- Email: [email protected]
ADMETox in zebrafish
Drug Discovery Today: Disease Models
DRUG DISCOVERY
TODAY
DISEASE
MODELS
Vol. 10, No. 1 2013
Editors-in-Chief Jan Tornell – AstraZeneca, Sweden Andrew McCulloch – University of California, SanDiego, USA
Zebrafish as a platform for in vivo drug discovery
ADMETox in zebrafish H. Diekmann*, A. Hill Evotec (UK) Ltd., 114 Milton Park, Abingdon OX14 4SA, UK1
To enable the widespread use of zebrafish larvae in drug discovery, it is required to define drug concentration at the target site and to assess metabolites at a resolution necessary for in vivo pharmacology screening. These questions are now being investigated using mass spec-
Section editors: Calum A. MacRae – Cardiovascular Division, Brigham and Women’s Hospital, Boston, MA, USA. Randall T. Peterson – Cardiovascular Research Center, Massachusetts General Hospital, Charlestown, MA, USA.
troscopy and contribute to our understanding of how the outcome of zebrafish toxicity, safety and efficacy studies can translate to rodent and human data.
Introduction Driven by the need to reduce attrition rates in drug development, information on disposition and metabolism of candidate drugs has become a crucial part of all aspects of the drug discovery process [1]. Beyond the optimization of drug candidates for potency and efficacy, it is fundamentally important to detect potentially detrimental toxic side effects within therapeutic doses. Zebrafish larvae share physiological, morphological and histological similarities with mammals and have been recognized as valuable models for evaluating drug candidates for toxicity and safety liabilities [2–5]. Their small size permit screening in microtiter plates with only single milligrams of compound for a full dose–response [6,7]. The zebrafish larva therefore lends itself to being the first in vivo vertebrate model in drug discovery for studying safety pharmacology and toxicity and has the potential to bridge the gap between traditional cell assays and regulated mammalian screens [6]. A key hurdle for this model, however, is the ability to determine the effective compound concentration in the zebrafish and to correlate this dose with rodent and human data.
*Corresponding author: H. Diekmann ([email protected]) 1 www.evotec.com. 1740-6757/$ ß 2012 Elsevier Ltd. All rights reserved.
DOI: 10.1016/j.ddmod.2012.02.005
Bioanalysis on zebrafish larvae Most zebrafish assays rely on aqueous exposure and compound uptake predominantly by diffusion through the skin [3,5]. Zebrafish larvae are typically placed in multi-well plates and compounds are directly added to the supporting medium in a suitable vehicle like DMSO (Fig. 1). Timing and length of exposure is optimized for each specific assay. The zebrafish larvae are then examined for morphological or behavioural phenotypes (e.g. Fig. 2). Exterior structures (including the eye, jaw and fins) and internal organs (such as heart, liver and gut) can be assessed without the need for dissection as zebrafish larvae develop ex utero and are virtually transparent (Fig. 2). Because compound uptake is dictated by the physicochemical properties of each compound, the potency of a compound cannot be predicted from the aqueous concentration or the log P value (Table 1) and [5,8]. Bioanalysis therefore needs to be performed to correlative any toxic phenotype with the actual concentration of compound within the larvae (Fig. 1) and [5,9]. This can be achieved using radio-labelled compound and liquid scintillation counting (LSC) [10,11] or radio high performance liquid chromatography (rHPLC) [12], similarly to ADME studies in rodents [13]. To avoid the use of radio-active material, larvae can alternatively be extracted with acetonitrile and then analysed using LCMS–MS alongside a calibration curve for quantification purposes [12]. This procedure provides the average weight of compound in the larva at the respective aqueous concentration and allows the ranking of compounds according to their potency based upon their body burden (uptake of compound by the larvae). These uptake values e31
Drug Discovery Today: Disease Models | Zebrafish as a platform for in vivo drug discovery
Vol. 10, No. 1 2013
912.9 913.7
Relative Abundance
100 80 60
930.7
40 931.7 20
935.9
484.8
176.7 278.9
409.6
507.7
671.6
0 200
400
600
1109.5 1207.5
698.2 801.7 800
1000
1200
m/z
Drug Discovery Today: Disease Models
Figure 1. Typical zebrafish assay set-up. Zebrafish embryos or larvae are arrayed in multi-well plates and compound is added to the surrounding medium. After a suitable incubation period, zebrafish are analysed for morphological or behavioural phenotypes. Thereafter, the compound body burden is determined using LC–MS technology.
eye
brain
otolith
jaw
heart
liver
muscles
yolk
gut
body length and shape
notochord
pigmentation
blood vessels
liver toxicity
jaw and brain Drug Discovery Today: Disease Models
Figure 2. Examples for toxicity phenotypes in zebrafish larvae. Morphological features such as eye, jaw and fins and internal organs such as heart, liver and gut are depicted in a 4 day old, untreated control zebrafish larvae (top picture). The bottom pictures give examples of phenotypic abnormalities after compound treatment.
