability of the zebrafish BBB has been studied to evaluate drug delivery to the CNS and correlate it with that observed in mice [46]. ...
... To determine if GNPr-PEG and GNPr-PEG-Ang2 had the ability to cross the BBB and reach the CNS, we performed in vivo assays using 5 dpf larvae, which have already developed their BBB [46]. It is important to mention that before these assays, we demonstrated that stability in E3 medium was not affected (Supplementary Material, Fig. S3.2). ...
One of main drawbacks for the treatment of neurodegenerative pathologies is ensuring the delivery of therapeutic agents into the central nervous system (CNS). Nowadays, gold nanoprisms (GNPr) have become an emerging nanomaterial with a localized surface plasmon resonance in the biological window, showing applications in both detection and treatment of diseases. In this work, GNPr were functionalized with polyethylene glycol (PEG) and Angipep-2 (Ang2) peptide to obtain a new highly stable nanomaterial and evaluate its toxicity and ability to cross the blood-brain barrier (BBB) in a zebrafish larvae model. The success in the functionalization was confirmed by a full characterization that showed the physicochemical changes at each step. In turn, the colloidal stability of GNPr-PEG-Ang2 in biologically relevant media also was demonstrated. The toxicity assays of GNPr-PEG-Ang2 performed on SH-SY5Y neuroblastoma cell line and on zebrafish larvae showed no effects both in vitro and in vivo. GNPr delivery to the CNS was studied in zebrafish larvae by immersion. We confirmed that functionalization with PEG-Ang2 improved the crossing through the BBB in this model compared with GNPr functionalized only with PEG. Notably, our nanomaterial was not detected in the CNS of zebrafish larvae 24 h after exposure that correlates with an adequate clearance of GNPr-PEG-Ang2 from the brain. This report is the first study of GNPr in the in vivo model of zebrafish larvae demonstrating that its functionalization with Ang2 allows the crossing of the BBB. Moreover, considering the stability achieved of the GNPr-PEG-Ang2 and the results of in vitro and in vivo studies, this work becomes in a high contribution to the design of new nanomaterials with potential biomedical applications for CNS-related diseases.
... Among these new models, the zebrafish (Danio rerio) has become a popular vertebrate model for drug screening due to high fecundity, easy husbandry, light transparency during early developmental stages (permitting developmental observations in vivo), and genetic homology to mammals, including the expression of similar hormones and neurotransmitters (Guo, 2004;Kalueff et al., 2014;Liu and Baraban, 2019). Our previous study clearly demonstrated that zebrafish are an effective model for the pharmacokinetic study of central nervous system (CNS) drugs after oral administration ( Kim et al., 2017). Also, we found similar bloodbrain barrier (BBB) partition coefficients in zebrafish and mice. ...
... Our study showed that the obtained blood to brain concentration ratio (K p,brain ), ranged from approximately 3.2 to 6.7 for zebrafish (Fig. 5C), was similar to those in mice, ranged from 2.7 to 3.0 (Fig. 5D, Fig. S5 and Table S2). Indeed, BPA prove as permeable across the zebrafish BBB, underscoring the utility of this model as previously reported method ( Kim et al., 2017). Moreover, brain accumulation resulted in broad disruption of neurotransmitter systems (Fig. 4 and Table 2), thereby accounting for the behavioral changes (Figs. 2 and 3). ...
Bisphenol A (BPA) is a chemical monomer widely used in the production of hard plastics for food containers and personal items. Through improper industrial control and disposal, BPA has become a pervasive environmental contaminant, and toxicological studies have shown potent xenobiotic endocrine disruptor activity. Prenatal exposure in particular can lead to infertility and nervous system disorders characterized by behavioral aggression, depression, and cognitive impairment, thus necessitating careful hazard assessment.
In this study, we evaluated BPA accumulation rate, blood-brain barrier (BBB) permeability, lethality, cardiotoxicity, behavioral effects, and impacts on multiple neurochemical pathways in zebrafish larvae. The bioconcentration factor (BCF) ranged from 1.95 to 10.0, resulting in a high rate of accumulation in the larval body. Also, high BBB permeability allowed BPA to accumulate at similar rates in both zebrafish and adult mouse (blood to brain concentration ratios of 3.2–6.7 and 1.8 to 5.5, respectively). In addition, BPA-exposed zebrafish larvae exhibited developmental deformities, reduced heart rate, and impaired behavioral patterns, including decreased total distance traveled, slower movement velocity, and altered color-preference. These impairments were associated with inhibition of the phenylalanine to dopamine synthesis pathway and an imbalance between excitatory and inhibitory neurotransmitter systems. Our results suggest that behavioral alteration in BPA-exposed zebrafish result from high accumulation and ensuing dysregulation of serotonergic, kynurenergic, dopaminergic, cholinergic, and GABAergic neurotransmitter systems. In conclusion, similarities in toxic responses to mammalian models highlight the utility of the zebrafish larva as a convenient model for screening environmental toxins.
... Direct measurement of drug absorption into larvae has revealed that some chemicals are almost completely excluded, whereas others are concentrated more than 10-fold in larval tissue, making it difficult to ascertain the effective dose applied to the larva [111,116]. Nevertheless, comparative studies between adult zebrafish and mice suggest that brain-to-plasma rations for panels of small molecules are very similar between the species suggesting phylogenetic conservation of blood-brain barrier (BBB) properties [117]. The extent to which the larval zebrafish BBB replicates the transport properties of the human blood-brain barrier for drug delivery to the CNS is currently unclear, but compounds that do not enter the CNS and therefore appear inactive in larval zebrafish screens are unlikely to be useful in mammalian models and this may add value to larval screening platforms. ...
Extensive phylogenetic conservation of molecular pathways and neuroanatomical structures, associated with efficient methods for genetic modification, has been exploited increasingly to generate zebrafish models of human disease. A range of powerful approaches can be deployed to analyze these models with the ultimate goal of elucidating pathogenic mechanisms and accelerating efforts to find effective treatments. Unbiased neurobehavioral assays can provide readouts that parallel clinical abnormalities found in patients, although some of the most useful assays quantify responses that are not routinely evaluated clinically, and differences between zebrafish and human brains preclude expression of the full range of neurobehavioral abnormalities seen in disease. Imaging approaches that use fluorescent reporters and standardized brain atlases coupled with quantitative measurements of brain structure offer an unbiased means to link experimental manipulations to changes in neural architecture. Together, quantitative structural and functional analyses allow dissection of the cellular and physiological basis underlying neurological phenotypes. These approaches are used increasingly as outputs in chemical modifier screens, which provide a major opportunity to exploit zebrafish models to identify small molecule modulators of pathophysiology that may be informative for understanding disease mechanisms and possible therapeutic approaches.
... Several studies have been performed to analyze the function of the BBB in larvae, with fluorescent tracers injected into the heart or caudal vein and assessed in the brain [41,42]. Adult zebrafish are also used to assess drug delivery into the brain using LC-MS/MS analysis [43]. We hypothesized that accumulated PS-NPs in the brain penetrate through the BBB. ...
Microplastics, small pieces of plastic derived from polystyrene, have recently become an ecological hazard due to their toxicity and widespread occurrence in aquatic ecosystems. In this study, we exposed zebrafish larvae to two types of fluorescent polystyrene nanoparticles (PS-NPs) to identify their size-dependent effects. PS-NPs of 50 nm, unlike 100 nm PS-NPs, were found to circulate in the blood vessels and accumulate in the brains of zebrafish larvae. Behavioral and electroencephalogram (EEG) analysis showed that 50 nm PS-NPs induce abnormal behavioral patterns and changes in EEG power spectral densities in zebrafish larvae. In addition, the quantification of endogenous neurochemicals in zebrafish larvae showed that 50 nm PS-NPs disturb dopaminergic metabolites, whereas 100 nm PS-NPs do not. Finally, we assessed the effect of PS-NPs on the permeability of the blood–brain barrier (BBB) using a microfluidic system. The results revealed that 50 nm PS-NPs have high BBB penetration compared with 100 nm PS-NPs. Taken together, we concluded that small nanoparticles disturb the nervous system, especially dopaminergic metabolites.
... Notably, zebrafish possess a vertebrate neural structural organisation, and all of the major structures are similar to the mammalian brain. Furthermore, zebrafish also possesses a functional Blood-Brain Barrier (BBB), similar to humans [18]. Many important neurotransmitters were detected in the neurotransmitter profile of zebrafish, which is very important for neuroscientific studies [19]. ...
Animal models of human neurodegenerative disease have been investigated for several decades. In recent years, zebrafish (Danio rerio) and medaka (Oryzias latipes) have become popular in pathogenic and therapeutic studies about human neurodegenerative diseases due to their small size, the optical clarity of embryos, their fast development, and their suitability to large-scale therapeutic screening. Following the emergence of a new generation of molecular biological technologies such as reverse and forward genetics, morpholino, transgenesis, and gene knockout, many human neurodegenerative disease models, such as Parkinson’s, Huntington’s, and Alzheimer’s, were constructed in zebrafish and medaka. These studies proved that zebrafish and medaka genes are functionally conserved in relation to their human homologues, so they exhibit similar neurodegenerative phenotypes to human beings. Therefore, fish are a suitable model for the investigation of pathologic mechanisms of neurodegenerative diseases and for the large-scale screening of drugs for potential therapy. In this review, we summarize the studies in modelling human neurodegenerative diseases in zebrafish and medaka in recent years.
... Zebrafish also express homologs of human ABCC2, ACBCC4 and ABCC5, which are localized to brain endothelial cell lumenal membranes in mammalian systems. Zebrafish express ABCC4 and ABCC5 in the brain, although localization by cell type has not been determined [75,76] . There is no evidence yet for expression of ABCC2 in the zebrafish brain. ...
The brain is protected from toxins by a tightly regulated network of specialized cells, including endothelial cells, pericytes, astrocyes, and neurons, known collectively as the blood-brain barrier (BBB). This selectively permeable barrier permits only the most crucial molecules essential for brain function to enter and employs a number of different mechanisms to prevent the entry of potentially harmful toxins and pathogens. In addition to a physical barrier comprised of endothelial cells that form tight junctions to restrict paracellular transport, there is an active protective mechanism made up of energy-dependent transporters that efflux compounds back into the bloodstream. Two of these ATP-binding cassette (ABC) transporters are highly expressed at the BBB: P-glycoprotein (P-gp, encoded by the ABCB1 gene) and ABCG2 (encoded by the ABCG2 gene). Although a number of in vitro and in vivo systems have been developed to examine the role that ABC transporters play in keeping compounds out of the brain, all have inherent advantages and disadvantages. Zebrafish (Danio rerio) have become a model of interest for studies of the BBB due to the similarities between the zebrafish and mammalian BBB systems. In this review, we discuss what is known about ABC transporters in zebrafish and what information is still needed before the zebrafish can be recommended as a model to elucidate the role of ABC transporters at the BBB.
... Although, animal data remains a requirement for a compound to progress into clinical stages initiatives by policy makers to replace, reduce and refine (3R) the use of animals in drug discovery has led researchers to use alternate in vivo models to study PK/ PD. For instance, zebrafish was found to be a good model to test for the permeability of small molecules (Kim et al. 2017). Herein, we discuss the benefits and limitations of applying alternative models such as zebra fish, invertebrates Drosophila melanogaster (fruit fly), and Caenorhabditis elegans (roundworm) in studying ADME properties during drug development. ...
