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Contemporary Chemical Approaches
for Green and Sustainable Drugs
Advances in Green and
Sustainable Chemistry
Contemporary
Chemical Approaches
for Green and
Sustainable Drugs
Series Editor
Béla Török
Timothy Dransfield
Edited by
Marianna Török
Elsevier
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Contents
List of contributors xiii
1. Using the zebrafish model system to identify the

health effects of pharmaceutical pollutants
Christina Kaucic, Anusha Lakshmi Dharmavathi
and Jennifer L. Freeman
1. Introduction 1
2. The zebrafish model system 2
3. Use of the zebrafish model system in drug discovery 3
4. Significance of pharmaceutical pollution 6
5. Use of the zebrafish model system to assess pharmaceutical
pollutant toxicity 7
6. Methods and approaches to assess pharmaceutical pollutant
toxicity using the zebrafish model system 13
6.1 Acute developmental toxicity assessments with the
developing zebrafish 13
6.2 High-throughput screenings (HTS) for developmental toxicity
assessments 14
6.3 Zebrafish developmental and adult behavioral assays to assess
pharmaceutical pollutant toxicity 16
6.4 Cellular and molecular assays to identify mechanisms of
7. Future directions for the use of zebrafish in defining
8. Conclusions 21
Acknowledgments and Funding sources 21
References 21
2. Analysis of pharmaceuticals in the environment

Aditya Kulkarni and Scott E. Miller
1. Introduction 27
2. Sources of pharmaceutical pollutants 28
2.1 Effects of trace level pharmaceutical pollutants on
humans 29
2.2 Effects of trace level pharmaceutical pollutants on
aquatic environments 30
v
vi Contents
3. Analytical methods for trace level analysis of water samples 32

3.1 Solid phase extraction (SPE) 33
3.2 High-performance liquid chromatography (HPLC) 35
3.3 Mass spectrometry (MS) 36
3.4 Other techniques 39
4. Risk management 41
5. Conclusion 41
List of abbreviations 41
References 42
3. Leaking of antibiotics in the aquatic environment

Indu, Manisha Sharma and Kashyap Kumar Dubey
1. Introduction 47
2. How antibiotics are reaching the aquatic environment? 49
2.1 From human use 49
2.2 From hospital waste 53
2.3 From animal use 56
2.4 From agricultural use 58
2.5 From pharmaceutical industry waste 59
3. Fate of antibiotics in aquatic environment 60
4. Conclusion 61
References 62
4. Advances in drug development with the

application of artificial intelligence
Manuela Souza Leite, Anderson Alles de Jesus,
Paulo Jardel Leite Araujo and Brunno Ferreira dos Santos
1. Machine learning and artificial intelligence in the
pharmaceutical industry: perspective 69
2. Data mining techniques 71
2.1 Statistics 72
2.2 Clustering 72
2.3 View 72
2.4 Decision tree 73
2.5 Neural networks 73
3. Artificial neural networks (ANN) in property prediction
to drug discovery 75
4. Support vector machines (SVM) in drug discovery and
development 78
5. Conclusion 80
Acknowledgments 81
References 81
Further reading 88
Contents vii
5. Virtual screening techniques in pharmaceutical

research
Justine C. Williams, Stanley Opare, Senthil Kumar Sugadoss,
Aravindhan Ganesan and Subha Kalyaanamoorthy
1. Introduction 89
2. Structure-based drug design (SBDD) 91
2.1 Protein structure prediction 92
3. Molecular docking 96
3.1 Search algorithms 96
3.2 Scoring functions 97
3.3 Target flexibility 101
3.4 De novo drug design 101
3.5 Binding energy estimation 103
3.6 Machine/deep learning methods in SBVS 104
4. Ligand-based drug discovery (LBDD) 107
4.1 Similarity searching 108
4.2 Ligand-based pharmacophore mapping 113
4.3 Quantitative structure-activity relationship (QSAR)
modeling 115
5. Summary and perspectives 117
References 119
6. In silico modeling of environmental toxicity

of drugs
Kabiruddin Khan and Kunal Roy
1. Introduction 129
2. Pharmaceutical ecotoxicity analysis: general considerations 129
2.1 Overview of concerns 130
3. Release of pharmaceuticals to the environment 131
3.1 The sources of pharmaceuticals pollution to environment 131
4. Assessment of ecotoxicity of APIs, limitations, and solutions:
a data scientist’s perspective 131
5. Current advancement in ecotoxicity modeling of
pharmaceuticals 132
5.1 In silico tools reported in different research articles 133
6. Online expert systems for ecotoxicity prediction 141
7. Conclusion 149
Nomenclature list 150
Acknowledgments 151
References 151
viii Contents
7. Sustainable separations in pharmaceutical

manufacturing
Gergo Ignacz, Robert Orkenyi, Arpad Konczol and Gyorgy Szekely
1. Introduction 155
1.1 Separation concepts in the pharmaceutical industry 156
1.2 Continuous and automated separation processes 158
2. Chromatography-based separations 159
2.1 Sustainable aspects of chromatography 159
2.2 Toward green chromatographic techniques 159
2.3 Strategies toward green liquid chromatography 162
2.4 Relevant industrial applications: case studies 166
2.5 Conclusions 167
3. Membrane based separations 167
3.1 Membrane separation in the pharmaceutical industry:
a liquid dominant sector 167
3.2 Organic solvent nanofiltration (OSN) 168
4. Continuous purification processes 174
4.1 Classification of the continuous flow purification
processes 174
4.2 Continuous crystallization (CC) 175
4.3 Centrifugal partition chromatography 177
4.4 Simulated moving bed chromatography 179
4.5 Comparison of the previously discussed continuous
purification methods 180
5. Forecasting the future of API separations 183
References 185
8. Green synthetic methods in drug discovery and