Table 1. Bioanalysis examples and compound ranking Compound
Toxic aqueous concentration (mM)
1
100
15.7
3.3
2
100
26.3
1.8
3
50
64.2
3.9
4
50
83.3
4.9
5
50
89.2
4.5
6
50
163.0
1.8
Absorption
7
50
178.8
2.8
Bioanalysis not only allows the comparative ranking of a defined set of compounds (Table 1), but is also necessary to
e32
www.drugdiscoverytoday.com
Body burden (ng/larva)
log P
can differ more than ten-fold even though the aqueous concentration that induced toxicity is similar (Table 1). For example, compound 1 in Table 1 is seemingly less toxic than compound 7, because morphological abnormalities are only observed at a higher aqueous concentration (100 mM for compound 1 versus 50 mM for compound 7). Bioanalysis, however, revealed a much lower body burden for compound 1 versus compound 7, therefore classifying it as more toxic. This hence demonstrates that compound ranking for toxicity or efficacy is unreliable if based purely on aqueous concentrations.
Vol. 10, No. 1 2013
Drug Discovery Today: Disease Models | Zebrafish as a platform for in vivo drug discovery
confirm and quantify compound uptake (body burden) in zebrafish larvae [8] and to identify false-negative results attributable to poor compound absorption [9,14–16]. For example, erythromycin and D-sotalol, which cause QT prolongation in mammals, did not change the heart rate in 3 dpf zebrafish larvae [14]. Bioanalysis, however, revealed very low body burdens of 0.53 and 0.02 ng/larvae, respectively, indicating that these compounds are not taken up into the fish sufficiently to elicit a toxic effect. Similarly, sodium valproate, which can cause acute liver failure in humans, did not induce liver abnormalities in zebrafish larvae [15,16]. The determination of the body burden again indicated insufficient absorption for this compound. Interestingly, exposure of zebrafish larvae to valproic acid did cause liver abnormalities as expected as it was better absorbed into zebrafish larvae [15,16]. Therefore, it can be advisable to test a given compound as different chemical forms to circumvent poor uptake issues. On the other hand, overt toxicity could occur when compounds are readily taken up and high exposure levels are reached; that is, non-toxic compounds exceeding a threshold limit value-ceiling (TLV-C) may result in abnormalities or lethality [9,15]. To distinguish between gross toxicity/morbidity and ‘true toxicity’ and therefore avoid false-positive classifications, a threshold concentration based on historical data (LOECs for non-toxic drugs in use by man) can be imposed to help estimate likely overdose levels for previously untested compounds. In one developmental toxicity study undertaken in 2008, a threshold concentration of 40 ng/larva was chosen by a consortium of pharmaceutical companies based on the LOECs of 18 undisclosed non-toxic compounds [17]. As such, compounds with an LOEC 40 ng/larva were not classified as embryotoxic, but instead the phenotypes observed were attributed to compound overdose and flagged for further investigation. For example, the non-teratogenic compound dextromethorphan only led to morphological abnormalities at a body burden of 123 ng/larva [17,18], whereas highly teratogenic compounds tended to cause adverse effects at a much lower body burden (typically between 1 and 20 ng/larva). Currently, this toxicity threshold is still only used as a guide since it might have to be adjusted according to the structural class of compounds tested, compound efficacy and the expected therapeutic dose. In addition, the TLV-C is likely to differ between assays types (e.g. cardiotoxicity, embryotoxicity, hepatotoxicity) as exposure length and zebrafish age might influence the uptake and therefore the toxicity of compounds. Our preliminary data show for example that the body burden for azetazolamide after a four day incubation period is almost twice as much at 7 dpf compared to 4 dpf (Evotec unpublished results).