Improved PK/PD prediction in early stages of drug development is essential to inform lead optimization strategies and reduce attrition rates. Recently, there have been significant advancements in the development of new in vitro and in vivo strategies to better characterize pharmacokinetic properties and efficacy of drug leads. Herein, we review advances in experimental and mathematical models for clearance predictions, advancements in developing novel tools to capture slowly metabolized drugs, in vivo model developments to capture human etiology for supporting drug development, limitations and gaps in these efforts, and a perspective on the future in the field.
... Likewise, this enzyme is a key factor for limiting moxidectin in the brain (Kiki-Mvouaka et al., 2010), and sensitivity to intoxication by MLs in mammals is attributed to deficient P-glycoprotein activity in the BBB (Dowling 2006;Geyer et al., 2009). The function and structure of the BBB in zebrafish are conserved, developing between 3 and 10 days post fertilization (dpf) (Fleming et al., 2013;Kim et al., 2017;O'Brown et al., 2018). However, in an investigation of BBB development in zebrafish embryos, Fleming et al. (2013) identified P-glycoprotein orthologs that show efflux transporter activity from 8 days post fertilization. ...
Moxidectin is an antiparasitic drug belonging to the class of the macrocyclic lactones, subgroup mylbemicins. It is used worldwide in veterinary practice, but little is known about its potential environmental risks. Thus, we used the zebrafish embryo as a model system to study the potential effects of moxidectin on aquatic non-target organisms. The analyses were performed in two experimental sets: (1) acute toxicity and apical endpoints were characterized, with biomarker assays providing information on the activity levels of catalase (CAT), glutathione S-transferase (GST), lactate dehydrogenase (LDH), and acetylcholinesterase (AChE); and (2) internal concentration and spatial distribution of moxidectin were determined using ultraperformance liquid chromatography quadrupole-time-of-flight mass spectrometry (UPLC-QToF-MS) and matrix-assisted laser desorption/ionization-MS imaging (MALDI-MSi). The acute toxicity to zebrafish embryos (96 hpf) appeared mainly as a decrease in hatching rates (EC50 = 20.75 μg/L). It also altered the enzymatic activity of biomarker enzymes related to xenobiotic processing, anaerobic metabolism, and oxidative stress (GST, LDH, and CAT, respectively) and strongly accumulated in the embryos, as internal concentrations were 4 orders of magnitude higher than those detected in exposure solutions. MALDI-MSi revealed accumulations of the drug mainly in the head and eyes of the embryos (72 and 96 hpf). Thus, our results show that exposure to moxidectin decreases hatching success by 96 h and alters biochemical parameters in the early life stages of zebrafish while accumulating in the head and eye regions of the animals, demonstrating the need to prioritize this compound for environmental studies.
... Although mice are widely used to study autophagy on the tissue barrier, zebrafish shows unique advantages in the research of cerebrovascular pathological changes due to their optically translucent larval body and the masses of transgenic lines with tissue-specific reporters. Zebrafish BBB develops as early as 3 days post-fertilization (dpf) and can be used to evaluate the permeability of the BBB against small molecules [17]. Zebrafish has also been proven as a powerful in vivo model to study autophagic activity in different biological and pathological processes, such as development [18], tissue regeneration [19], infection [20], and neurodegeneration [21]. ...
Blood-brain barrier (BBB) disruption is a key event in triggering secondary damage to the central nervous system (CNS) under stroke, and is frequently associated with abnormal macroautophagy/autophagy in brain microvascular endothelial cells (BMECs). However, the underlying mechanism of autophagy in maintaining BBB integrity remains unclear. Here we report that in BMECs of patients suffering stroke, CLDN5 (claudin 5) abnormally aggregates in the cytosol accompanied by autophagy activation. In vivo zebrafish and in vitro cell studies reveal that BBB breakdown is partially caused by CAV1 (caveolin 1)-mediated redistribution of membranous CLDN5 into the cytosol under hypoxia. Meanwhile, autophagy is activated and contributes mainly to the degradation of CAV1 and aggregated CLDN5 in the cytosol of BMECs, therefore alleviating BBB breakdown. Blockage of autophagy by genetic methods or chemicals aggravates cytosolic aggregation of CLDN5, resulting in severer BBB impairment. These data demonstrate that autophagy functions in the protection of BBB integrity by regulating CLDN5 redistribution and provide a potential therapeutic strategy for BBB disorder-related cerebrovascular disease.
Abbreviations: BBB: blood-brain barrier; BECN1: beclin 1; BMEC: brain microvascular endothelial cell; CAV1: caveolin 1; CCA: common carotid artery; CLDN5: claudin 5; CNS: central nervous system; CQ: chloroquine; HIF1A: hypoxia inducible factor 1 subunit alpha; MCAO: middle cerebral artery occlusion-reperfusion; OCLN: occludin; ROS: reactive oxygen species; STED: stimulated emission depletion; TEER: trans-endothelial electrical resistance; TEM: transmission electron microscopy; TJ: tight junction; TJP1: tight junction protein 1; UPS: ubiquitin-proteasome system
... The neurotransmitter profile such as GABA, glutamate, dopamine, noradrenaline, serotonin, histamine and acetylcholine are present in the zebrafish, whereby altered neurotransmitter system is reported to be affected in human diseases [55]. In addition, zebrafish possesses a functional Blood-Brain Barrier (BBB) similar to humans, with the expression of tight junction proteins ZO-1 and claudin-5 in the endothelial vascular cells, as well as the transporter protein permeability glycoprotein/ATP-binding cassette transporter subfamily B 4/5 within the brain [56]. These factors attributed to the wide use of zebrafish in neuroscience studies, from neurodegenerative diseases to ageing, autism, sleep disorders, cognitive deficits, depression, psychoses and addiction [50]. ...
Background and Objective
Parkinson disease (PD) is one of the commonest neurodegenerative disorders that affects the motor system, and includes cardinal motor symptoms such as resting tremor, cogwheel rigidity, bradykinesia and postural instability. Its prevalence is increasing worldwide due to increase in life span. Although, two centuries since the first description of the disease, no proper cure with regard to treatment strategies and control of symptoms could be reached. One of the major challenges faced by the researchers is to have a suitable research model.
Results
Rodents are the most common PD models used but no single model can replicate the true nature of PD. In this review, we aim to discuss another animal model, the zebrafish (Danio rerio) which is gaining popularity. Zebrafish brain has all the major structures found in the mammalian brain, complete with neurotransmitters systems, and possesses a functional blood-brain barrier similar to humans. From the perspective of PD research, the zebrafish possesses the ventral diencephalon which is thought to be homologous to the mammalian substantia nigra. We summarize the various zebrafish models available to study PD, namely chemical-induced and genetic models.
Conclusion
The zebrafish can complement to the use of other animal models for the mechanistic study of PD and help in the screening of new potential therapeutic compounds.
... Since the complex psychopharmacology of amitriptyline remains poorly understood, further in-vivo research is needed to examine the effects of this TCA on behavior and physiology. The zebrafish (Danio rerio) has recently become a valuable model organism in biomedicine [13,24,31,35] and neuroscience research, including developmental neuroscience, neural circuits imaging and neuropharmacology [19,23,47,48]. Earlier studies in zebrafish have revealed their high sensitivity to acute amitriptyline action, evoking robust anxiolytic-like effects at low doses and toxic/ataxic effects at high doses [8]. ...
Amitriptyline is a commonly used tricyclic antidepressant (TCA) inhibiting serotonin and norepinephrine reuptake. The exact CNS action of TCAs remains poorly understood, necessitating new screening approaches and novel model organisms. Zebrafish (Danio rerio) are rapidly emerging as a promising tool for pharmacological research of antidepressants, including amitriptyline. Here, we examine the effects of chronic 2-week exposure to 10 and 50 μg/L amitriptyline on zebrafish behavior and monoamine neurotransmitters. Overall, the drug at 50 μg/L evoked pronounced anxiolytic-like effects in the novel tank test (assessed by more time in top, fewer transition and shorter latency to enter the top). Like other TCAs, amitriptyline reduced serotonin turnover, but also significantly elevated whole-brain norepinephrine and dopamine levels. The latter effect was not reported in this model previously, and accompanied higher brain expression of tyrosine hydroxylase (a rate-limiting enzyme of catecholamine biosynthesis), but unaltered expression of dopamine-β-hydroxylase and monoamine oxidase (the enzymes of dopamine metabolism). This response may underlie chronic amitriptyline action on dopamine and norepinephrine neurotransmission, and contribute to the complex CNS profile of this drug observed both clinically and in animal models. Collectively, these findings also confirm the important role of monoamine modulation in the regulation of anxiety-related behavior in zebrafish, and support the utility of this organism as a promising in-vivo model for CNS drug screening.
... At 5days post-fertilization (dpf), zebrafish larvae show spontaneous swimming and their visual system is fully developed. As many reports suggest that the behavioral responses of zebrafish are similar to those of mammals (Noyes et al., 2015;Demin et al., 2017;Kim et al., 2017), it seems appropriate to use zebrafish for testing of exercise, learning, and memory in behavioral evaluation. ...
Most third- and fourth-generation cephalosporins, such as cefotaxime, cefmenoxime, cefepime, and cefpirome, contain an aminothiazoyl ring at the C-7 position. Drug impurity, which may be produced either during synthesis or upon degradation, can induce adverse effects. Various reports have indicated that neurotoxicity is a side effect of cephalosporin. In this study, we developed methods for assessing the free-swimming activities and behaviors in zebrafish larvae in response to continuous darkness and stimulation of light-to-dark photoperiod transition by chemical treatments. We also performed transcriptome analysis to identify differentially expressed genes (DEGs). Gene ontology analysis revealed that various processes related to nervous system development were significantly enriched by DEGs. We integrated 16 DEGs with protein–protein interaction networks and identified that neuroactive ligand–receptor interaction [e.g., λ-aminobutyric acid and glutamate receptor, metabotropic 1a (GRM1A)] pathway was regulated by the compounds. Our findings suggested that neurobehavioral effects mainly depend on the mother nucleus structure 7-aminocephalosporanic acid and the substitution at the C-3 position. In addition, gad2, or111-4, or126-3, grm1a, opn8c, or111-5, or113-2, and or118-3 may potentially be utilized as novel biomarkers for this class of cephalosporins, which causes neurotoxicity. This study provides neurological behavior, transcriptome, and docking information that could be used in further investigations of the structures and developmental neurotoxicity relationship of chemicals.
Limited studies on neurotoxicity following chronic exposure to butyl-paraben (BuP) have been conducted. In this study, neurobehavior in zebrafish adults was assessed using a novel tank test, photomotor response tests, and T-maze tests after exposure to BuP for 28 days at the concentrations of 0, 0.01, 0.1, and 1.0 mg/L. To comprehensively understand the underlying molecular perturbations in the brain, alterations in transcripts, neurotransmitters, and neurosteroids were measured. We found that BuP penetrated the blood-brain barrier and impaired neurobehavior in photosensitivity at 1.0 mg/L and in memory at 0.1 and 1.0 mg/L. RNA-seq analysis showed that phototransduction, tight junctions, and neuroactive ligand receptor activity were significantly affected, which explains the observed abnormal neurobehaviors. Neurosteroid analysis revealed that BuP increased cortisol levels in a concentration-dependent manner and specifically reduced allopregnanolone levels at all tested concentrations, suggesting that cortisol and allopregnanolone are significant neurosteroid markers associated with photosensitivity and memory deficits. Collectively, we demonstrated that BuP can cross the blood-brain and modulate the levels of transcripts, associated with phototransduction and circadian rhythm, and neurosteroidal cortisol and allopregnanolone, resulting in abnormal neurobehavioral responses to light stimulation and learning and memory.