development
Guoshu Xie, Rita Bernadett Vlocskó and Béla Török
1. Introduction 201
2. Catalysis 203
2.1 Homogeneous catalysis 204
2.2 Heterogeneous catalysis 217
3. Nontraditional activation methods and energy efficiency of
chemical processes 238
3.1 Microwave-assisted organic synthesis 238
3.2 Ultrasonic activation 242
3.3 Photochemical activation 248
3.4 Electrochemical activation 256
4. Conclusions 260
References 261
Contents ix
9. Characterizing the environmentally benign nature of

chemical processes: green chemistry metrics
David Daggett, Yizhou Shi and Béla Török
1. Introduction 281
2. Emergence of green chemistry 282
3. Sustainable production of pharmaceuticals and their building
blocks: quantitative green metrics to evaluate chemical
processes 282
3.1 Mass-related metrics 283
3.2 Energy-related metrics 294
3.3 Greenhouse gas emission and ozone creation metrics 296
3.4 Solvent-related metrics 298
3.5 Life cycle assessment (LCA) 300
4. Conclusions 300
References 301
10. Green chemistry approaches to drugs that treat

epidemic and pandemic diseases
Berkeley W. Cue
1. Introduction 307
2. Antibacterial drugs 309
2.1 Penicillin and cephalosporin antibiotics 309
2.2 Macrolide antibiotics 310
2.3 Fluoroquinolone antibiotics: ciprofloxacin 313
3. Drugs to treat malaria 313
3.1 Amodaquine 314
3.2 Arteminisin 315
3.3 Hydroxychloroquine 317
3.4 Piperaquine 318
4. Drugs to treat HIV/AIDS 318
4.1 Nevirapine 319
4.2 Dolutegravir 321
5. Antivirals to treat COVID-19 322
5.1 Remdesivir 322
5.2 Molnupiravir (EIDD-2801, MK-4482) 323
6. Conclusions 325
References 326
Further reading 330
11. Dynamic effects of organic molecules for drug

delivery in micelles
Debanjana Ghosh, Ria Ramoutar and Shainaz Landge
1. Introduction 333
2. Drug solubilization through drug delivery vehicles 335
2.1 Micelles 335
2.2 Liposomes 336
x Contents
3. Molecular drug delivery by organized self-assemblies 337

3.1 Small molecule-surfactant based drug delivery 337
3.2 Liposomal system as drug delivery vehicle 347
3.3 Reverse micelles nanocarriers for drug delivery 360
4. Conclusion 370
References 373
12. Antibody-drug conjugates for targeted delivery

Garima Pandey, Sunil K. Tripathi and Vivek Bulbule
1. Introduction 377
1.1 What is an antibody-drug conjugate? 377
1.2 Importance of ADCs in targeted drug delivery 379
1.3 Current ADCs approved or in clinical trials 380
2. Composition 385
2.1 Target and antibody 385
2.2 Linkers 386
2.3 Payloads 395
3. Antibody-drug conjugation 396
3.1 Through side chain lysine residue 397
3.2 Through side chain cysteine residue 398
3.3 Drug antibody ratio (DAR) 400
3.4 Site-specific conjugation 400
4. Physical stability of antibody-drug conjugates 403
5. Chemical stability of antibody-drug conjugate 404
6. Formulation development of antibody-drug conjugate 405
7. Future aspects of ADCs 406
References 408
13. Toward the green synthesis of peptides and

peptidic drugs
Dóra Bogdán, Levente Kárpáti and István M. Mándity
1. Peptides and peptide synthesis: the formation of peptide
bond 421
1.1 Historical perspective of solution phase peptide synthesis 421
1.2 Solid-phase peptide synthesis (SPPS) 422
1.3 Solid-phase fragment/segment condensation in the 1980s 424
1.4 Automatization of the SPPS 424
2. Improvements in the solid-phase method 426
2.1 Optimization of the linkers and polymeric supports in
SPPS 426
2.2 Development of the N-terminal amino protecting
groups 427
2.3 On resin monitoring of the coupling efficiency 428
Contents xi
2.4 Development of coupling agents and coupling

methodologies 428
3. Novel methods for dissolving limitations of SPPS 430
3.1 Native chemical ligation (NCL) 430
3.2 Continuous-flow solid-phase peptide synthesis (CF-SPPS) 431
3.3 Solution-phase continuous-flow peptide synthesis 435
3.4 Microwave-assisted peptide synthesis 436
4. Large-scale peptide synthesis methods 439
4.1 Continuous-flow large scale peptide synthesis 439
4.2 Solution-phase large-scale peptide synthesis 440
4.3 Solid-phase large-scale peptide synthesis 440
4.4 Hybrid large-scale peptide synthesis 441
5. Green chemistry aspects of peptide synthesis 441
6. Summary 445
Acknowledgments 447
References 448
14. The multitarget approach as a green tool in

medicinal chemistry
Rita Bernadett Vlocskó, Sinem Apaydın, Béla Török
and Marianna Török
1. Introduction 457
2. The green impact of multitarget drug discovery 460
2.1 Green syntheses in multitarget drug development (MTDD) 462
2.2 Combining in silico and experimental methods:
inherently green improvement to design and
testing efficiency 464
3. Multitarget lead generationdscreening-versus
knowledge-based approaches 465
3.1 Knowledge-based rational design 466
3.2 Screening-based multitarget lead generation 468
3.3 Natural product-inspired scaffolds for designing MTDs 472
4. Selected case studies with multitarget focus 476
4.1 Neurodegenerative diseasesdAlzheimer’s disease 476
4.2 Infectious diseases 477
4.3 Cancer 478
4.4 Epigenetic polypharmacology 479
5. Challenges and limitations in multitarget drug design and
development 480
6. Conclusions 481
References 483
xii Contents
15. Directed evolution: a new powerful tool in drug