Pharmacokinetics Bioanalysis cannot only be performed on larval homogenates, but also on the well solutions to potentially obtain
additional pharmacokinetic information. The concentration of compound left in solution at the end of the incubation period can be compared against a ‘blank standard’ solution. This standard does not contain zebrafish embryos/larvae and demonstrates how much compound may have degraded or stuck to the plastic (assay plate) during the course of the experiment (Evotec unpublished results). Since the fish volume (1–2 ml per larva) is negligible compared to the assay solution (0.5–1 ml), the compound concentration in the assay solution containing fish should be comparable to the blank standard in case of equimolar uptake. However, if the compound is readily absorbed and has significantly accumulated in the zebrafish larvae, the compound concentration of the assay solution will be reduced accordingly. Significantly reduced assay concentration compared to the blank standard indicates rapid compound metabolism if mass balance is not retained. For example, only 4% of the nominal start concentration of midazolam was detected in the solution at the end of an experiment, even though the amount of compound detected in the zebrafish larvae was only 3%. This suggested that most of the compound had been metabolized in the course of the experiment [12]. Owing to the excess of compound available in the dosing solution, compound absorption has been expected to reach a maximum steady state level in zebrafish embryos and larvae and the kinetics of compound uptake have so far been largely neglected for toxicity assessments. Typically, zebrafish embryos/larvae are exposed to compound continually throughout the assay and the body burden is determined at the end of the exposure period (Fig. 1) and [5,9,15]. However, the effective concentration to cause a certain phenotype might differ significantly from the body burden at the end of a longterm exposure experiment. In a zebrafish teratogenicity assay, for example, embryos are exposed to compound at 2–4 h post fertilization (hpf) and analysed for morphological abnormalities at 96–120 hpf [18–20]. If compounds are continuously accumulating over this 4–5 day period, the body burden at the end of the assay would overestimate the effective concentration, as teratogenic effects are expected to occur during embryogenesis at 2–48 hpf. On the other hand, the measured body burden could underestimate the effective concentration for compounds rapidly metabolized during the later stages of the experiment. For example, preliminary data showed a steady increase in body burden over a four day incubation period for compound 1 (Fig. 3). Compound 2, however, was taken up to a greater extend and reached a steady state level by the third day of exposure (Fig. 3). Although generalized conclusions cannot yet be drawn from this limited data set, it looks advisable to determine the body burden at earlier time points to avoid confounding results due to the aforementioned scenarios. Alternatively, bioanalysis samples could be taken at different times during an experiment, similarly to plasma concentration time course studies in rodents [1]. www.drugdiscoverytoday.com
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Drug Discovery Today: Disease Models | Zebrafish as a platform for in vivo drug discovery
and desloratidine showed lower compound concentrations in the head versus the trunk. This is in agreement with mammalian studies showing poor BBB penetration for these compounds [27]. Finally, one important factor to consider is the presence of prominent yolk during embryonic stages of development. Hydrophobic compounds may accumulate disproportionately in this lipid-rich environment compared to the rest of the body. As this could lead to an overestimation for the effective concentrations required to induce organ toxicity, it should be tested whether the removal of the yolk would alter bioanalysis results [28].
60 body burden (ng/larva)
compound 1 compound 2 40
20
0 1
2
3
4
dpe Drug Discovery Today: Disease Models
Figure 3. Compound uptake kinetics. Zebrafish larvae were immersed in compound and the body burden determined at 1, 2, 3 and 4 days post exposure (dpe). Compound 1 was taken up linearly during the exposure period while compound 2 reached a plateau level at 3 dpe.
Distribution The distribution of compounds within zebrafish has not yet been extensively studied. Compounds are usually added to the fish support medium in a suitable vehicle (e.g. 0.5–1% DMSO) and thought to be taken up into zebrafish embryos and larvae by passive diffusion through the skin and gills (aqueous exposure) [3,4]. Larvae could also ingest compound from the dosing solution, therefore making the gastrointestinal tract another key site for absorption. Alternatively, compounds can be injected into the yolk sac or the vasculature, from where they get distributed throughout the body [21]. Whether effective levels are reached within the target tissues by either method is dictated by the physicochemical properties of the compound and is generally only proven by a positive assay readout for the organ of interest [4,5]. Tissue-specific bioanalysis is rather infeasible due to the small size of the larvae, although distribution and accumulation in specific organs could potentially be visualized using radioactively labelled compounds and quantitative whole body autoradiography [22,23]. As zebrafish larvae develop a functional blood brain barrier (BBB) between 3 and 8 dpf [24,25], it is possible that compounds may not reach the central nervous system (CNS) in older zebrafish larvae, although anti-epileptic compounds have been successfully tested in 6 dpf zebrafish [26]. In an attempt to show the exclusion of drugs from the zebrafish brain and therefore to prove the existence of a functional BBB in zebrafish larvae, bioanalysis was performed separately on heads and trunks of larvae that were immersed in various drugs [27]. Diphenhydramine, haloperidol and scopolamine, which cross the BBB in mammals, were equally distributed between head and body in zebrafish larvae. By contrast, scopolamine N-butyl bromide e34
Vol. 10, No. 1 2013
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Excretion Compound secretion should be irrelevant for most zebrafish assays using continuous aqueous exposure since a stable equilibrium between compound absorption and secretion should be established within the experiment. Compound excretion could nevertheless be analysed using pulsed exposure and is particularly important for microinjection experiments. For instance, several compounds hepatotoxic to human such as diclofenac and amiodarone are known to not cause liver abnormalities upon microinjection into 3 dpf zebrafish larvae. In the case of diclofenac, bioanalysis revealed a body burden of 9.2 ng/larva at 1 h post injection (hpi), demonstrating successful compound delivery, but no diclofenac could be detected in larvae at 24 and 48 h postinjection (hpi) (Evotec unpublished results). At these time points, however, diclofenac was detectable in the well solutions, indicating fast and complete excretion or diffusion of the compound from the larvae. Amiodarone, on the other hand, was detected at 156 ng/larva (1 hpi), 132 ng/larva (24hpi) and 85 ng/larva (48 hpi) with no compound appearing in the well solution. Amiodarone therefore seemed to be slowly metabolized by the zebrafish larvae (Evotec unpublished results).