Increasing evidence has demonstrated that cadmium accumulation in the blood increases the risk of neurological diseases. However, how cadmium breaks through the blood-brain barrier (BBB) and is transferred from the blood circulation into the central nervous system is still unclear. In this study, we examined the toxic effect of cadmium chloride (CdCl2) on the development and function of BBB in zebrafish. CdCl2 exposure induced cerebral hemorrhage, increased BBB permeability and promoted abnormal vascular formation by promoting VEGF production in zebrafish brain. Furthermore, in vivo and in vitro experiments showed that CdCl2 altered cell-cell junctional morphology by disrupting the proper localization of VE-cadherin and ZO-1. The potential mechanism involved in the inhibition of protein tyrosine phosphatase (PTPase) mediated by cadmium-induced ROS was confirmed with diphenylene iodonium (DPI), a ROS production inhibitor. Together, these data indicate that BBB is a critical target of cadmium toxicity and provide in vivo etiological evidence of cadmium-induced neurovascular disease in a zebrafish BBB model.
Epilepsy is a common chronic neurological disease characterized by recurrent epileptic seizures. A seizure is an uncontrolled electrical activity in the brain that can cause different levels of behavior, emotion, and consciousness. One-third of patients fail to receive sufficient seizure control, even though more than fifty FDA-approved anti-seizure drugs (ASDs) are available. In this study, we attempted small molecule screening to identify potential therapeutic agents for the treatment of seizures using seizure-induced animal models. Through behavioral phenotype-based screening, 4-(2-chloro-4-fluorobenzyl)-3-(2-thienyl)-1,2,4-oxadiazol-5(4H)-one (GM-90432) was identified as a prototype. GM-90432 treatment effectively decreased seizure-like behaviors in zebrafish and mice with chemically induced seizures. These results were consistent with decreased neuronal activity through immunohistochemistry for pERK in zebrafish larvae. Additionally, electroencephalogram (EEG) analysis revealed that GM-90432 decreases seizure-specific EEG events in adult zebrafish. Moreover, we revealed the preferential binding of GM-90432 to voltage-gated Na⁺ channels using a whole-cell patch clamp technique. Through pharmacokinetic analysis, GM-90432 effectively penetrated the blood-brain barrier and was distributed into the brain. Taken together, we suggest that GM-90432 has the potential to be developed into a new ASD candidate.
Human health are to be monitored for glucose level, blood pressure level and intimated at the right time for taking the necessary pills or food. Defense forces, Diabetes victim and victim with heart attack must be given alert on their BP level and accordingly they can react for taking the appropriate pill or food. A victim may go low in his /her BP level while sleeping more vulnerable to this kind of situation which requires salient tracking and alerting system. Smart polymers combined with Biomimetic Elastomers forms the Injectable Polymer hydrogels. Moreover, many IoT devices have been discovered. A study throws light on a fact that most of continuous tracking observations, human skin suffers a bone disorder called Osteoporosis which ultimately leads to deformation of bones. But hydration loss must be reducible for a bi tissue to be discovered; thereby bypassing the drawbacks caused due to standalone IoT sensors. Injectable Polymer hydrogels is already used for plants and animals for avoiding damage to them during research experiments. The polymers must be molded to the required consistency for synthesizing wearable sensors eliminating health hazards. The conventional IoT system is substituted with the biotechnological solution to enhance continuous monitoring and alert system for victim and elderly aged people. Injectable Polymer hydrogels an artificial skin protein constitutes the composition of nutrients and proteins available in the form of powder can be used for manufacturing the wearable devices to maintain the conductivity range in such a way that it does not affect the human skin. It is proposed to synthesize the artificial skin gel in a relative state that it produces accurate results with sensor data transfer also. The solution proposed here provides the safe usage of irremovable monitoring and tracking of health status.
The blood-brain barrier (BBB) poses a serious impediment to the delivery of effective therapies to the central nervous system (CNS). Over time, various model systems have been crafted and used to evaluate the complexities of the BBB, which includes an impermeable physical barrier and a series of energy-dependent efflux pumps. Models of the BBB have mainly sought to assess changes in endothelial cell permeability, the role of ATP-dependent efflux transporters in drug disposition, and alterations in communication between BBB cells and the microenvironment. In the context of disease, various animal models have been utilized to examine real time BBB drug permeability, CNS dynamic changes, and overall treatment response. In this review, we outline the use of these in vitro and in vivo blood-brain barrier model systems to study normal physiology and diseased states. These current models each have their own advantages and disadvantages for studying the response of biologic processes to physiological and pathological conditions. Additional models are needed to mimic more closely the dynamic quality of the BBB, with the goal focused on potential clinical applications.
Ionocytes are specialized cells in the epidermis of embryonic zebrafish (Danio rerio)that play important roles in ion homeostasis and have functional similarities to mammalian renal cells. Here, we examined whether these cells might also share another functional similarity with renal cells, which is the presence of efflux transporter activities useful for elimination of toxic small molecules. Xenobiotic transporters (XTs), including the ATP-Binding Cassette (ABC)family, are a major defense mechanism against diffusible toxic molecules in aquatic embryos, including zebrafish, but their activity in the ionocytes has not previously been studied. Using fluorescent small molecule substrates of XT, we observed that specific populations of ionocytes uptake and efflux fluorescent small molecules in a manner consistent with active transport. We specifically identified a P-gp/ABCB1 inhibitor-sensitive efflux activity in the H ⁺ -ATPase-rich (HR)ionocytes, and show that these cells exhibit enriched expression of the ABCB gene, abcb5. The results extend our understanding of the functional significance of zebrafish ionocytes and indicate that these cells could play an important role in protection of the fish embryo from harmful small molecules.
Clostridium perfringens epsilon toxin (ETX) is hypothesized to mediate blood-brain barrier (BBB) permeability by binding to the myelin and lymphocyte protein (MAL) on the luminal surface of endothelial cells (ECs). However, the kinetics of this interaction and a general understanding of ETX's behavior in a live organism have yet to be appreciated. Here we investigate ETX binding and BBB breakdown in living Danio rerio (zebrafish). Wild-type zebrafish ECs do not bind ETX. When zebrafish ECs are engineered to express human MAL (hMAL), proETX binding occurs in a time-dependent manner. Injection of activated toxin in hMAL zebrafish initiates BBB leakage, hMAL downregulation, blood vessel stenosis, perivascular edema, and blood stasis. We propose a kinetic model of MAL-dependent ETX binding and neurovascular pathology. By generating a humanized zebrafish BBB model, this study contributes to our understanding of ETX-induced BBB permeability and strengthens the proposal that MAL is the ETX receptor.
To ensure tissue homeostasis the brain needs to be protected from blood-derived fluctuations or pathogens that could affect its function. Therefore, the brain capillaries develop tissue-specific properties to form a selective blood-brain barrier (BBB), allowing the passage of essential molecules to the brain and blocking the penetration of potentially harmful compounds or cells. Previous studies reported the presence of this barrier in zebrafish. The intrinsic features of the zebrafish embryos and larvae in combination with optical techniques, make them suitable for the study of barrier establishment and maturation. In this review, we discuss the most recent contributions to the development and formation of a functional zebrafish BBB. Moreover, we compare the molecular and cellular characteristic of the zebrafish and the mammalian BBB.
The interactions of nanomedicines with biological environments is heavily influenced by their physicochemical properties. Formulation design and optimization are therefore key steps towards successful nanomedicine development. Unfortunately, detailed assessment of nanomedicine formulations, at a macromolecular level, in rodents is severely limited by the restricted imaging possibilities within these animals. Moreover, rodent in vivo studies are time consuming and expensive, limiting the number of formulations that can be practically assessed in any one study. Consequently, screening and optimisation of nanomedicine formulations is most commonly performed in surrogate biological model systems, such as human-derived cell cultures. However, despite the time and cost advantages of classical in vitro models, these artificial systems fail to reflect and mimic the complex biological situation a nanomedicine will encounter in vivo. This has acutely hampered the selection of potentially successful nanomedicines for subsequent rodent in vivo studies. Recently, zebrafish have emerged as a promising in vivo model, within nanomedicine development pipelines, by offering opportunities to quickly screen nanomedicines under in vivo conditions and in a cost-effective manner so as to bridge the current gap between in vitro and rodent studies. In this review, we outline several advantageous features of the zebrafish model, such as biological conservation, imaging modalities, availability of genetic tools and disease models, as well as their various applications in nanomedicine development. Critical experimental parameters are discussed and the most beneficial applications of the zebrafish model, in the context of nanomedicine development, are highlighted.
Research on zebrafish embryonic development has already contributed to major breakthroughs in our understanding of how the cardiovascular system forms and functions. Zebrafish embryos are transparent, allowing noninvasive in vivo imaging. The advancements of high-resolution imaging and image analysis software, combined with the generation of tissue-specific transgenic lines and forward genetic screens, enabled the study of endothelial development at cellular resolution. Also, zebrafish embryos are not fully dependent on a functional cardiovascular system during the first few days of development since they get enough oxygen by passive diffusion. This advantage allowed the deciphering of the interplay between cardiac form and function as well as the identification of severe mutations of the heart and vessels. In this chapter, we highlight the experimental approaches and disease models used in zebrafish to study different aspects of the cardiovascular system.
The present study explores the potential of 10-day-old zebrafish (Danio rerio) as a predictive blood-brain-barrier model using a set of 7 pharmaceutical agents. For this purpose, zebrafish were incubated with each of these 7 drugs separately via the route of immersion and the concentration reaching the brain was determined by applying a brain extraction procedure allowing isolation of the intact brain from the head of the zebrafish larvae. Sample analysis was performed utilizing capillary ultra-high performance liquid chromatography (cap-UHPLC) on a Pepmap RSLC C18 capillary column (150 mm × 300 μm, dp = 2 μm) coupled to a variable wavelength UV detector. Gradient separation was performed in 28 min at a flow rate of 5 μL/min and the optimal injection volume was determined to be 1 μL. The brain extraction procedure was established for the zebrafish strain TG898 exhibiting red fluorescence of the brain, allowing control of the integrity of the extracted parts. Quantitative experiments carried out on pooled samples of six zebrafish (n = 6) demonstrated the selective semipermeable nature of the blood-brain barrier after incubating the zebrafish at the maximum tolerated concentration for the investigated pharmaceuticals. The obtained brain-to-trunk ratios ranged between 0.3 for the most excluded compound and 1.2 for the pharmaceutical agent being most accumulated in the brain of the fish.
Workflow of brain extraction to study the uptake of pharmaceuticals in the brain of zebrafish larvae
Introduction:
Adult zebrafish pharmacology is evolving rapidly for creating efficacy and safety models for drug discovery. However, there is very limited research in understanding pharmacokinetics (PK) in adult zebrafish. Methods for understanding PK will help in conducting pharmacokinetic - pharmacodynamic (PK-PD) correlations and improving the quality and applicability of data obtained using zebrafish.
Methods:
We conducted adult zebrafish PK and brain penetration studies on two known compounds (irinotecan and lorcaserin) with distinct PK and brain penetration properties using validated LCMS/MS method. Irinotecan was studied at a dose of 100mg/kg i.p. and levels of the parent drug and active metabolite SN-38 were measured. Loracserin was studies at a dose of 10mg/kg by two routes i.p. and p.o.