development
Andrea Baier and Ryszard Szyszka
1. Introduction 493
2. Methods in directed evolution 494
2.1 Methods for gene manipulation 495
2.2 Methods for screening and selection of enzyme libraries 501
3. Application of directed evolution in drug development 502
3.1 Simvastatin synthase LovD 502
3.2 Transaminase ATA-17 in the synthesis of sitagliptin 504
3.3 Candida antarctica lipase A (CALA) in the synthesis of
ibuprofen 505
4. Perspectives 506
References 507
16. Conventional and advanced treatment methods

for the removal of pharmaceuticals and related
compounds in wastewater
James A. Noblet
1. Introduction 511
2. Overview of conventional wastewater treatment 513
3. Effectiveness of conventional wastewater treatment in
removing pharmaceuticals 514
4. Advanced treatment processes for the removal of
pharmaceuticals from municipal wastewater 516
4.1 Overview 516
4.2 Ozonation 516
4.3 Activated carbon methods 517
4.4 Membrane-based technologies 518
4.5 Newer methods: advanced oxidative processes 521
5. Case Study #1: San Bernardino municipal water
department 524
5.1 Rapid infiltration and extraction (RIX) facility 524
6. Case Study #2: the Orange County Sanitation District,
Fountain Valley, CA 529
6.1 Ground water replenishment system (GWRS) 529
7. Conclusion 529
References 532
Further reading 533
Index 535
List of contributors
Anderson Alles de Jesus, Coordination of the Interdisciplinary Bachelor of Science

and Technology, Universidade Federal do Maranhão, Balsas-MA, Brazil
Sinem Apaydın, Department of Chemistry, University of Massachusetts Boston,
Boston, MA, United States
Paulo Jardel Leite Araujo, ICON Educacional, Aracaju-SE, Brazil
Andrea Baier, The John Paul II Catholic University of Lublin, Lublin, Poland
Dóra Bogdán, Department of Organic Chemistry, Faculty of Pharmacy, Semmelweis
University, Budapest, Hungary; TTK Lendület Artificial Transporters Research
Group, Institute of Materials and Environmental Chemistry, Research Centre for
Natural Sciences, Budapest, Hungary
Vivek Bulbule, Adesis Inc., New Castle, DE, United States
Berkeley W. Cue, BWC Pharma Consulting, LLC, Nottingham, NH, United States;
Department of Chemistry, University of Massachusetts Boston, Boston, MA,
United States
David Daggett, Department of Chemistry, University of Massachusetts Boston, Boston,
MA, United States
Brunno Ferreira dos Santos, Department of Chemical and Materials Engineering
(DEQM), Pontifical Catholic University of Rio de Janeiro (PUC-Rio), Rio de
Janeiro, Brazil
Jennifer L. Freeman, School of Health Sciences, Purdue University, West Lafayette,
IN, United States
Aravindhan Ganesan, ArGan’s Lab, School of Pharmacy, University of Waterloo,
Waterloo, ON, Canada
Debanjana Ghosh, Department of Chemistry and Biochemistry, Georgia Southern
University, Statesboro, GA, United States
Gergo Ignacz, Advanced Membranes and Porous Materials Center, Physical Science
and Engineering Division (PSE), King Abdullah University of Science and
Technology (KAUST), Thuwal, Saudi Arabia
Indu, Bioprocess Engineering Laboratory, Central University of Haryana,
Mahendergarh, Haryana, India
Subha Kalyaanamoorthy, Department of Chemistry, University of Waterloo,
Waterloo, ON, Canada
xiii
xiv List of contributors
Levente Kárpáti, Department of Organic Chemistry, Faculty of Pharmacy, Semmel-

weis University, Budapest, Hungary
Christina Kaucic, School of Health Sciences, Purdue University, West Lafayette, IN,
United States
Kabiruddin Khan, Department of Pharmaceutical Technology, Jadavpur University,
Kolkata, West Bengal, India
Arpad Konczol, RotaChrom Technologies LLC, Kecskemet, Hungary
Aditya Kulkarni, 908 Devices, Boston, MA, United States
Kashyap Kumar Dubey, Bioprocess Engineering Laboratory School of Biotech-
nology, Jawaharlal Nehru University, New Delhi, India
Anusha Lakshmi Dharmavathi, School of Health Sciences, Purdue University, West
Lafayette, IN, United States
Shainaz Landge, Department of Chemistry and Biochemistry, Georgia Southern
István M. Mándity, Department of Organic Chemistry, Faculty of Pharmacy, Sem-
melweis University, Budapest, Hungary; TTK Lendület Artificial Transporters
Research Group, Institute of Materials and Environmental Chemistry, Research
Centre for Natural Sciences, Budapest, Hungary
Scott E. Miller, 908 Devices, Boston, MA, United States
James A. Noblet, Department of Chemistry and Biochemistry, California State
University San Bernardino, San Bernardino, CA, United States
Stanley Opare, Department of Chemistry, University of Waterloo, Waterloo, ON,
Canada
Robert Orkenyi, RotaChrom Technologies LLC, Kecskemet, Hungary; Evonetix Ltd,
Cambridge, United Kingdom
Garima Pandey, Organix Inc., Woburn, MA, United States
Ria Ramoutar, Department of Chemistry and Biochemistry, Georgia Southern
Kunal Roy, Department of Pharmaceutical Technology, Jadavpur University, Kolkata,
West Bengal, India
Manisha Sharma, Bioprocess Engineering Laboratory, Central University of Haryana,
Mahendergarh, Haryana, India
Yizhou Shi, Department of Chemistry, University of Massachusetts Boston, Boston,
MA, United States
Manuela Souza Leite, Graduate Program in Process Engineering, Tiradentes
University, Aracaju-SE, Brazil; Institute of Technology and Research, Aracaju-SE,
Brazil
Senthil Kumar Sugadoss, Department of Chemistry, University of Waterloo, Water-
loo, ON, Canada
List of contributors xv
Gyorgy Szekely, Advanced Membranes and Porous Materials Center, Physical Science
and Engineering Division (PSE), King Abdullah University of Science and Tech-
nology (KAUST), Thuwal, Saudi Arabia
Ryszard Szyszka, The John Paul II Catholic University of Lublin, Lublin, Poland
Béla Török, Department of Chemistry, University of Massachusetts Boston, Boston,
MA, United States
Marianna Török, Department of Chemistry, University of Massachusetts Boston,
Boston, MA, United States
Sunil K. Tripathi, University of Michigan, Ann Arbor, MI, United States
Rita Bernadett Vlocskó, Department of Chemistry, University of Massachusetts
Boston, Boston, MA, United States
Justine C. Williams, Department of Chemistry, University of Waterloo, Waterloo, ON,
Canada
Guoshu Xie, Department of Chemistry, University of Massachusetts Boston, Boston,
MA, United States
Chapter 1
Using the zebrafish model