Metabolism In addition to mere detection of compound absorption, bioanalysis also allows the investigation of zebrafish metabolism. In particular, extensive metabolism of parent compound could lead to reduced activity (detoxification) whereas creation of new active metabolites could lead to increased or off-target toxicity [12]. For example, only 35% of testosterone was recovered as unchanged parent compound after just 4 h exposure, as the majority of compound had been metabolized and was detected as testosterone glucuronide in larvae and well solution [12]. Likewise, danozol and tamoxifen, whose metabolites have been shown to induce hepatotoxicity, also cause liver abnormalities in zebrafish larvae [15,16]. Zebrafish embryos and larvae have the ability to perform both phase I (oxidation, n-demethylation, o-demethylation and n-dealkylation) and phase II (sulfation and glucuronidation) metabolism reactions and the metabolic enzymes
Vol. 10, No. 1 2013
Drug Discovery Today: Disease Models | Zebrafish as a platform for in vivo drug discovery
involved in these processes are highly conserved compared to mammals [[see references in 12 and 29]]. In particular, many human cytochrome P450 (CYP) enzymes, which are essential for the activation or inactivation of many endogenous and exogenous chemicals, have direct orthologs in zebrafish [30], indicating that zebrafish metabolic profiles may be similar to mammals. For example, zebrafish larvae have been shown to de-ethylate phenacetin to paracetamol or to demethylate dextromethorphan to dextrorphan [12]. In addition, several verapamil metabolites have been identified that are also formed in mammals [12]. However, differences to mammalian metabolic pathways have been identified with other compounds. Cisapride, for example, was mainly metabolized to cisapride N-sulfate in zebrafish larvae, which is only a minor metabolite in mammals [12]. Terfenadine is almost completely metabolized to azacyclonol and terfenadine alcohol in zebrafish larvae and not to fexofenadine as in humans (Evotec unpublished results). Further studies are therefore necessary to evaluate the similarities and differences in metabolic pathways between human and zebrafish.
Conclusion Zebrafish larvae have gained increasing popularity for toxicity testing over the past decade. Possible issues with compound absorption and metabolism potentiated by the typical aqueous exposure have benefited from the addition of bioanalysis to the screening assays. Using LCMS technology, any observed toxic phenotypes can be correlated with actual compound body burden and potential false-negative and false-positive results can be identified. The determination of effective concentrations should help facilitate investigations into how zebrafish results translate to rodent and human toxicity data and contribute to the global acceptance of zebrafish assays in drug discovery.
Conflict of interest Evotec offers zebrafish screening services to pharmaceutical industries.
Acknowledgements We thank A. Dodd, M. Jones and M Richardson for sharing data prior to publication.
References 1 Humphreys, W.G. (2008) Drug metabolism research as an integral part of the drug discovery process. In Drug Metabolism in Drug Design and Development (Zhang, D., Zhu, M., Humphreys, W.G., eds), pp. 239–260, Wiley-Interscience 2 Grunwald, D.J. and Eisen, J.S. (2002) Headwaters of the zebrafishemergence of a new model vertebrate. Nat. Rev. Genet. 3, 717–724 3 Barros, T.P. et al. (2008) Zebrafish: an automated high content screening platform for safety pharmacology. J. Pharmacol. Toxicol. Methods 58, 150 4 Rubinstein, A.L. (2006) Zebrafish assays for drug toxicity screening. Expert Opin. Drug Metab. Toxicol. 2, 231–240
5 Berghmans, S. et al. (2008) Zebrafish based assays for the assessment of cardiac, visual and gut function-potential safety screens for early drug discovery. J. Pharmacol. Toxicol. Methods 58, 59–68 6 Hill, A.J. (2008) Zebrafish in drug discovery: bridging the gap between in vitro and in vivo methodologies. Preclinical World 121–123 7 Eimon, P.M. and Rubinstein, A.L. (2009) The use of in vivo zebrafish assays in drug toxicity screening. Expert Opin. Drug Metab. Toxicol. 5, 393–401 8 Doshna, C. et al. (2009) Multi-phase analysis of uptake and toxicity in zebrafish: relationship to compound physical–chemical properties. Toxicol. Sci. 108 (S1), 78 9 Van den Bulck, K. et al. (2011) Zebrafish developmental toxicity assay: a fishy solution to reproductive toxicity screening, or just a red herring? Reprod. Toxicol. 32, 213–219 10 Henry, T.R. et al. (1997) Early life stage toxicity of 2,3,7,8tetrachlorodibenzo-p-dioxin in zebrafish (Danio rerio). Toxicol. Appl. Pharmacol. 