Results:
Zebrafish PK and brain penetration profiles for both compounds were very similar to that of higher mammals including humans. Irinotecan was metabolised to SN-38 in ratios similar to ratios seen in other species and the compound had long half life with very low brain penetration in our studies. Loracasin was highly permeable in brain as compared to the exposure in blood, with long half life and high relative bioavailability, similar to other mammalian species including humans.
Discussion:
Adult zebrafish PK studies are relatively an unexplored area of zebrafish research. The zebrafish data for key parameters of irinotecan and loracserin shows a high correlation to the data from higher species, including human. This report explores and discusses the use of adult zebrafish as a predictive PK tool for higher animal studies.
Zebrafish are a unique cell to behavior model for studying the basic biology of human inherited neurological conditions. Conserved vertebrate genetics and optical transparency provide in vivo access to the developing nervous system as well as high-throughput approaches for drug screens. Here we review zebrafish modeling for two broad groups of inherited conditions that each share genetic and molecular pathways and overlap phenotypically: cognitive neurodevelopmental disorders such as Autism Spectrum Disorders, Intellectual Disability and Schizophrenia, and neurodegenerative diseases, such as Cerebellar Ataxia, Hereditary Spastic Paraplegia and Charcot-Marie Tooth disease. We also conduct a small meta-analysis of zebrafish orthologues of high confidence neurodevelopmental disorder and neurodegenerative disease genes by looking at duplication rates and relative protein sizes. In the past zebrafish genetic models of these neurodevelopmental disorders and neurodegenerative diseases have provided insight into cellular, circuit and behavioral level mechanisms contributing to these conditions. Moving forward, advances in genetic manipulation, live imaging of neuronal activity and automated high-throughput molecular screening promise to help delineate the mechanistic relationships between different types of neurological conditions and accelerate discovery of therapeutic strategies.
Significance
Overcoming resistance to targeted kinase inhibitors is a major clinical challenge in oncology. Development of crizotinib resistance through the emergence of a secondary ROS1 mutation, ROS1 G2032R , was observed in patients with ROS1 fusion-positive lung cancer. In addition, a novel ROS1 fusion recently has been identified in glioblastoma. A new agent with robust activity against the ROS1 G2032R mutation and with CNS activity is needed to address these unmet medical needs. Here we report the identification of PF-06463922, a ROS1/anaplastic lymphoma kinase (ALK) inhibitor, with exquisite potency against ROS1 fusion kinases, capable of inhibiting the ROS1 G2032R mutation and FIG-ROS1–driven glioblastoma tumor growth in preclinical models. PF-06463922 demonstrated excellent therapeutic potential against ROS1 fusion-driven cancers, and it currently is undergoing phase I/II clinical trial investigation.
The currently available antiepileptic drugs are typically administered via oral or intravenous (IV) routes which commonly exhibit high systemic distribution into non-targeted tissues, leading to peripheral adverse effects and limited brain uptake. In order to improve the efficacy and tolerability of the antiepileptic drug therapy, alternative administration strategies have been investigated. The purpose of the present study was to assess the pharmacokinetics of carbamazepine administered via intranasal (IN) and IV routes to mice, and to investigate whether a direct transport of the drug from nose to brain could be involved. The similar pharmacokinetic profiles obtained in all matrices following both administration routes indicate that, after IN delivery, carbamazepine reaches quickly and extensively the bloodstream, achieving the brain predominantly via systemic circulation. However, the uneven biodistribution of carbamazepine through the brain regions with higher concentrations in the olfactory bulb and frontal cortex following IN instillation, in comparison with the homogenous brain distribution pattern after IV injection, strongly suggests the involvement of a direct transport of carbamazepine from nose to brain. Therefore, it seems that IN delivery represents a suitable and promising alternative route to administer carbamazepine not only for the chronically use of the drug but also in emergency conditions.
The efflux transporter P-glycoprotein, expressed at high levels at the blood-brain barrier, exerts a profound effect on the disposition of various therapeutic compounds in the brain. A rapid and efficient modulation of this efflux transporter could enhance the distribution of its substrates and thereby improve central nervous system pharmacotherapies. This study explored the impact of the intravenous co-administration of two P-glycoprotein modulators, tariquidar and elacridar, on the pharmacokinetics and brain distribution of loperamide, a P-glycoprotein substrate probe, in rats. After one hour post-dosing, tariquidar and elacridar, both at a dose of 1.0 mg/kg, increased loperamide levels in the brain by 2.3- and 3.5-fold, respectively. However, the concurrent administration of both P-glycoprotein modulators, each at a dose of 0.5 mg/kg, increased loperamide levels in the brain by 5.8-fold and resulted in the most pronounced opioid-induced clinical signs. This phenomenon may be the result of a combined non-competitive modulation by tariquidar and elacridar. Besides, the simultaneous administration of elacridar and tariquidar did not significantly modify the pharmacokinetic parameters of loperamide. This observation potentially allows the concurrent use of low but therapeutic doses of P-gp modulators to achieve full inhibitory effects.
Zebrafish are becoming increasingly popular as an organism in which to model human disease and to study the effects of small molecules on complex physiological and pathological processes. Since larvae are no more than a few millimetres in length, and can live in volumes as small as 100 microliters, they are particularly amenable to high-throughput and high content compound screening in 96 well plate format. There is a growing literature providing evidence that many compounds show similar pharmacological effects in zebrafish as they do in mammals, and in particular humans. However, a major question regarding their utility for small molecule screening for neurological conditions is whether a molecule will reach its target site within the central nervous system. Studies have shown that Claudin-5 and ZO-1, tight-junction proteins which are essential for blood-brain barrier (BBB) integrity in mammals, can be detected in some cerebral vessels in zebrafish from 3 days post-fertilisation (d.p.f.) onwards and this timing coincides with the retention of dyes, immunoreactive tracers and fluorescent markers within some but not all cerebral vessels. Whilst these findings demonstrate that features of a BBB are first present at 3 d.p.f., it is not clear how quickly the zebrafish BBB matures or how closely the barrier resembles that of mammals. Here, we have combined anatomical analysis by transmission electron microscopy, functional investigation using fluorescent markers and compound uptake using liquid chromatography/tandem mass spectrometry to demonstrate that maturation of the zebrafish BBB occurs between 3 d.p.f. and 10 d.p.f. and that this barrier shares both structural and functional similarities with that of mammals.
The zebrafish has become an important in vivo model in biomedical research. Effective methods must be developed and utilized to deliver compounds or agents in solutions for scientific research. Current methods for administering compounds orally to adult zebrafish are inaccurate due to variability in voluntary consumption by the fish. A gavage procedure was developed to deliver precise quantities of infectious agents to zebrafish for study in biomedical research. Adult zebrafish over 6 months of age were anesthetized with 150 mg/L of buffered MS-222 and gavaged with 5 μl of solution using flexible catheter implantation tubing attached to a cut 22-G needle tip. The flexible tubing was lowered into the oral cavity of the zebrafish until the tip of the tubing extended past the gills (approximately 1 cm). The solution was then injected slowly into the intestinal tract. This method was effective 88% of the time, with fish recovering uneventfully. This procedure is also efficient as one person can gavage 20-30 fish in one hour. This method can be used to precisely administer agents for infectious diseases studies, or studies of other compounds in adult zebrafish.
Background
Successful delivery of compounds to the brain and retina is a challenge in the development of therapeutic drugs and imaging agents. This challenge arises because internalization of compounds into the brain and retina is restricted by the blood–brain barrier (BBB) and blood-retinal barrier (BRB), respectively. Simple and reliable in vivo assays are necessary to identify compounds that can easily cross the BBB and BRB.
Methods
We developed six fluorescent indoline derivatives (IDs) and examined their ability to cross the BBB and BRB in zebrafish by in vivo fluorescence imaging. These fluorescent IDs were administered to live zebrafish by immersing the zebrafish larvae at 7-8 days post fertilization in medium containing the ID, or by intracardiac injection. We also examined the effect of multidrug resistance proteins (MRPs) on the permeability of the BBB and BRB to the ID using MK571, a selective inhibitor of MRPs.
Results
The permeability of these barriers to fluorescent IDs administered by simple immersion was comparable to when administered by intracardiac injection. Thus, this finding supports the validity of drug administration by simple immersion for the assessment of BBB and BRB permeability to fluorescent IDs. Using this zebrafish model, we demonstrated that the length of the methylene chain in these fluorescent IDs significantly affected their ability to cross the BBB and BRB via MRPs.
Conclusions
We demonstrated that in vivo assessment of the permeability of the BBB and BRB to fluorescent IDs could be simply and reliably performed using zebrafish. The structure of fluorescent IDs can be flexibly modified and, thus, the permeability of the BBB and BRB to a large number of IDs can be assessed using this zebrafish-based assay. The large amount of data acquired might be useful for in silico analysis to elucidate the precise mechanisms underlying the interactions between chemical structure and the efflux transporters at the BBB and BRB. In turn, understanding these mechanisms may lead to the efficient design of compounds targeting the brain and retina.
Caffeine is the most widely consumed centralnervous-system stimulant. Three main mechanisms of action of caffeine on the central nervous system have been described. Mobilization of intracellular calcium and inhibition of specific phosphodiesterases only occur at high non-physiological concentrations of caffeine. The only likely mechanism of action of the methylxanthine is the antagonism at the level of adenosine receptors. Caffeine increases energy metabolism throughout the brain but decreases at the same time cerebral blood flow, inducing a relative brain hypoperfusion. Caffeine activates noradrenaline neurons and seems to affect the local release of dopamine. Many of the alerting effects of caffeine may be related to the action of the methylxanthine on serotonine neurons. The methylxanthine induces dose-response increases in locomotor activity in animals. Its psychostimulant action on man is, however, often subtle and not very easy to detect. The effects of caffeine on learning, memory, performance and coordination are rather related to the methylxanthine action on arousal, vigilance and fatigue. Caffeine exerts obvious effects on anxiety and sleep which vary according to individual sensitivity to the methylxanthine. However, children in general do not appear more sensitive to methylxanthine effects than adults. The central nervous system does not seem to develop a great tolerance to the effects of caffeine although dependence and withdrawal symptoms are reported.
Acetylcholinesterase (AChE) inhibitors are widely used as pesticides and drugs. Their primary effect is the overstimulation of cholinergic receptors which results in an improper muscular function. During vertebrate embryonic development nerve activity and intracellular downstream events are critical for the regulation of muscle fiber formation. Whether AChE inhibitors and related neurotoxic compounds also provoke specific changes in gene transcription patterns during vertebrate development that allow them to establish a mechanistic link useful for identification of developmental toxicity pathways has, however, yet not been investigated. Therefore we examined the transcriptomic response of a known AChE inhibitor, the organophosphate azinphos-methyl (APM), in zebrafish embryos and compared the response with two non-AChE inhibiting unspecific control compounds, 1,4-dimethoxybenzene (DMB) and 2,4-dinitrophenol (DNP). A highly specific cluster of APM induced gene transcripts was identified and a subset of strongly regulated genes was analyzed in more detail. The small heat shock protein hspb11 was found to be the most sensitive induced gene in response to AChE inhibitors. Comparison of expression in wildtype, ache and sop(fixe) mutant embryos revealed that hspb11 expression was dependent on the nicotinic acetylcholine receptor (nAChR) activity. Furthermore, modulators of intracellular calcium levels within the whole embryo led to a transcriptional up-regulation of hspb11 which suggests that elevated intracellular calcium levels may regulate the expression of this gene. During early zebrafish development, hspb11 was specifically expressed in muscle pioneer cells and Hspb11 morpholino-knockdown resulted in effects on slow muscle myosin organization. Our findings imply that a comparative toxicogenomic approach and functional analysis can lead to the identification of molecular mechanisms and specific marker genes for potential neurotoxic compounds.