system to identify the health
effects of pharmaceutical
pollutants
Christina Kaucic, Anusha Lakshmi Dharmavathi and Jennifer L. Freeman
School of Health Sciences, Purdue University, West Lafayette, IN, United States
1. Introduction
With all eyes shifting toward the impacts of global climate change and
pollution on human and ecological health, sustainability and environmental
safety are eminent topics that need to be addressed. Specifically, there is much
focus toward how pharmaceutical waste and buildup in the environment may
affect the general health and wellness of exposed organisms.1 Pharmaceutical
pollution, which refers to the presence of synthetic drugs and their metabolites
in the environment, is largely unregulated and has the potential to be detri-
mental to human and ecological health. In the past, environmental toxicology
studies, including those assessing pharmaceutical pollution, have relied pri-
marily on rodent or other mammalian models to perform toxicity assessments.
Limitations and high economic costs of large-scale mammalian models for
assessing environmental toxicity have led to an expansion of other vertebrate
model systems being applied in this research area. As selecting the optimal
animal model is vital to understanding environmental toxicology, researchers
have enthusiastically turned to the zebrafish model to study the effects of
environmental pollutants including pharmaceutical pollution as an animal
model complementary to the traditional mammalian assays. In this chapter, we
discuss the advantages, strengths, and limitations for utilizing the zebrafish
model system in toxicological studies and in particular, those studies assessing
the adverse effects of pharmaceutical pollution. In addition, we detail common
zebrafish assays employed to assess developmental toxicity, morphology,
behavior, and molecular perturbations induced by pharmaceutical pollution
exposure.
Contemporary Chemical Approaches for Green and Sustainable Drugs

https://doi.org/10.1016/B978-0-12-822248-5.00011-5
Copyright © 2022 Elsevier Inc. All rights reserved. 1
2 Contemporary Chemical Approaches for Green and Sustainable Drugs
2. The zebrafish model system

Zebrafish (Danio rerio) are a notable and popular vertebrate model in devel-
opmental and molecular biology due to their suitability for genomic studies
and transparent embryos, which allow for easy developmental assessment.2
Based on a number of strengths, their value as a complementary model system
for toxicity studies has steadily increased over the past 20 years including
those addressing environmental pollution and their use in drug development.3,4
In addition to being a small, economical model in comparison with
mammalian counterparts, the zebrafish has high fecundity, easy husbandry, is
near-transparent during the early stages of development, and, perhaps most
importantly, has a high degree of molecular synteny with humans. In fact, 71%
of all human proteins and 82% of disease-causing proteins have an ortholog in
the zebrafish.5 Coupled to the well-conserved physiology with humans, the
zebrafish is an ideal model for human drug discovery and toxicological
studies.
A few of the most simplistic benefits of using the zebrafish as a toxico-
logical model are their small size and husbandry. Unlike other model species,
adult zebrafish are fully grown at 1e1.5 inches long. This alone allows for an
increased sample size in laboratories with limited space for housing and
breeding. The small size of the fish also allows for smaller quantities of dosing
solutions in toxicological studies which, in turn, minimizes laboratory costs
and chemical waste. Additionally, zebrafish mating pairs have a high fecundity
and produce many small embryos that can increase sample sizes and number
of replicates in dose-response assays and high-throughput toxicity testing.
Major reasons for the popularity of the zebrafish model in developmental
biology are based on their high fecundity and embryos that have transparent
chorions. Transparency allows for developmental staging studies and identi-
fication of phenotypic abnormalities at the earliest developmental stages. The
transparent chorions of zebrafish embryos prove even more valuable when
used with in situ hybridization and immunohistochemistry techniques. In situ
hybridization and immunohistochemistry are typically used in developmental
biology to investigate gene and protein expression in embryonic tissues; the
clarity of the zebrafish embryo allows labeled probes to be easily identified. In
addition, development of transgenic zebrafish models with fluorescent tags
(e.g., green fluorescent protein, GFP) (Fig. 1.1) also permits assessment of
gene expression patterns, tissue/organ development, cellular localization of
proteins, and other applications at the earliest developmental stages.6,7 The use
of these techniques has spurred questions about the relevance of gene and
protein expression studies in the zebrafish and the relationship to the human
genome. Understanding this syntenic relationship between the two species
allows for human-disease model studies to take place using the zebrafish.
The zebrafish genome is complex with a high degree of similarity to the
human genome.8 In a study investigating the differences between the human
Using the zebrafish model system to identify Chapter | 1 3
FIGURE 1.1 Zebrafish fli1 [(Tg(fli1:eGFP)y1] transgenic larva. This transgenic line involves a
transgenic insertion of enhanced green fluorescent protein (eGFP) in the vasculature under control
of the fli1 promoter. This transgenic line is used to easily visualize vascular defects in zebrafish
following chemical exposures.
genome and the zebrafish genome, it was discovered that around 80% of genes
and expressed sequence tags are conserved.9 In a latter more extensive study,
results showed 82% of human disease genes have at least one zebrafish
ortholog.5 Moreover, gene mutations in zebrafish have been shown to produce
less embryonic lethality than the same gene mutations in mammalian models,
largely due to a genome duplication event.10 As such, researchers are better
able to study the affected gene function and the associated signaling pathways
in pathogenesis. In toxicological studies, this fact is vital as we are able to
study developmental changes and the molecular mechanisms of the xenobiotic
exposures.
3. Use of the zebrafish model system in drug discovery