142, 56–68 11 Heiden, T.K. et al. (2005) Accumulation, Tissue distribution, and maternal transfer of dietary 2,3,7,8-tetrachlorodibenzo-p-dioxin: impacts on reproductive success of zebrafish. Toxicol. Sci. 87, 497–507 12 Alderton, W. et al. (2010) Accumulation and metabolism of drugs and CYP probe substrates in zebrafish larvae. Xenobiotica 40, 547–557 13 Marathe, et al. (2004) The use of radiolabeled compounds for ADME studies in discovery and exploratory development. Curr. Pharm. Des. 10, 2991–3008 14 Hill, A.J. et al. (2009) Cardiotoxicity testing in zebrafish: relevance of bioanalysis. J. Pharmacol. Toxicol. Methods 62, e22–e23 15 Hill, A.J. (in press) Hepatotoxicity testing in larval zebrafish. In Zebrafish: Methods for Assessing Drug Safety and Toxicity (McGrath, P., ed), WileyBlackwell (4 Nov 2011) 16 Jones, M. et al. (2009) Comparison between zebrafish and Hep G2 assays for the predictive identification of hepatotoxins. Toxicology 262, 13–14 17 Gustafson, A.L. et al. (2008) Validation of zebrafish as a model for screening teratogenicity. 47th Annual Meeting of the Society of Toxicology, March 16–20, 2008. Seattle, Washington, USA. Toxicological Sciences 102 (S-1), 407 #1980 18 Hill, A.J. et al. (2010) A review of developmental toxicity screening using zebrafish larvae. 31st Annual Meeting of the American College of Toxicology, November 7–10, Baltimore, MD, USA 19 Brannen, K.C. et al. (2010) Development of a zebrafish embryo teratogenicity assay and quantitative prediction model. Birth Defects Res. B Dev. Reprod. Toxicol. 89, 66–77 20 Selderslaghs, I.W. et al. (2009) Development of a screening assay to identify teratogenic and embryotoxic chemicals using the zebrafish embryo. Reprod. Toxicol. 28, 308–320 21 Hentschel, D.M. et al. (2005) Acute renal failure in zebrafish: a novel system to study a complex disease. Am. J. Physiol.-Renal Physiol. 288, F923–F929 22 Bourrachot, S. et al. (2008) The effects of waterborne uranium on the hatching success, development, and survival of early life stages of zebrafish (Danio rerio). Aquat. Toxicol. 90, 29–36 23 Solon, et al. (2002) Whole-body autoradiography in drug discovery. Curr. Drug Metab. 3, 451–462 24 Jeong, J.Y. et al. (2010) Functional and developmental analysis of the blood–brain barrier in zebrafish. Brain Res. Bull. 75, 619–628 25 Xie, J. et al. (2010) A novel transgenic zebrafish model for blood-brain and blood-retinal barrier development. BMC Dev. Biol. 23, 76 26 Bate, J. et al. (2010) Zebrafish as an alternative model system to identify compounds with anti-epileptic activity. 49th Annual Meeting of the Society of Toxicology, March 7–11, 2010, Salt Lake City, UT, USA 27 Alderton, W. et al. (2007) Characterisation of the maturation of the blood brain barrier in zebrafish. 5th European Zebrafish Genetics and Development Meeting, 12–15 July 2007, Amsterdam, The Netherlands 28 Link, V. et al. (2006) Proteomics of early zebrafish embryos. BMC Dev. Biol. 6, 1–9 29 Hill, A.J. (2011) Use of zebrafish in drug discovery: safety assessment. Drug Discov. Eval. (in press) 30 Goldstone, J.V. et al. (2010) Identification and developmental expression of the full complement of cytochrome P450 genes in zebrafish. BMC Genomics 11, 643–663
www.drugdiscoverytoday.com
e35
DRUG DISCOVERY
TODAY
DISEASE
MODELS
Vol. 10, No. 1 2013
Editors-in-Chief Jan Tornell – AstraZeneca, Sweden Andrew McCulloch – University of California, SanDiego, USA
Zebrafish as a platform for in vivo drug discovery
ADMETox in zebrafish H. Diekmann*, A. Hill Evotec (UK) Ltd., 114 Milton Park, Abingdon OX14 4SA, UK1
To enable the widespread use of zebrafish larvae in drug discovery, it is required to define drug concentration at the target site and to assess metabolites at a resolution necessary for in vivo pharmacology screening. These questions are now being investigated using mass spec-
Section editors: Calum A. MacRae – Cardiovascular Division, Brigham and Women’s Hospital, Boston, MA, USA. Randall T. Peterson – Cardiovascular Research Center, Massachusetts General Hospital, Charlestown, MA, USA.
troscopy and contribute to our understanding of how the outcome of zebrafish toxicity, safety and efficacy studies can translate to rodent and human data.
Introduction Driven by the need to reduce attrition rates in drug development, information on disposition and metabolism of candidate drugs has become a crucial part of all aspects of the drug discovery process [1]. Beyond the optimization of drug candidates for potency and efficacy, it is fundamentally important to detect potentially detrimental toxic side effects within therapeutic doses. Zebrafish larvae share physiological, morphological and histological similarities with mammals and have been recognized as valuable models for evaluating drug candidates for toxicity and safety liabilities [2–5]. Their small size permit screening in microtiter plates with only single milligrams of compound for a full dose–response [6,7]. The zebrafish larva therefore lends itself to being the first in vivo vertebrate model in drug discovery for studying safety pharmacology and toxicity and has the potential to bridge the gap between traditional cell assays and regulated mammalian screens [6]. A key hurdle for this model, however, is the ability to determine the effective compound concentration in the zebrafish and to correlate this dose with rodent and human data.