Epilepsy is a complex brain disorder with multiple underlying causes and poorly understood pathogenetic mechanisms. Animal models have been indispensable tools in experimental epilepsy research. Zebrafish (Danio rerio) are rapidly emerging as a promising model organism to study various brain disorders. Seizure-like behavioral and neurophysiological responses can be evoked in larval and adult zebrafish by various pharmacological and genetic manipulations, collectively emphasizing the growing utility of this model for studying epilepsy. Here, we discuss recent developments in using zebrafish models to study the seizure-like behavior involved in epilepsy, outlining current challenges and strategies for further translational research in this field.
Nilotinib has been shown to be a more potent inhibitor of BCR-ABL than imatinib. We evaluated the efficacy and safety of nilotinib, as compared with imatinib, in patients with newly diagnosed Philadelphia chromosome-positive chronic myeloid leukemia (CML) in the chronic phase.
In this phase 3, randomized, open-label, multicenter study, we assigned 846 patients with chronic-phase Philadelphia chromosome-positive CML in a 1:1:1 ratio to receive nilotinib (at a dose of either 300 mg or 400 mg twice daily) or imatinib (at a dose of 400 mg once daily). The primary end point was the rate of major molecular response at 12 months.
At 12 months, the rates of major molecular response for nilotinib (44% for the 300-mg dose and 43% for the 400-mg dose) were nearly twice that for imatinib (22%) (P<0.001 for both comparisons). The rates of complete cytogenetic response by 12 months were significantly higher for nilotinib (80% for the 300-mg dose and 78% for the 400-mg dose) than for imatinib (65%) (P<0.001 for both comparisons). Patients receiving either the 300-mg dose or the 400-mg dose of nilotinib twice daily had a significant improvement in the time to progression to the accelerated phase or blast crisis, as compared with those receiving imatinib (P=0.01 and P=0.004, respectively). No patient with progression to the accelerated phase or blast crisis had a major molecular response. Gastrointestinal and fluid-retention events were more frequent among patients receiving imatinib, whereas dermatologic events and headache were more frequent in those receiving nilotinib. Discontinuations due to aminotransferase and bilirubin elevations were low in all three study groups.
Nilotinib at a dose of either 300 mg or 400 mg twice daily was superior to imatinib in patients with newly diagnosed chronic-phase Philadelphia chromosome-positive CML. (ClinicalTrials.gov number, NCT00471497.)
Osteoporosis is a disease of low bone mass most often caused by an increase in bone resorption that is not sufficiently compensated for by a corresponding increase in bone formation. As gut-derived serotonin (GDS) inhibits bone formation, we asked whether hampering its biosynthesis could treat osteoporosis through an anabolic mechanism (that is, by increasing bone formation). We synthesized and used LP533401, a small molecule inhibitor of tryptophan hydroxylase-1 (Tph-1), the initial enzyme in GDS biosynthesis. Oral administration of this small molecule once daily for up to six weeks acts prophylactically or therapeutically, in a dose-dependent manner, to treat osteoporosis in ovariectomized rodents because of an isolated increase in bone formation. These results provide a proof of principle that inhibiting GDS biosynthesis could become a new anabolic treatment for osteoporosis.
Lumen expansion driven by hydrostatic pressure occurs during many morphogenetic processes. Although it is well established that members of the Claudin family of transmembrane tight junction proteins determine paracellular tightness within epithelial/endothelial barrier systems, functional evidence for their role in the morphogenesis of lumenized organs has been scarce. Here, we identify Claudin5a as a core component of an early cerebral-ventricular barrier system that is required for ventricular lumen expansion in the zebrafish embryonic brain before the establishment of the embryonic blood-brain barrier. Loss of Claudin5a or expression of a tight junction-opening Claudin5a mutant reduces brain ventricular volume expansion without disrupting the polarized organization of the neuroepithelium. Perfusion experiments with the electron-dense small molecule lanthanum nitrate reveal that paracellular tightness of the cerebral-ventricular barrier decreases upon loss of Claudin5a. Genetic analyses show that the apical neuroepithelial localization of Claudin5a depends on epithelial cell polarity and provide evidence for concerted activities between Claudin5a and Na(+),K(+)-ATPase during luminal expansion of brain ventricles. These data establish an essential role of a barrier-forming Claudin in ventricular lumen expansion, thereby contributing to brain morphogenesis.
Chronic myeloid leukemia (CML) in Asia has an incidence rather lower than in Western countries yet tends to afflict a younger population. As in the West, imatinib mesylate (IM, Glivec) has supplanted busulphan, hydroxyurea and interferon-alpha as first-line treatment. Its use has resulted in a dramatic decline in the number of hematopoietic stem cell transplantations (HSCT) performed. Although it is expensive, IM induces a complete cytogenetic response in 60-90% of newly diagnosed patients, and up to 10% for those in blastic phase. The standard dose of 400 mg is well tolerated by most patients, although adverse events have been observed, including drug-induced cytopenia. Through the Glivec International Patient Assistance Program, the majority of CML patients has access to IM and can expect prolonged survival, even in the absence of HSCT. However, just as in Western countries, resistance to imatinib has emerged in Asian countries. They will require the novel tyrosine kinase inhibitors (dasatinib, nilotinib) becoming available through either clinical trials or market approval. This review examines the available data on CML in China, Hong Kong, India, the Philippines, Singapore, South Korea, Taiwan and Thailand.
In summary, recent studies over the last ten years have concentrated on what the blood-brain barrier does rather than what it is. This focus has changed the concept of this important membrane from a passive, relatively immutable structure to a dynamic interface between blood and brain. Further understanding of the molecular cell physiology of the brain capillary endothelium will undoubtedly lead to new insights into both drug action at the BBB and drug delivery through this barrier.
The zebrafish gastrointestinal system matures in a manner akin to higher vertebrates. We describe nine mutations that perturb development of these organs. Normally, by the fourth day postfertilization the digestive organs are formed, the epithelial cells of the intestine are polarized and express digestive enzymes, the hepatocytes secrete bile, and the pancreatic islets and acini generate immunoreactive insulin and carboxypeptidase A, respectively. Seven mutations cause arrest of intestinal epithelial development after formation of the tube but before cell polarization is completed. These perturb different regions of the intestine. Six preferentially affect foregut, and one the hindgut. In one of the foregut mutations the esophagus does not form. Two mutations cause hepatic degeneration. The pancreas is affected in four mutants, all of which also perturb anterior intestine. The pancreatic exocrine cells are selectively affected in these four mutations. Exocrine precursor cells appear, as identified by GATA-5 expression, but do not differentiate and acini do not form. The pancreatic islets are spared, and endocrine cells mature and synthesize insulin. These gastrointestinal mutations may be informative with regard to patterning and crucial lineage decisions during organogenesis, and may be relevant to diabetes, congenital dysmorphogenesis and disorders of cell proliferation.
Statins have been reported to lower the risk of developing Alzheimer's disease; however, the mechanism of this potentially important neuroprotective action is not understood. Lowering cholesterol levels does not appear to be the primary mechanism. Statins have pleiotropic effects in addition to lowering cholesterol, and statins may act on several different pathways involving distinct gene expression patterns that would be difficult to determine by focusing on a few genes or their products in a single study. In addition, gene expression patterns may be specific to a particular statin. To understand the molecular targets of statins in brain, DNA microarrays were used to identify gene expression patterns in the cerebral cortex of mice chronically treated with lovastatin, pravastatin, and simvastatin. Furthermore, brain statin levels were determined using liquid chromatography/tandem mass spectrometry. These studies revealed 15 genes involved in cell growth and signaling and trafficking that were similarly changed by all three statins. Overall, simvastatin had the greatest influence on expression as demonstrated by its ability to modify the expression of 23 genes in addition to those changed by all three drugs. Of particular interest was the expression of genes associated with apoptotic pathways that were altered by simvastatin. Reverse transcription-polymerase chain reaction experiments confirmed the microarray findings. All three drugs were detected in the cerebral cortex, and acute experiments revealed that statins are relatively rapidly removed from the brain. These results provide new insight into possible mechanisms for the potential efficacy of statins in reducing the risk of Alzheimer's disease and lay the foundation for future studies.
The blood-brain barrier (BBB) remains one of the greatest challenges for the discovery and development of treatments for CNS disorders, which to this day remains one of the riskiest disease areas in terms of clinical success rates. Although the BBB is currently seen predominantly as a permeability obstacle for CNS drug delivery, it is becoming increasingly clear that the BBB has many more implications for the pharmaceutical industry impacting on CNS pharmacology and pathology, CNS pharmacokinetics and pharmacodynamics, and adverse CNS effects, to name but a few areas. The present review does not intend to summarize the activities in the field of BBB research per se, which has been accomplished by a number of excellent recent reviews, but instead to provide an overview of the role of BBB studies from a pharmaceutical industry perspective. This review will elaborate on the specific needs in terms of BBB-related issues across the different drug discovery and development phases, i.e. target identification and validation, lead generation and optimization, candidate selection and profiling, preclinical development and clinical studies. The specific approaches taken will be discussed in terms of specific requirements, questions to be asked, feasibility, interpretability, and impact. It becomes clear that few of the existing BBB models fully meet the requirements of the industrialized drug discovery process, highlighting the need for an array of new or modified tools and approaches that are more effective in helping make decisions which are more specifically tailored to the various stages of the lengthy process from target to the clinic. In looking at the numerous ongoing activities in the area of BBB research from the drug discovery and development point of view, an attempt has been made to place a stronger emphasis on the applicability of particular techniques and approaches, to identify gaps and areas for future activities. In order to materialize the considerable knowledge gained in recent years, the review is intended to foster an increased awareness of the need to better integrate basic academic research with the specific requirements of the pharmaceutical industry for the search of effective and safe new CNS medicines.
The tight junction (TJ) or zona occludens (ZO) proteins TJP1/ZO-1, TJP2/ZO-2 and TJP3/ZO-3 belong to the membrane associated guanylate kinase-like (MAGUK) protein family and link TJ integral membrane proteins to the actin cytoskeleton. TJPs also serve as scaffolds for signaling proteins and transcription factors that regulate vesicular traffic and cell proliferation and differentiation. Here, we report the identification of two tjp1/zo-1 (tjp1.1 and tjp1.2) and tjp2/zo-2 (tjp2.1 and tjp2.2) genes each and one tjp3/zo-3 gene, and characterize their tissue specific distribution and developmental expression in zebrafish embryos. Transcripts for all five TJPs are maternally supplied and localized expression due to embryonic transcription is observed following the midblastula transition stage of development. In addition to a widespread distribution, individual genes show tissue specific expression patterns and a dynamic regulation during the developmental stages from 2 cells to 4 dpf analyzed. The most noticeable differences in expression patterns are observed in the posterior part of the embryo during somitogenesis. While all TJPs are expressed in the pronephric ducts and epidermis by 18 hpf, tjp1.1 is highly expressed in the hypochord and blood island, tjp1.2 in the somites and the posterior part of the notochord, tjp2.1 in the somites and the ventral part of the spinal cord, and tjp2.2 in the somites only. Individual TJPs are also strongly expressed in different layers of the eye and, at later stages, in central nervous system (CNS) tissues. Interestingly, the differential tissue and developmental expression of the two tjp1 and tjp2 genes indicates that the duplicated genes have been adapted for distinct transcriptional regulations during evolution.