The scientific discovery of new drugs being manufactured from pharmaceu-
tical companies has declined over the years, while the production costs have
steadily increased. A major cause is the tedious testing that is needed in order
to put a new drug on the market. Usually, numerous in vitro biochemical and
cell assays followed by in vivo testing with mammalian models are required
before human trials can commence. This process often leads to only a few
drugs on the market. These prospective drugs can get terminated at any phase
of drug development due to adverse health outcomes, lack of efficacy, or
excessive toxicity problems. Most of the failures of drug toxicity come at the
level of using animal models. The causes of drug toxicity observed are
organized into mechanism-based (on-target), immune hypersensitivity, off-
target toxicity, and covalent modification. Therefore, drug development is an
important process that serves as an essential way to evaluate the safety and
toxicity of potential harmful drugs that can be found in our environment.
Although current methods work in preventing the toxic compounds from

the clinical setting, there are still false negatives observed during preclinical
toxicity. This is seen because drug safety is initially evaluated preclinically in
animal models. This increase in preclinical toxicity as well as the adverse
events that come from human toxicity account for one-third of the cases of
attrition for prospective drugs. As such, it is important to identify toxic
compounds accurately to reduce potential toxicity effects. Steps in the drug-
development paradigm support that early intervention with removing toxic
compounds can help save resources and reduce costs (Fig. 1.2). Additionally,
in the early phase of drug development between 40% and 80% of the com-
pounds are halted in development due to safety concerns,4 supporting the
investigation of this time-point. In general, the incidence of adverse drug re-
actions varies with the type of drug and dosage, supporting that drug toxicity
can be a major limiting factor to the efficacy of widely used agents.
Drug toxicity has provided scientific and practical applications, but
three major discrepancies still exist that can help reduce the toxic effects of
the chemicals through investigation. These three major areas of focus are
finding useful biomarkers of toxicity, establishing useful in vitro/in vivo re-
lationships, and linking animal models with human toxicity more accurately.11
Through drug discovery, we have been able to find methods to approach these
three main issues in order to assess drug safety and toxicity. An important
in vivo relationship that allows us to improve overall toxic effects in drug
FIGURE 1.2 The generalized

drug discovery and development
pathway before a novel drug passes
through the US Food and Drug
Administration (FDA) approval for
safety testing.
development was accomplished by expanding the use of animal models

beyond mammals including utilization of the zebrafish model. The zebrafish is
showing great potential to eliminate unsafe compounds during the early stages
of drug development and evaluate the preclinical toxicity of new drugs at a
lower overall economic cost.4
Toxicity is a major attrition in drug development leading to the withdrawal
of certain drugs. As such, research has been done to accurately find the po-
tential adverse health outcomes that may occur. Cardiotoxicity is one of the
major concerns for pharmaceutical companies along with hepatic, oncological,
and neurological drugs due to their unfavorable complications. Cardiotoxicity
has indicated its relevance particularly in children and adolescents as they are
generally more susceptible to toxic effects. Cardiotoxicity has remained an
issue as unforeseen heart attacks, strokes, or arrhythmias may occur as was
seen for Rofecoxib, an antiarthritic drug, and Propulsid, a gastrointestinal
prokinetic agent.12 In order to understand this toxicity, the zebrafish model
system was applied as a reliable cardiotoxicological tool. Despite some of the
physiological differences between the zebrafish and mammalian heart, use of
zebrafish presents an optimal assessment of organ development. The heart is
the first organ that develops in zebrafish during the early stages of embryo-
genesis supporting high conservation and exhibiting similar functional char-
acteristics, which allows to more accurately link animal models with human
toxicity. This is why zebrafish are particularly suitable for determining
developmental toxicity, general toxicity, and to perform initial high-throughput
drug screenings in order to accomplish and validate drug development studies.
Additional past studies have evaluated cardiotoxicity and compared the
zebrafish with other in vivo and in vitro models. The data have concluded
support for the zebrafish model system as an efficient way to evaluate drug
toxicity and give comprehensive knowledge for new generations of drugs.13
A second example of the application of the zebrafish model system in
addressing concerns during drug development revolves around drug-induced
liver injury (DILI). In clinical medicine, a major challenge occurs from the
deliberate overdose of acetaminophen. In terms of drug development, DILI
remains a problem because of safety concerns. The overdose from acet-
aminophen is predictable because it is dose-dependent, whereas idiosyncratic
liver toxicity occurs in the later phases of drug development making it hard to
predict during the early stages of development. This challenge was addressed
by utilizing the zebrafish model system in high-throughput screenings (HTS)
to advance the further study of DILI and showed mechanistic similarity for
DILI in humans.14 These methods can be useful to identify new mediators for
predicting human DILI with potential therapeutic compounds and early
identification of hepatotoxic compounds to help reduce costs in the process of
drug development.15,16
In recent years, many drugs have been withdrawn from the market due to
safety concerns and the costs of new drug development have risen. The use of
biomarkers can address several major problems associated with drug devel-
opment. Biomarkers enhance determination of whether drug candidates are
promising early in development as well as reduce the costs of drug develop-
ment. Use of biomarkers can also identify applicable drugs with enhanced
safety and find treatments for those patients who need the best balance of risk
and benefit. Moreover, translational biomarkers between fish and mammalian
models can be established to further improve the accuracy and our under-
standing of preclinical toxicity during drug development. As done with the
DILI studies, biomarkers show to be a useful tool that may have great value in
drug discovery.14,16 Biomarkers can provide a bridge between fish, rodents,
and humans. Thus, it is important to expand upon the use of biomarkers in the
process of drug discovery when working with animal models. This expansion
can help detect future drug toxicity issues in humans, which can make the
process more efficient and lower the costs toward a more successful outcome
in the future. Overall, these applications support the use of the zebrafish in
future studies progressing toward promising drugs during the early develop-
mental phase of drug discovery. Furthermore, these studies provide confidence
for application of the zebrafish to address the environmental toxicity questions
surrounding pharmaceutical pollution, providing a connection between the
overall toxicity of these pharmaceuticals whether in the early stages of drug
discovery or at the latter stages evaluating the toxicity of pharmaceutical
pollution.
4. Significance of pharmaceutical pollution