*Corresponding author: H. Diekmann ([email protected]) 1 www.evotec.com. 1740-6757/$ ß 2012 Elsevier Ltd. All rights reserved.
DOI: 10.1016/j.ddmod.2012.02.005
Bioanalysis on zebrafish larvae Most zebrafish assays rely on aqueous exposure and compound uptake predominantly by diffusion through the skin [3,5]. Zebrafish larvae are typically placed in multi-well plates and compounds are directly added to the supporting medium in a suitable vehicle like DMSO (Fig. 1). Timing and length of exposure is optimized for each specific assay. The zebrafish larvae are then examined for morphological or behavioural phenotypes (e.g. Fig. 2). Exterior structures (including the eye, jaw and fins) and internal organs (such as heart, liver and gut) can be assessed without the need for dissection as zebrafish larvae develop ex utero and are virtually transparent (Fig. 2). Because compound uptake is dictated by the physicochemical properties of each compound, the potency of a compound cannot be predicted from the aqueous concentration or the log P value (Table 1) and [5,8]. Bioanalysis therefore needs to be performed to correlative any toxic phenotype with the actual concentration of compound within the larvae (Fig. 1) and [5,9]. This can be achieved using radio-labelled compound and liquid scintillation counting (LSC) [10,11] or radio high performance liquid chromatography (rHPLC) [12], similarly to ADME studies in rodents [13]. To avoid the use of radio-active material, larvae can alternatively be extracted with acetonitrile and then analysed using LCMS–MS alongside a calibration curve for quantification purposes [12]. This procedure provides the average weight of compound in the larva at the respective aqueous concentration and allows the ranking of compounds according to their potency based upon their body burden (uptake of compound by the larvae). These uptake values e31
Drug Discovery Today: Disease Models | Zebrafish as a platform for in vivo drug discovery
Vol. 10, No. 1 2013
912.9 913.7
Relative Abundance
100 80 60
930.7
40 931.7 20
935.9
484.8
176.7 278.9
409.6
507.7
671.6
0 200
400
600
1109.5 1207.5
698.2 801.7 800
1000
1200
m/z
Drug Discovery Today: Disease Models
Figure 1. Typical zebrafish assay set-up. Zebrafish embryos or larvae are arrayed in multi-well plates and compound is added to the surrounding medium. After a suitable incubation period, zebrafish are analysed for morphological or behavioural phenotypes. Thereafter, the compound body burden is determined using LC–MS technology.
eye
brain
otolith
jaw
heart
liver
muscles
yolk
gut
body length and shape
notochord
pigmentation
blood vessels
liver toxicity
jaw and brain Drug Discovery Today: Disease Models
Figure 2. Examples for toxicity phenotypes in zebrafish larvae. Morphological features such as eye, jaw and fins and internal organs such as heart, liver and gut are depicted in a 4 day old, untreated control zebrafish larvae (top picture). The bottom pictures give examples of phenotypic abnormalities after compound treatment.
Table 1. Bioanalysis examples and compound ranking Compound
Toxic aqueous concentration (mM)
1
100
15.7
3.3
2
100
26.3
1.8
3
50
64.2
3.9
4
50
83.3
4.9
5
50
89.2
4.5
6
50
163.0
1.8
Absorption
7
50
178.8
2.8
Bioanalysis not only allows the comparative ranking of a defined set of compounds (Table 1), but is also necessary to
e32
www.drugdiscoverytoday.com
Body burden (ng/larva)
log P
can differ more than ten-fold even though the aqueous concentration that induced toxicity is similar (Table 1). For example, compound 1 in Table 1 is seemingly less toxic than compound 7, because morphological abnormalities are only observed at a higher aqueous concentration (100 mM for compound 1 versus 50 mM for compound 7). Bioanalysis, however, revealed a much lower body burden for compound 1 versus compound 7, therefore classifying it as more toxic. This hence demonstrates that compound ranking for toxicity or efficacy is unreliable if based purely on aqueous concentrations.