Two independent high-throughput zebrafish behavioral screens of tens of thousands of compounds identify the 'finazines', a novel group of antipsychotics, and their endogenous genetic target, the σ1 receptor.
Introduction:
The brain-blood ratio is an important model correlating the brain-targeting ability of neurotherapeutics with the CNS pharmacokinetics, which need to be presented before the scientific community for exploration of its scientific worth. The purpose of this article is to bring this key concept and its precise discussion to the attention of the researchers. Areas covered: Three major points are discussed herein: First, the significance of brain-blood ratio with respect to investigational neurotherapeutics, and carrier systems and correlation of its research findings with the brain targeting efficiency. Second, the various factors influencing the brain-blood ratio. Third, the various strategies for enhancing the brain-blood ratio. In addition, the benchmark criteria for CNS-likeness of drug molecules and the correlation of brain-blood ratio with brain targeting ability of neurotherapeutics have been tabulated. Expert opinion: The brain-blood ratio (also referred to as the brain-plasma ratio) represents one of the tools available today for estimation of CNS pharmacokinetics. It is preferred over other complicated techniques (in situ brain perfusion and microdialysis) due to its ease of use and practicality. We are optimistic that the brain-blood ratio offers an excellent way of evaluating brain-targeting efficiency of neurotherapeutics effectively. In our opinion, it is a very fundamental aspect of brain bioavailability and needs to be presented in a precise way.
The presence of carcinogenic compounds in the aquatic environment is a recognized problem. ABC transporters are well known players in the multidrug-resistance (MDR) phenomenon in mammals associated with resistance to chemotherapy, however little is known in fish species. Thus, the aim of this study was to induce hepatic tumours and evaluate long-term effects on P-glycoprotein (P-gp) and proliferating cell nuclear antigen (PCNA) proteins in Danio rerio liver, after exposure to diethylnitrosamine (DEN). Several hepatic histopathological alterations were observed in zebrafish after exposure to DEN including pre-neoplastic lesions 6 and 9 months post-exposure. After 3, 6 and 9 months of exposure to DEN, P-gp and PCNA proteins expression were up-regulated. In conclusion, this study has shown that zebrafish ABC transporters can play a similar role as in human disease, hence zebrafish can be used also as a biological model to investigate in more deep mechanisms involved in disease processes.
Preclinical ResearchCentral nervous system (CNS) disorders represent a major problem of health in developed countries, with important consequences in disability and health economics. Recent findings in CNS genomics and pharmacogenomics suggest that the introduction of pharmacogenomic procedures in clinical practice may help to optimize therapeutics (efficacy and safety issues). The genes involved in the pharmacogenomics of CNS drugs fall into five categories: (i) genes associated with CNS pathogenesis; (ii) genes associated with the mechanism of action of drugs; (iii) genes associated with drug metabolism; (iv) genes associated with drug transporters; and (v) pleiotropic genes involved in multifaceted cascades and metabolic reactions. Pharmacogenomics accounts for 30–90% variability in pharmacokinetics and pharmacodynamics. Only 20–30% of the Caucasian population processes normally approximately 60% of the current drugs that are metabolized via CYP2D6, CYP2C9, and CYP2C19. Clinical pharmacogenomics may contribute to personalizing pharmacological treatment, predicting patient/drug‐dose selection, minimizing drug interactions, increasing drug efficacy, and reducing unnecessary costs.
Motor neuron diseases (MNDs) are an etiologically heterogeneous group of disorders of neurodegenerative origin, which result in degeneration of lower (LMNs) and/or upper motor neurons (UMNs). Neurodegenerative MNDs include pure hereditary spastic paraplegia (HSP), which involves specific degeneration of UMNs, leading to progressive spasticity of the lower limbs. In contrast, spinal muscular atrophy (SMA) involves the specific degeneration of LMNs, with symmetrical muscle weakness and atrophy. Amyotrophic lateral sclerosis (ALS), the most common adult-onset MND, is characterized by the degeneration of both UMNs and LMNs, leading to progressive muscle weakness, atrophy, and spasticity. A review of the comparative neuroanatomy of the human and zebrafish motor systems showed that, while the zebrafish was a homologous model for LMN disorders, such as SMA, it was only partially relevant in the case of UMN disorders, due to the absence of corticospinal and rubrospinal tracts in its central nervous system. Even considering the limitation of this model to fully reproduce the human UMN disorders, zebrafish offer an excellent alternative vertebrate model for the molecular and genetic dissection of MND mechanisms. Its advantages include the conservation of genome and physiological processes and applicable in vivo tools, including easy imaging, loss or gain of function methods, behavioral tests to examine changes in motor activity, and the ease of simultaneous chemical/drug testing on large numbers of animals. This facilitates the assessment of the environmental origin of MNDs, alone or in combination with genetic traits and putative modifier genes. Positive hits obtained by phenotype-based small-molecule screening using zebrafish may potentially be effective drugs for treatment of human MNDs.
We describe a method for obtaining pharmacokinetics (PK) and pharmacology data from adult zebrafish in terms of mg/kg using a novel method of oral administration. Using carbamazepine (CBZ) as a test drug, we employed dried blood spot (DBS) cards to enable drug quantification for PK; and we evaluated the pharmacological anxiolytic effect using novel tank test.
The PK study confirmed the presence of CBZ in both blood and brain and the behavioural study showed dose dependent anxiolytic effect. The reproducibility of oral dosing was confirmed by the fact that the results obtained in both the experiments had negligible errors.
This report enables a novel approach for optimizing the utility of zebrafish in drug discovery and drug delivery research
The blood-brain barrier (BBB) represents a functional interface between the blood stream and the neuronal microenvironment. Distinct cellular and molecular features of brain microvessel endothelial cells result in barrier and carrier functions that guarantee exclusion of adverse components such as neurotoxic metabolites on the one hand and selective passage of essential nutrients on the other hand. Circumventricular organs are brain structures that lack BBB characteristics, allowing for hormone-mediated interactions between e.g. the pituitary and distant organs. The retina, as an integral part of the central nervous system, is enclosed by the pigment epithelium, which functions as a barrier interface between the systemic blood vessels of the neighboring choroid and the retina. Various BBB-specific markers, tight junction components, and carrier systems including amino acid and saccharide transporters have been cloned. P-glycoprotein has been of special interest because this efflux pump counteracts entry of numerous therapeutically relevant drugs into the nervous system. Various in vitro systems of the retinal pigment epithelium (RPE) have been established and employed to analyze pharmacological aspects and pathological cell interactions. The most advanced systems are organotypic cultures and acute preparations of the RPE, i.e. fully intact tissue sheets that can be used as in vivo-like BBB models for transport studies and drug profiling.
Introduction:
The blood-brain barrier (BBB) is a selectively permeable micro-vascular unit which prevents many central nervous system (CNS)-targeted compounds from reaching the brain. A significant problem in CNS drug development is the ability to model BBB permeability in a timely, reproducible and cost-effective manner. Through the years, several models have been used such as artificial membranes, cell culture and animal models.
Areas covered:
In this focused review, the authors cover novel models which have been developed or are in the process of being developed which can be used in modeling BBB. These models can either be used to determine BBB permeability or whether a compound may be disrupting the BBB. Many of these models lend themselves to high-throughput screening. The main model organisms covered here are the grasshopper (Locusta migratoria), fruit fly (Drosophila melanogaster) and zebrafish (Danio rerio).
Expert opinion:
Many of the models covered here have only recently been utilized for BBB studies and still needs to be fully studied for its impact on reducing costs during drug development. The strength of these models lay in the fact that a whole organism experiment can be done in high throughput fashion as compared with classical vertebrate models such as micro-dialysis.
The objective of the present study was to examine the cassette dosing method in determination of brain-to-plasma concentration ratio (area under the concentration-time profiles for plasma/area under the concentration-time profiles for brain, K(p)). Eleven model compounds, amprenavir, citalopram, digoxin, elacridar, imatinib, (3S,6S,12aS)-1,2,3,4,6,7,12,12a-octahydro-9-methoxy-6-(2-methylpropyl)-1,4-dioxopyrazino[1',2':1,6]pyrido[3,4-b]indole-3-propanoic acid 1,1-dimethylethyl ester (Ko143), loperamide, prazosin, quinidine, sulfasalazine, and verapamil, were selected to compare their K(p) determined from discrete dosing in wild-type mice and their K(p) from cassette dosing in wild-type, Mdr1a/1b(-/-), Bcrp1(-/-), and Mdr1a/1b(-/-)/Bcrp1(-/-) mice at 1 to 3 mg/kg. The mice brain and plasma were collected at 0.25, 1, and 3 h and were analyzed using high-performance liquid chromatography-tandem mass spectrometry methods. The K(p) determined from discrete dosing versus cassette dosing in the wild-type mice were within 2-fold for all the compounds except sulfasalazine and Ko143. The brain concentrations of sulfasalazine and Ko143 and the plasma concentrations of Ko143 were below the lower limit of quantitation. In addition, the K(p) values estimated by mass spectrometry responses, namely the ratio of compound peak area to internal standard peak area, were within 2-fold of the K(p) observed from the actual concentrations. Furthermore, the ratios of K(p) in Mdr1a/1b(-/-), Bcrp1(-/-), and Mdr1a/1b(-/-)/Bcrp1(-/-) mice versus the K(p) in the wild-type mice from cassette dosing were consistent with the ones reported in the literature where the compounds were dosed discretely. These results demonstrate that drug-drug interactions at the blood-brain barrier are unlikely at a subcutaneous dose of 1 to 3 mg/kg and support the use of the cassette dosing approach to assess brain penetration in drug discovery.
There remains an urgent need for therapeutic agents that provide improved symptomatic treatment and attenuate disease progression in patients with Alzheimer's disease (AD). 5-HT(4) receptors are widely expressed in those CNS areas which receive substantial cholinergic input and are involved in cognition. The ability of 5-HT(4) receptor agonists to increase acetylcholine (ACh) release and reduce cognitive impairment in both animals and humans has been demonstrated. In addition, 5-HT(4) receptor agonist modulation of levels of the amyloid precursor protein (APP) derived peptides, soluble amyloid precursor protein (sAPPα) and amyloid beta protein (Aβ) in the CNS has been reported. In this study, the preclinical properties of three structurally-distinct 5-HT(4) receptor selective agonists, PRX-03140, velusetrag and TD-8954, were studied to assess their potential for symptomatic and disease-modifying benefit in the treatment of AD. All three compounds exhibited high affinity for the rat 5-HT(4) receptor but could be discriminated on the basis of their agonist activity. In cAMP accumulation and sAPPα secretion assays using recombinant HEK293f-5-HT(4(d))-APP(695) cells, velusetrag and TD-8954 were potent, full agonists, relative to 5-HT, whereas PRX-03140 was a partial agonist (intrinsic activity 18%, relative to 5-HT). In a guinea pig colon isolated tissue preparation, TD-8954 exhibited lower intrinsic activity than velusetrag, and PRX-03140 had negligible agonist activity. In the rat Morris water maze (MWM) cognition test, velusetrag and TD-8954 (0.1 mg/kg), but not PRX-03140 (0.03-1 mg/kg), significantly reversed the scopolamine-induced spatial learning deficit via activation of 5-HT(4) receptors. Coadministration of subefficacious doses of the acetylcholinesterase inhibitor (AChEi), donepezil (0.1 mg/kg, i.p.), and either velusetrag or TD-8954 (0.01 mg/kg i.p.) resulted in reversal of the scopolamine-induced cognitive deficit. Pharmacokinetic data indicated that the CNS penetration for all three 5-HT(4) receptor agonists was relatively low. However, the pharmacodynamic-pharmacokinetic relationships in the MWM model for velusetrag and TD-8954 were consistent with their respective receptor pharmacology (binding affinity and intrinsic efficacy) and CNS penetration properties. Collectively, these findings support a potential role for potent and efficacious 5-HT(4) receptor agonists in the treatment of AD.