Pharmaceuticals are a huge part of modern health care. In fact, statistical
analysis of the global pharmaceutical industry shows that, in 2017, the
worldwide market was worth $934.8 billion USD and was projected to reach
$1170 billion USD by 2021,17 but in fact was greater than $1250 billion USD
by 2019.17 There are a few reasons for this significant growth. First and
foremost, technological advancements in industrialized countries have made
drug design and development easier and much less time-consuming. Aging
societies and advances in research have also led to a significant increase in
global pharmaceutical consumption. This may seem encouraging to some as it
is difficult to picture modern medicine and any advancements within modern
medicine without the use of pharmaceuticals.18 However, drug design,
development, and distribution are not without consequences. The occurrence
of both human and veterinary pharmaceuticals in the environment has been a
concern since the early 1990s. Since then, a myriad of studies have demon-
strated the potential dangers that the presence of pharmaceuticals in the
environment may have on the health of wildlife and humans.19
At this point, there is no question about the presence of pharmaceuticals in
the environment. Studies have demonstrated the occurrence of significant
concentrations of many pharmaceuticals in surface water, subsurface water,
groundwater, and wastewater.1,20 It is known that most often human phar-

maceuticals enter the environment via effluent from wastewater treatment
plants. In a 2007 study, samples of effluent from a wastewater treatment plant
in India were collected.21 Drugs such as Enrofloxacin, Aspirin, Citalopram,
and Ranitidine were just a few examples of those present in the samples. Not
only were these pharmaceuticals present, but they were present in “the highest
levels of pharmaceuticals reported in any effluent.” Concentrations ranged
from 90 to 31,000 parts per billion (ppb; mg/L). Another study monitored
samples of influent and effluent collected from wastewater treatment plants in
Greece. Using LC-MS analysis, researchers identified Furosemide, the beta-
blockers Atenolol and Metoprolol, analgesics including paracetamol (acet-
aminophen) and Nimesulide, salicylic acid, Trimethoprim, Ciprofloxacin,
Diclofenac, and caffeine.22 Other studies of wastewater treatment plants from
around the world have identified the presence of ibuprofen, methylparaben,
Naproxen, tetrahydrocannabinol (THC), Diazepam, tetracycline, erythro-
mycin, and Cefoxitin to name a few.23 As the drinking water supply for most
communities is obtained from surface water or groundwater, many have
questioned what substances are present in their own water supplies and what
effects low dose exposures have on health.
5. Use of the zebrafish model system to assess

pharmaceutical pollutant toxicity
Aiming to provide answers to toxicity questions related to pharmaceutical
pollution, toxicologists have focused research in the past decade on studying
how both terrestrial and aquatic organisms are influenced by pharmaceutical
pollution. This work has expanded to include animal models of human health
including rodent and zebrafish studies. As discussed above, it is now
commonly understood and accepted that the zebrafish exhibits similar physi-
ological responses to mammals in toxicity testing and that zebrafish are being
used in the drug discovery pipeline. Thus, it is logical to apply the zebrafish to
define the adverse human health effects of low-dose exposure to pharmaceu-
ticals and environmental pollutants due to the high genome and physiological
function conservation.2,3 Since human and veterinary pharmaceuticals are
designed to elicit specific physiological responses at low doses, the zebrafish is
an ideal model system for pharmaceutical pollution studies. While there is an
abundance of studies using zebrafish to address various aspects of drug
toxicity, in this section we specifically focus on example studies where
zebrafish were used to address questions surrounding pharmaceutical pollution
demonstrating feasibility (Table 1.1). These studies have included various
experimental endpoints in both developing and adult zebrafish, which are
further detailed in the next sections covering methods and approaches.
In multiple studies, Galus et al.24e26 investigated organ-specific and/or
developmental effects of exposure to pharmaceutical and personal care
TABLE 1.1 Summary of example studies defining pharmaceutical pollutant toxicity using the zebrafish model.
Pharmaceuticals Exposure parameters Results Implications References