Vol. 10, No. 1 2013
Drug Discovery Today: Disease Models | Zebrafish as a platform for in vivo drug discovery
confirm and quantify compound uptake (body burden) in zebrafish larvae [8] and to identify false-negative results attributable to poor compound absorption [9,14–16]. For example, erythromycin and D-sotalol, which cause QT prolongation in mammals, did not change the heart rate in 3 dpf zebrafish larvae [14]. Bioanalysis, however, revealed very low body burdens of 0.53 and 0.02 ng/larvae, respectively, indicating that these compounds are not taken up into the fish sufficiently to elicit a toxic effect. Similarly, sodium valproate, which can cause acute liver failure in humans, did not induce liver abnormalities in zebrafish larvae [15,16]. The determination of the body burden again indicated insufficient absorption for this compound. Interestingly, exposure of zebrafish larvae to valproic acid did cause liver abnormalities as expected as it was better absorbed into zebrafish larvae [15,16]. Therefore, it can be advisable to test a given compound as different chemical forms to circumvent poor uptake issues. On the other hand, overt toxicity could occur when compounds are readily taken up and high exposure levels are reached; that is, non-toxic compounds exceeding a threshold limit value-ceiling (TLV-C) may result in abnormalities or lethality [9,15]. To distinguish between gross toxicity/morbidity and ‘true toxicity’ and therefore avoid false-positive classifications, a threshold concentration based on historical data (LOECs for non-toxic drugs in use by man) can be imposed to help estimate likely overdose levels for previously untested compounds. In one developmental toxicity study undertaken in 2008, a threshold concentration of 40 ng/larva was chosen by a consortium of pharmaceutical companies based on the LOECs of 18 undisclosed non-toxic compounds [17]. As such, compounds with an LOEC 40 ng/larva were not classified as embryotoxic, but instead the phenotypes observed were attributed to compound overdose and flagged for further investigation. For example, the non-teratogenic compound dextromethorphan only led to morphological abnormalities at a body burden of 123 ng/larva [17,18], whereas highly teratogenic compounds tended to cause adverse effects at a much lower body burden (typically between 1 and 20 ng/larva). Currently, this toxicity threshold is still only used as a guide since it might have to be adjusted according to the structural class of compounds tested, compound efficacy and the expected therapeutic dose. In addition, the TLV-C is likely to differ between assays types (e.g. cardiotoxicity, embryotoxicity, hepatotoxicity) as exposure length and zebrafish age might influence the uptake and therefore the toxicity of compounds. Our preliminary data show for example that the body burden for azetazolamide after a four day incubation period is almost twice as much at 7 dpf compared to 4 dpf (Evotec unpublished results).
Pharmacokinetics Bioanalysis cannot only be performed on larval homogenates, but also on the well solutions to potentially obtain
additional pharmacokinetic information. The concentration of compound left in solution at the end of the incubation period can be compared against a ‘blank standard’ solution. This standard does not contain zebrafish embryos/larvae and demonstrates how much compound may have degraded or stuck to the plastic (assay plate) during the course of the experiment (Evotec unpublished results). Since the fish volume (1–2 ml per larva) is negligible compared to the assay solution (0.5–1 ml), the compound concentration in the assay solution containing fish should be comparable to the blank standard in case of equimolar uptake. However, if the compound is readily absorbed and has significantly accumulated in the zebrafish larvae, the compound concentration of the assay solution will be reduced accordingly. Significantly reduced assay concentration compared to the blank standard indicates rapid compound metabolism if mass balance is not retained. For example, only 4% of the nominal start concentration of midazolam was detected in the solution at the end of an experiment, even though the amount of compound detected in the zebrafish larvae was only 3%. This suggested that most of the compound had been metabolized in the course of the experiment [12]. Owing to the excess of compound available in the dosing solution, compound absorption has been expected to reach a maximum steady state level in zebrafish embryos and larvae and the kinetics of compound uptake have so far been largely neglected for toxicity assessments. Typically, zebrafish embryos/larvae are exposed to compound continually throughout the assay and the body burden is determined at the end of the exposure period (Fig. 1) and [5,9,15]. However, the effective concentration to cause a certain phenotype might differ significantly from the body burden at the end of a longterm exposure experiment. In a zebrafish teratogenicity assay, for example, embryos are exposed to compound at 2–4 h post fertilization (hpf) and analysed for morphological abnormalities at 96–120 hpf [18–20]. If compounds are continuously accumulating over this 4–5 day period, the body burden at the end of the assay would overestimate the effective concentration, as teratogenic effects are expected to occur during embryogenesis at 2–48 hpf. On the other hand, the measured body burden could underestimate the effective concentration for compounds rapidly metabolized during the later stages of the experiment. For example, preliminary data showed a steady increase in body burden over a four day incubation period for compound 1 (Fig. 3). Compound 2, however, was taken up to a greater extend and reached a steady state level by the third day of exposure (Fig. 3). Although generalized conclusions cannot yet be drawn from this limited data set, it looks advisable to determine the body burden at earlier time points to avoid confounding results due to the aforementioned scenarios. Alternatively, bioanalysis samples could be taken at different times during an experiment, similarly to plasma concentration time course studies in rodents [1]. www.drugdiscoverytoday.com
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and desloratidine showed lower compound concentrations in the head versus the trunk. This is in agreement with mammalian studies showing poor BBB penetration for these compounds [27]. Finally, one important factor to consider is the presence of prominent yolk during embryonic stages of development. Hydrophobic compounds may accumulate disproportionately in this lipid-rich environment compared to the rest of the body. As this could lead to an overestimation for the effective concentrations required to induce organ toxicity, it should be tested whether the removal of the yolk would alter bioanalysis results [28].
60 body burden (ng/larva)
compound 1 compound 2 40
20
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2
3
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Figure 3. Compound uptake kinetics. Zebrafish larvae were immersed in compound and the body burden determined at 1, 2, 3 and 4 days post exposure (dpe). Compound 1 was taken up linearly during the exposure period while compound 2 reached a plateau level at 3 dpe.