The zebrafish, Danio rerio, has been established as an excellent vertebrate model for the study of developmental biology and gene function. It also has proven to be a valuable model to study human diseases. Here, we reviewed recent publications using zebrafish to study the pathology of human neurodegenerative diseases including Parkinson's, Huntington's, and Alzheimer's. These studies indicate that zebrafish genes and their human homologues have conserved functions with respect to the etiology of neurodegenerative diseases. The characteristics of the zebrafish and the experimental approaches to which it is amenable make this species a useful complement to other animal models for the study of pathologic mechanisms of neurodegenerative diseases and for the screening of compounds with therapeutic potential.
Statins are widely being used for the treatment of a variety of conditions beyond their original indication for lowering cholesterol. We have previously reported that simvastatin affected the dopaminergic system in the rat brain. This study aims to investigate locomotor and anxiety effects along with the regional changes of N-methyl-d-aspartate (NMDA) receptors in the rat brain after 4-week administration of simvastatin. Hyperlocomotive and anxiolytic-like activities in the rat were observed after chronic administration of high dose simvastatin (10 mg/kg/day). Distributions and alterations of NMDA receptors in the post-mortem rat brain were detected by [(3)H] MK-801 binding autoradiography. Simvastatin increased [(3)H] MK-801 binding, predominantly in the prefrontal cortex (20%, p=0.003), primary motor cortex (20%, p<0.001), cingulate cortex (28%, p<0.001), hippocampus (41%, p<0.001), caudate putamen (30%, p=0.029), nucleus accumbens (27%, p=0.035) and amygdala (45%, p<0.001) compared to controls. Significant positive correlations were identified between hyperlocomotive as well as anxiolytic-like activities and the upregulation of NMDA receptors in different brain regions. Our results also provide strong evidence that chronic high dose simvastatin administration is to exhibit NMDA antagonist-like effects, which would partially explain the anxiolytic and hyperlocomotor activities. These findings contribute to a better understanding of the critical roles of simvastatin in modulating psycho-neurodegenerative disorders, via NMDA receptors.
The biosynthesis of cholesterol is mainly regulated by 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase. Because the liver is the major site of cholesterol synthesis, it is the primary target of the class of drugs known as HMG-CoA reductase inhibitors. Simvastatin (SV) is a lactone prodrug which undergoes reversible metabolism. In the hydroxy acid form (SVA) it is a potent inhibitor of HMG-CoA reductase. SV is well absorbed by rats, dogs, and humans. After an oral dose of SV, tissue distribution studies were consistent with high hepatic extraction of SV and relatively poor tissue penetration of SVA. The majority of a radioactive dose of SV is eliminated in bile. A high portal/systemic gradient for 6'-OH-SVA, an active biliary metabolite, suggests its probable reentry and indicates potential for prolongation of HMG-CoA reductase inhibition. AUC comparisons in dogs after simultaneous iv (3H) and intraportal (14C) infusions indicate that hepatic extraction is high with only 8% of SV reaching the systemic circulation unchanged. Approximately 98% and 96% of SV was bound to human and dog plasma protein, respectively. The physiological disposition of SV in dog appears to be a suitable paradigm for man. Because of its high hepatic extraction SV should be both specific and selective with respect to the inhibition of HMG-CoA reductase.
A new H1 antihistamine, cetirizine, was studied to determine its effects on mediators and cellular infiltration during the cutaneous late-phase response (LPR). Ten ragweed-allergic subjects, who had previously demonstrated a cutaneous LPR, were examined in a double-blind, crossover study. Either cetirizine, 20 mg, or placebo was administered orally once daily for 2 days before and the morning of placement of a skin chamber overlying an unroofed heat/suction-induced blister to which was added antigen or buffer. Skin test erythema was significantly reduced by cetirizine at 15 minutes, 2 hours, and 4 hours by 56%, 40%, and 39%, respectively (all, p less than or equal to 0.01), but by 6 and at 8 hours, the cutaneous erythema was not significantly lessened. Histamine release was not altered by cetirizine treatment, but prostaglandin D2 (PGD2) production, which peaked at 3 to 5 hours, was clearly reduced by cetirizine treatment, being lower at all time points during the reaction; this was significant by analysis of variance (p less than or equal to 0.04). The inhibition was most marked during the fifth hour of the reaction when there was a 50% suppression of the PGD2 level by cetirizine (0.193 ng/ml to 0.075 ng/ml [p less than or equal to 0.03]). The most dramatic effect of cetirizine was attenuation of the inflammatory cell migration into the chamber. Eosinophil infiltration was decreased by about 75% during hours 6, 7, and 8 (p less than or equal to 0.04), whereas the number of neutrophils was reduced by the same magnitude at the same times (p less than or equal to 0.04).(ABSTRACT TRUNCATED AT 250 WORDS)
The antidiarrheal opiates loperamide, fluperamide, diphenoxylate and fetoxylate inhibited binding of [3H]nitrendipine to membranes from guinea-pig cerebral cortex with Ki values of 0.5 to 10 microM. Loperamide and fluperamide reversed the tiapamil elicited lowering of [3H]nitrendipine binding with IC50 values of 0.2 to 0.5 microM, indicating a verapamil-like action of these drugs. An oral dose of 1 mg/kg of loperamide reduced gastrointestinal motility and gave concentrations of 0.45 +/- 0.19, 0.38 +/- 0.22 and 0.49 +/- 0.25 microM in the duodenum, jejunum and ileum, respectively. The apparent Ki for loperamide in preventing calcium-induced contractions of guinea-pig ileum depolarized with 80 mM potassium was 0.10 microM. We propose that calcium channel antagonism is responsible at least in part for the antidiarrheal actions of loperamide and related agents. Evidence includes the calcium antagonist actions of loperamide at antidiarrheal doses, the constipating effects of certain calcium antagonists and the failure of opiate antagonists to prevent some intestinal effects of loperamide.
The presence of the blood-brain barrier (BBB), reduces the brain uptake of many drugs, peptides and other solutes from blood. Strategies for increasing the uptake of drugs and peptide-based drugs include; structural modifications to increase plasma half-life; improving passive penetration of the BBB by increasing the lipophilicity of the molecule; designing drugs which react with transporters present in the BBB; and reducing turnover and efflux from the central nervous system (CNS).
Donepezil hydrochloride (Aricept) is a drug for the treatment of Alzheimer's disease. The absorption, distribution, metabolism, and excretion of donepezil were investigated in male Sprague-Dawley rats after a single oral administration. Orally administered (14)C-labeled donepezil was absorbed rapidly. The plasma level of unchanged donepezil declined more rapidly than that of radioactivity, and the brain level of radioactivity declined almost in parallel with the plasma level of unchanged donepezil. The ratio of donepezil to total radioactivity in brain was 86.9 to 93.0%, indicating low permeability of the metabolites through the blood-brain barrier. No heterogeneous localization of radioactivity was recognized in the brain and the concentration in each part of the brain was 1.74 to 2.24 times the plasma concentration. Cumulative biliary, urinary, and fecal excretion of radioactivity in bile duct-cannulated rats was 72.9, 24.4, and 8.84%, respectively, of the administered radioactivity at 48 h after administration. These results indicate that the absorption of donepezil is almost complete, and that its metabolites are mainly excreted into feces through the bile and some of them are subject to enterohepatic circulation. The metabolism of donepezil was extensive in rats and involved O-demethylation, aromatic hydroxylation, N-dealkylation, N-oxidation, and glucuronide conjugation of O-demethylate. The structures of the metabolites were determined by mass spectrometry and (1)H-NMR analysis. In plasma, urine, and bile, O-glucuronides accounted for the majority of the radioactivity, and in brain, unchanged donepezil was mostly detected. No metabolites were found in brain. There was no notable accumulation of radioactivity in whole blood and tissues.
Although the antidiarrheal loperamide is a potent opiate, it does not produce opioid central nervous system effects at usual doses in patients. On the basis of in vitro studies demonstrating that loperamide is a substrate for the adenosine triphosphate-dependent efflux membrane transporter P-glycoprotein, we postulated that inhibition of P-glycoprotein with quinidine would increase entry of loperamide into the central nervous system with resultant respiratory depression.
To test this hypothesis, a 16-mg dose of loperamide was administered to eight healthy male volunteers in the presence of either 600 mg quinidine, a known inhibitor of P-glycoprotein, or placebo. Central nervous system effects were measured by evaluation of the respiratory response to carbon dioxide rebreathing as a measure of opiate-induced respiratory depression.
Loperamide produced no respiratory depression when administered alone, but respiratory depression occurred when loperamide (16 mg) was given with quinidine at a dose of 600 mg (P < .001). These changes were not explained by increased plasma loperamide concentrations.
This study therefore demonstrates first the potential for important drug interactions to occur by a new mechanism, namely, inhibition of P-glycoprotein, and second that the lack of respiratory depression produced by loperamide, which allows it to be safely used therapeutically, can be reversed by a drug causing P-glycoprotein inhibition, resulting in serious toxic and abuse potential.
Despite considerable advances in the pharmacotherapy of epilepsy, about 30% of epileptic patients are refractory to antiepileptic drugs (AEDs). In most cases, a patient who is resistant to one major AED is also refractory to other AEDs, although these drugs act by different mechanisms. The mechanisms that lead to drug resistance in epilepsy are not known. Recently, over-expression of multidrug transporters, such as P-glycoprotein (PGP) and multidrug resistance-associated protein (MRP), has been reported in surgically resected epileptogenic human brain tissue and suggested to contribute to the drug resistance of epilepsy. However, it is not known to what extent multidrug transporters such as PGP or MRP are involved in transport of AEDs. In the present study, we used in vivo microdialysis in rats to study whether the concentration of carbamazepine in the extracellular fluid of the cerebral cortex can be enhanced by inhibition of PGP or MRP, using the PGP inhibitor verapamil and the MRP inhibitor probenecid. Local perfusion with verapamil or probenecid via the microdialysis probe increased the extracellular concentration of carbamazepine. The data indicate that both PGP and MRP participate in the regulation of extracellular brain concentrations of the major AED carbamazepine.