n Acetaminophen Adult zebrafish (6 weeks n adult female oocyte atresia and altered Low concentration chronic 24
n Carbamazepine duration) or embryos ovarian histology with all compounds exposure to pharmaceuticals
n Gemfibrozil (1e72 hpf) n altered kidney tubule morphology in both results in negative impacts to
n Venlafaxine 0.5 or 10 ppb single sexes with all compounds multiple organ systems
exposure of each n altered liver histology in both sexes with including reproductive
pharmaceutical exposure to acetaminophen or Gemfibrozil system, kidney, and liver,
n decreased plasma 11-ketotestosterone in along with embryo
both sexes with exposure to Carbamazepine mortality.
n embryonic acetaminophen exposure
increased developmental abnormalities
n increased mortality in embryonic exposure
with all compounds at low concentration
(0.5 ppb)
n Acetaminophen Adult zebrafish (6 weeks n adult female oocyte atresia and altered Environmentally relevant 25
n Carbamazepine duration) or embryos (1 ovarian histology in all exposures concentrations of these
n Gemfibrozil e72 hpf) n altered kidney tubule morphology in both pharmaceuticals in mixture
n Venlafaxine Mixture of four sexes with all exposures results in multiple organ
pharmaceuticals at 0.5 or n developmental abnormalities in high system impacts including
10 ppb or 5% or 25% exposure concentration ovary in females and kidney
diluted wastewater effluent n increased embryo mortality in high in both sexes, along with
concentration of mixture mortality in embryos.
n Carbamazepine Adult zebrafish (6 weeks n decreased reproductive output in adult Parental chronic low-dose 26
n Gemfibrozil duration) then crossed to offspring from parents with pharmaceutical pharmaceutical exposure
produce F1 generation, treatment was sufficient to cause
which was reared in control n reciprocal crosses indicated more impacts to significant reproductive
water and then assessed as males with drug-specific effects on sperm effects in male offspring.
adults morphology (Carbamazepine: longer
10 ppb single exposure of midpieces; Gemfibrozil: shorter head
each pharmaceutical lengths and midpieces)
n no effects on organ pathology of liver,
kidney, or gonads
n Carbamazepine Developmental exposure (6 n no significant effects on mortality, hatching Single and mixture 27
n Dilitiazem e144 hpf) rate, or morphology treatments representing
n Fluoxetine Exposures included single n Carbamazepine resulted in a significant pharmaceutical pollution
n Gemfibrozil pharmaceutical at sub-acute increase in all three genes (cyp19a2, er-a, can use gene expression
n Metformin concentrations (0.001, 0.01, and gnrh3), but only at concentrations biomarkers to provide
0.1, or 1 ppb) or a mixture of greater than those found in the water location and mixture-
5 pharmaceuticals, along samples (>0.1 ppb) specific toxicity profiling.
with negative (embryo n Diltiazem and Fluoxetine increased
media), solvent (0.001% expression of cyp19a2 in all treatment
ethanol), and positive (17b- concentrations, while er-a and gnrh3
estradiol) controls expression alterations were only at higher
concentrations (>0.1 ppb)
Using the zebrafish model system to identify Chapter | 1

n Gemfibrozil only impacted cyp19a2
expression at 0.01 and 1 ppb
n Metformin caused significant expression
changes of cyp19a2 and gnrh3 at 1 ppb
and greater and of er-a at 10 ppb and greater
n mixture studies representing different
locations in Lake Michigan showed a
significant increase in the expression of all
three genes for five of the seven different
chemical mixtures tested
n Caffeine Larval exposure for 96 h (72 n decreased expression of cyp1a in all single No additive toxicity was 28
n Ibuprofen e168 hpf) pharmaceutical treatments, but increased observed, but differences
n Carbamazepine Exposures included single expression in high mixture treatment seen in between the
n Tamoxifen pharmaceuticals (Caffeine, n expression of vtg decreased in Tamoxifen individual and mixture
ibuprofen, or treatments treatments signifying need to
Carbamazepine at 0.05 or n EROD activity decreased in lower ibuprofen include mixture toxicity
5 mM; Tamoxifen at 0.003 or treatment and increased in the high mixture assessments.
0.3 mM) or a low or high treatment
mixture treatment with
negative (embryo media),
solvent (0.001% ethanol),
and positive (17a-
9
ethinylestradiol or
phenanthrene) controls
Continued
TABLE 1.1 Summary of example studies defining pharmaceutical pollutant toxicity using the zebrafish model.dcont’d
Pharmaceuticals Exposure parameters Results Implications References
n Bezafibrate Embryonic exposure (2e50 n no visible acute toxicity for any single or Delayed behavioral 29
n Caffeine hpf) mixture treatments alterations were observed
n Carbamazepine Single pharmaceutical n delayed hatch at 72 hpf for Triclosan (1 ppb) for several pharmaceuticals
n Clarithromycin exposure at 0.01, 0.1, 1, 10, and ibuprofen (100 ppb) and the high mixture
n Diclofenac or 100 ppb n decreased swimming speed at 100 ppb of treatment.
n Ibuprofen Low (1X), medium (10X), or Triclosan, Clarithromycin, and the high
n Sulfamethoxazole high (100X) mixture of all 8 mixture treatment at 118 hpf
n Triclosan pharmaceuticals n increased swimming speed at 100 ppb of
Carbamazepine at 118 hpf
n paracetamol Developmental exposure n nondose response morphological alterations Environmentally relevant 30
(acetaminophen) (<3e96 hpf) at 48, 72, and 96 hpf for paracetamol concentrations of these
n Ciprofloxacin Paracetamol at 5, 25, 125, n hyper- and/or hypoactivity observed at 144 pharmaceuticals resulted in
625, or 3125 ppb hpf for paracetamol and Ciprofloxacin behavioral and biomarker
Ciprofloxacin at 0.005, dependent on phase and concentration alterations, while
0.013, 0.031, 0.078, 0.195, n paracetamol caused increase in AChE, total morphological and DNA
or 0.488 ppb GPx, and GST activity; alterations in GSH methylation changes were
Treatment water refreshed at metabolism; and increased lipid observed only for
48 hpf peroxidation paracetamol.
n Ciprofloxacin resulted in increased AChE
activity and a decrease in catalase activity
and lipid peroxidation
n increased DNA methylation following
paracetamol exposure
AChE: acetylcholinesterase; cyp1a: cytochrome P450 1a; cyp19a2: cytochrome P450 19a2; er-a: estrogen receptor-alpha; EROD: ethoxyresorufin-O-deethylase; gnrh3:
gonadotropin releasing hormone; GPx: glutathione peroxidase; GST: glutathione transferase; hpf: hours postfertilization; ppb: parts per billion (mg/L); vtg: vitellogenin.
products in singular or mixture exposures of acetaminophen, Carbamazepine,