Distribution The distribution of compounds within zebrafish has not yet been extensively studied. Compounds are usually added to the fish support medium in a suitable vehicle (e.g. 0.5–1% DMSO) and thought to be taken up into zebrafish embryos and larvae by passive diffusion through the skin and gills (aqueous exposure) [3,4]. Larvae could also ingest compound from the dosing solution, therefore making the gastrointestinal tract another key site for absorption. Alternatively, compounds can be injected into the yolk sac or the vasculature, from where they get distributed throughout the body [21]. Whether effective levels are reached within the target tissues by either method is dictated by the physicochemical properties of the compound and is generally only proven by a positive assay readout for the organ of interest [4,5]. Tissue-specific bioanalysis is rather infeasible due to the small size of the larvae, although distribution and accumulation in specific organs could potentially be visualized using radioactively labelled compounds and quantitative whole body autoradiography [22,23]. As zebrafish larvae develop a functional blood brain barrier (BBB) between 3 and 8 dpf [24,25], it is possible that compounds may not reach the central nervous system (CNS) in older zebrafish larvae, although anti-epileptic compounds have been successfully tested in 6 dpf zebrafish [26]. In an attempt to show the exclusion of drugs from the zebrafish brain and therefore to prove the existence of a functional BBB in zebrafish larvae, bioanalysis was performed separately on heads and trunks of larvae that were immersed in various drugs [27]. Diphenhydramine, haloperidol and scopolamine, which cross the BBB in mammals, were equally distributed between head and body in zebrafish larvae. By contrast, scopolamine N-butyl bromide e34
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Excretion Compound secretion should be irrelevant for most zebrafish assays using continuous aqueous exposure since a stable equilibrium between compound absorption and secretion should be established within the experiment. Compound excretion could nevertheless be analysed using pulsed exposure and is particularly important for microinjection experiments. For instance, several compounds hepatotoxic to human such as diclofenac and amiodarone are known to not cause liver abnormalities upon microinjection into 3 dpf zebrafish larvae. In the case of diclofenac, bioanalysis revealed a body burden of 9.2 ng/larva at 1 h post injection (hpi), demonstrating successful compound delivery, but no diclofenac could be detected in larvae at 24 and 48 h postinjection (hpi) (Evotec unpublished results). At these time points, however, diclofenac was detectable in the well solutions, indicating fast and complete excretion or diffusion of the compound from the larvae. Amiodarone, on the other hand, was detected at 156 ng/larva (1 hpi), 132 ng/larva (24hpi) and 85 ng/larva (48 hpi) with no compound appearing in the well solution. Amiodarone therefore seemed to be slowly metabolized by the zebrafish larvae (Evotec unpublished results).
Metabolism In addition to mere detection of compound absorption, bioanalysis also allows the investigation of zebrafish metabolism. In particular, extensive metabolism of parent compound could lead to reduced activity (detoxification) whereas creation of new active metabolites could lead to increased or off-target toxicity [12]. For example, only 35% of testosterone was recovered as unchanged parent compound after just 4 h exposure, as the majority of compound had been metabolized and was detected as testosterone glucuronide in larvae and well solution [12]. Likewise, danozol and tamoxifen, whose metabolites have been shown to induce hepatotoxicity, also cause liver abnormalities in zebrafish larvae [15,16]. Zebrafish embryos and larvae have the ability to perform both phase I (oxidation, n-demethylation, o-demethylation and n-dealkylation) and phase II (sulfation and glucuronidation) metabolism reactions and the metabolic enzymes
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involved in these processes are highly conserved compared to mammals [[see references in 12 and 29]]. In particular, many human cytochrome P450 (CYP) enzymes, which are essential for the activation or inactivation of many endogenous and exogenous chemicals, have direct orthologs in zebrafish [30], indicating that zebrafish metabolic profiles may be similar to mammals. For example, zebrafish larvae have been shown to de-ethylate phenacetin to paracetamol or to demethylate dextromethorphan to dextrorphan [12]. In addition, several verapamil metabolites have been identified that are also formed in mammals [12]. However, differences to mammalian metabolic pathways have been identified with other compounds. Cisapride, for example, was mainly metabolized to cisapride N-sulfate in zebrafish larvae, which is only a minor metabolite in mammals [12]. Terfenadine is almost completely metabolized to azacyclonol and terfenadine alcohol in zebrafish larvae and not to fexofenadine as in humans (Evotec unpublished results). Further studies are therefore necessary to evaluate the similarities and differences in metabolic pathways between human and zebrafish.
Conclusion Zebrafish larvae have gained increasing popularity for toxicity testing over the past decade. Possible issues with compound absorption and metabolism potentiated by the typical aqueous exposure have benefited from the addition of bioanalysis to the screening assays. Using LCMS technology, any observed toxic phenotypes can be correlated with actual compound body burden and potential false-negative and false-positive results can be identified. The determination of effective concentrations should help facilitate investigations into how zebrafish results translate to rodent and human toxicity data and contribute to the global acceptance of zebrafish assays in drug discovery.
Conflict of interest Evotec offers zebrafish screening services to pharmaceutical industries.
Acknowledgements We thank A. Dodd, M. Jones and M Richardson for sharing data prior to publication.
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