Relative plasma, brain and cerebrospinal fluid (CSF) exposures and unbound fractions in plasma and brain were examined for 18 proprietary compounds in rats. The relationship between in vivo brain-to-plasma ratio and in vitro plasma-to-brain unbound fraction (fu) was examined. In addition, plasma fu and brain fu were examined for their relationship to in vivo CSF-to-plasma and CSF-to-brain ratios, respectively. Findings were delineated based on the presence or absence of active efflux. Finally, the same comparisons were examined in FVB vs. MDR 1a/1b knockout mice for a selected P-glycoprotein (Pgp) substrate. For the nine compounds without indications of active efflux, predictive correlations were observed between ratios of brain-to-plasma exposure and plasma-to-brain fu (r(2) = 0.98), CSF-to-brain exposure vs. brain fu (r(2) = 0.72), and CSF-to-plasma exposure vs. plasma fu (r(2) = 0.82). For the nine compounds with indications of active efflux, nonspecific binding data tended to over predict the brain-to-plasma and CSF-to-plasma exposure ratios. Interestingly, CSF-to-brain exposure ratio was consistently under predicted by brain fu for this set. Using a select Pgp substrate, it was demonstrated that the brain-to-plasma exposure ratio was identical to that predicted by plasma-to-brain fu ratio in MDR 1a/1b knockout mice. In FVB mice, plasma-to-brain fu over predicted brain-to-plasma exposure ratio to the same degree as the difference in brain-to-plasma exposure ratio between MDR 1a/1b and FVB mice. Consistent results were obtained in rats, suggesting a similar kinetic behavior between species. These data illustrate how an understanding of relative tissue binding (plasma, brain) can allow for a quantitative examination of active processes that determine CNS exposure. The general applicability of this approach offers advantages over species- and mechanism-specific approaches.
Donepezil and rivastigmine are acetylcholinesterase (AChE) inhibitors used to improve cholinergic neurotransmission and cognitive function in Alzheimer's disease (AD). This study examined direct effects of these drugs on AChE activity in the frontal, temporal, and parietal cortices in AD. Six AD patients were scanned with positron emission tomography before and after 3 months of treatment with donepezil (10 mg/day), and five AD patients were scanned before and after 3 to 5 months of treatment with rivastigmine (9 mg/day). Healthy unmedicated controls were imaged twice to evaluate the reproducibility of the method. A specific AChE tracer, [methyl-11C]N-methyl-piperidyl-4-acetate, and a 3D positron emission tomography system with MRI coregistration were used for imaging. Treatment with donepezil reduced the AChE activity (k3 values) in the AD brain by 39% in the frontal (p < 0.001, Bonferroni corrected), 29% in the temporal (p = 0.02, corrected) and 28% in the parietal cortex (p = 0.05, corrected). The corresponding levels of inhibition for rivastigmine were 37% (p = 0.003, corrected), 28% (p = 0.03, uncorrected) and 28% (p = 0.05, corrected). When the treatment groups were combined, the level of AChE inhibition was significantly greater in the frontal cortex compared to the temporal cortex (p = 0.03, corrected). The test-retest analysis with healthy subjects indicated good reproducibility for the method, with a nonsignificant 0% to 7% intrasubject variability between scans. The present study provides first evidence for the effect of rivastigmine on cortical AChE activity. Our results indicate that the pooled effects of donepezil and rivastigmine on brain AChE are greater in the frontal cortex compared to the temporal cortex in AD. This regional difference is probably related to the prominent temporoparietal reduction of AChE in AD. We hypothesize that the clinical improvement in behavioral and attentional symptoms of AD due to AChE inhibitors is associated with the frontal AChE inhibition.
The present study evaluates the impact of P-glycoprotein (P-gp) on plasma-brain disposition and transepithelial transport of sedating versus nonsedating H1-antagonists using multidrug-resistant (mdr) gene 1a and 1b (mdr1a/b) knockout (KO) mice and human MDR1-transfected Madin-Darby canine kidney (MDCK) cells. Three nonsedating (cetirizine, loratadine, and desloratadine) and three sedating (diphenhydramine, hydroxyzine, and triprolidine) H1-antagonists were tested. Each compound was administered to KO and wild-type (WT) mice intravenously at 5 mg/kg. Plasma and brain drug concentrations were determined by liquid chromatography-mass spectrometry analysis. Mean pharmacokinetic parameters (CL, V(ss), and t(1/2)) were obtained using WinNonlin. In addition, certirizine, desloratadine, diphenhydramine, and triprolidine (2 microM) were tested as substrates for MDR1 using MDR1-MDCK cells. The bidirectional apparent permeability was determined by measuring the amount of compound at the receiving side at 5 h. The brain-to-plasma area under the curve (AUC) ratio was 4-, 2-, and >14-fold higher in KO compared with WT mice for cetirizine, loratadine, and desloratadine, respectively. In contrast, the brain-to-plasma AUC ratio between KO and WT was comparable for hydroxyzine, diphenhydramine, and triprolidine. Likewise, the efflux ratio between basolateral to apical and apical to basolateral was 4.6- and 6.6-fold higher in MDR1-MDCK than the parental MDCK for certirizine and desloratadine, respectively, whereas it was approximately 1 for diphenhydramine and triprolidine. Our results demonstrate that sedating H1-antagonists hydroxyzine, diphenhydramine, and triprolidine are not P-gp substrates. In contrast, nonsedating H1-antagonists cetirizine, loratadine, and desloratadine are P-gp substrates. Affinity for P-gp at BBB may explain the lack of central nervous system side effects of modern H1-antagonists.
Although the development of the digestive system of humans and vertebrate model organisms has been well characterized, relatively little is known about how the zebrafish digestive system forms. We define developmental milestones during organogenesis of the zebrafish digestive tract, liver, and pancreas and identify important differences in the way the digestive endoderm of zebrafish and amniotes is organized. Such differences account for the finding that the zebrafish digestive system is assembled from individual organ anlagen, whereas the digestive anlagen of amniotes arise from a primitive gut tube. Despite differences of organ morphogenesis, conserved molecular programs regulate pharynx, esophagus, liver, and pancreas development in teleosts and mammals. Specifically, we show that zebrafish faust/gata-5 is a functional ortholog of gata-4, a gene that is essential for the formation of the mammalian and avian foregut. Further, extraembryonic gata activity is required for this function in zebrafish as has been shown in other vertebrates. We also show that a loss-of-function mutation that perturbs sonic hedgehog causes defects in the development of the esophagus that parallel those associated with targeted disruption of this gene in mammals. Perturbation of sonic hedgehog also affects zebrafish liver and pancreas development, and these effects occur in a reciprocal fashion, as has been described during mammalian liver and ventral pancreas development. Together, these data define aspects of digestive system development necessary for the characterization of zebrafish mutants. Given the similarities of teleost and mammalian digestive physiology and anatomy, these findings have implications for developmental and evolutionary studies as well as research of human diseases, such as diabetes, liver cirrhosis, and cancer.
Unbound fractions in mouse brain and plasma were determined for 31 structurally diverse central nervous system (CNS) drugs and two active metabolites. Three comparisons were made between in vitro binding and in vivo exposure data, namely: 1) mouse brain-to-plasma exposure versus unbound plasma-to-unbound brain fraction ratio (fu(plasma)/fu(brain)), 2) cerebrospinal fluid-to-brain exposure versus unbound brain fraction (fu(brain)), and 3) cerebrospinal fluid-to-plasma exposure versus unbound plasma fraction (fu(plasma)). Unbound fraction data were within 3-fold of in vivo exposure ratios for the majority of the drugs examined (i.e., 22 of 33), indicating a predominately free equilibrium across the blood-brain and blood-CSF barriers. Some degree of distributional impairment at either the blood-CSF or the blood-brain barrier was indicated for 8 of the 11 remaining drugs (i.e., carbamazepine, midazolam, phenytoin, sulpiride, thiopental, risperidone, 9-hydroxyrisperidone, and zolpidem). In several cases, the indicated distributional impairment is consistent with other independent literature reports for these drugs. Through the use of this approach, it appears that most CNS-active agents freely equilibrate across the blood-brain and blood-CSF barriers such that unbound drug concentrations in brain approximate those in the plasma. However, these results also support the intuitive concept that distributional impairment does not necessarily preclude CNS activity.
This work examines the inter-relationship between the unbound drug fractions in blood and brain homogenate, passive membrane permeability, P-glycoprotein (Pgp) efflux ratio, and log octanol/water partition coefficients (cLogP) in determining the extent of central nervous system (CNS) penetration observed in vivo. The present results demonstrate that compounds often considered to be Pgp substrates in rodents (efflux ratio greater than 5 in multidrug resistant Madin-Darby canine kidney cells) with poor passive permeability may still exhibit reasonable CNS penetration in vivo; i.e., where the unbound fractions and nonspecific tissue binding act as a compensating force. In these instances, the efflux ratio and in vitro blood-brain partition ratio may be used to predict the in vivo blood-brain ratio. This relationship may be extended to account for the differences in CNS penetration observed in vivo between mdr1a/b wild type and knockout mice. In some instances, cross-species differences that might initially seem to be related to differing transporter expression can be rationalized from knowledge of unbound fractions alone. The results presented in this article suggest that the information exists to provide a coherent picture of the nature of CNS penetration in the drug discovery setting, allowing the focus to be shifted away from understanding CNS penetration toward the more important aspect of understanding CNS efficacy.
Vertebrate evolution has been largely driven by the duplication of genes that allow for the acquisition of new functions. The ATP-binding cassette (ABC) proteins constitute a large and functionally diverse family of membrane transporters. The members of this multigene family are found in all cellular organisms, most often engaged in the translocation of a wide variety of substrates across lipid membranes. Because of the diverse function of these genes, their large size, and the large number of orthologs, ABC genes represent an excellent tool to study gene family evolution. We have identified ABC proteins from the sea squirt (Ciona intestinalis), zebrafish (Danio rerio), and chicken (Gallus gallus) and, using phylogenetic analysis, identified those genes with a one-to-one orthologous relationship to human ABC proteins. All ABC protein subfamilies found in Ciona and zebrafish correspond to the human subfamilies, with the exception of a single ABCH subfamily gene found only in zebrafish. Multiple gene duplication and deletion events were identified in different lineages, indicating an ongoing process of gene evolution. As many ABC genes are involved in human genetic diseases, and important drug transport phenotypes, the understanding of ABC gene evolution is important to the development of animal models and functional studies.
The P-glycoprotein (P-gp)-deficient mouse model is used to assess the influence of P-gp-mediated efflux on the central nervous system (CNS) distribution of drugs. The steady-state unbound plasma/unbound brain concentration ratio ([plasma],(u)/[brain],(u)) is an alternative method for assessing CNS distribution of drugs independent of the mechanism(s) involved. The objective of this study was to compare the degree of CNS distributional impairment determined from the in vivo P-gp efflux ratio with that determined from the [plasma],(u)/[brain],(u) ratio. CNS distribution of 34 drugs, including opioids, triptans, protease inhibitors, antihistamines, and other clinically relevant drugs with either poor CNS distribution or blood-brain barrier efflux, was studied. Plasma and brain unbound fractions were determined by equilibrium dialysis. K(p,brain) and the P-gp efflux ratio were obtained from the literature or determined experimentally. The P-gp efflux ratio and the [plasma],(u)/[brain],(u) ratio were in concurrence (<3-fold difference) for 21 of the 34 drugs. However, the [plasma],(u)/[brain],(u) ratio exceeded the P-gp efflux ratio substantially (>4-fold) for 10 of the 34 drugs, suggesting that other, non-P-gp-mediated mechanism(s) may limit the CNS distribution of these drugs. The P-gp efflux ratio exceeded the [plasma],(u)/[brain],(u) ratio by more than 3-fold for three drugs, suggesting the presence of active uptake mechanism(s). These observations indicate that when mechanisms other than P-gp affect CNS distribution (non-P-gp-mediated efflux, poor passive permeability, cerebrospinal fluid bulk flow, metabolism, or active uptake), the P-gp efflux ratio may underestimate or overestimate CNS distributional impairment. The [plasma],(u)/[brain],(u) ratio provides a simple mechanism-independent alternative for assessing the CNS distribution of drugs.