Gemfibrozil, or Venlafaxine in adult and embryonic zebrafish to mimic
environmentally relevant concentrations (Table 1.1). First, singular exposures
of the four pharmaceuticals were completed in adult zebrafish and resulted in
oocyte atresia and altered ovarian histology in females and altered kidney
histology in both sexes.24 Liver histology was altered by exposure to acet-
aminophen or Gemfibrozil, while exposure to Carbamazepine decreased
plasma 11-ketotestosterone in both sexes. No alterations were observed in
developmental abnormalities in offspring from parental exposure, but em-
bryonic exposure to acetaminophen caused developmental abnormalities and
increased mortality was observed for all four pharmaceuticals at 0.5 ppb. In a
second study by the same group,25 a mixture of the four pharmaceuticals or
diluted effluent from wastewater where these four pharmaceuticals were the
most prevalent was investigated again in zebrafish adults and embryos
(Table 1.1). Exposure to these solutions resulted in many of the same per-
turbations observed by the single pharmaceutical exposures in the previous
study24 including impacts to ovarian histology in females and altered kidney
proximal tubule morphology in both sexes. Direct exposure in embryos caused
developmental abnormalities, specifically spinal malformations and pericar-
dial edema with increased embryo mortality in the highest concentration of the
pharmaceutical mixture.25 Furthermore, in a third study, chronic parental
exposure to Carbamazepine or Gemfibrozil at 10 ppb resulted in a reduction in
the number of clutches in adult offspring (F1) with a drug-specific effect on
sperm morphology of adult male offspring26 (Table 1.1). This study showed
that a parental exposure was sufficient to result in reproductive effects in their
offspring. An additional study with the same exposure parameters (i.e.,
parental exposure for 6 weeks to 10 ppb Carbamazepine or Gemfibrozil)
further supported the multigenerational finding indicating paternal influence
on sexual differentiation and male reproductive effects in the F1 offspring.31
The male reproductive effects were reported to be limited to the F1 generation
with no transgenerational effects observed in subsequent F2, F3, or F4 gen-
erations with the 10 ppb Gemfibrozil exposure (i.e., 6 weeks in the F0 gen-
eration only).32
Developmental toxicity assessments including morphometrics are being
coupled to molecular endpoints to link gene expression alterations and
phenotypic perturbations. For example, estrogen-related transcript analysis
was applied to assess the developmental toxicity of five pharmaceuticals
(Carbamazepine, Diltiazem, Fluoxetine, Gemfibrozil, and Metformin) reported
to be the most common and highly concentrated in water samples collected
from various locations in Lake Michigan.27,33 Assessments were completed
for singular pharmaceutical exposures and in mixtures. No significant mor-
tality or morphological alterations were observed for any pharmaceutical or
pharmaceutical mixtures, but significant transcript expression alterations of
estrogen receptor-alpha (er-a), brain aromatase (cyp19a2), and gonadotropin
releasing hormone 3 (gnrh3) were observed.27 Specifically, Carbamazepine

exposure resulted in a significant increase in all three genes, but only at
concentrations greater than those found in the water samples (>0.1 ppb).
Diltiazem and Fluoxetine increased expression of cyp19a2 in all treatment
concentrations, while er-a and gnrh3 expression alterations were only at
higher concentrations (>0.1 ppb). Exposure to Gemfibrozil only impacted
expression of cyp19a2 at 0.01 and 1 ppb (highest concentration tested), while
Metformin exposure caused significant expression changes of cyp19a2 and
gnrh3 at 1 ppb and greater and of er-a at 10 ppb and greater. Mixture studies
representing seven different locations in Lake Michigan also showed a sig-
nificant increase in the expression of all three genes for five of the seven
different chemical mixtures tested. This study demonstrated that zebrafish
toxicity testing can be used to determine estrogen-related transcript alterations
of location and mixture-specific pharmaceutical pollution (Table 1.1). A
similar study assessed impacts to transcript expression of cytochrome P450 1a
(cyp1a) and vitellogenin (vtg) and ethoxyresorufin-O-deethylase (EROD) ac-
tivity following exposure to caffeine, ibuprofen, Carbamazepine, or Tamoxifen
as a single pharmaceutical or in a low or high mixture treatment through 96 h
postfertilization (hpf).28 Treatment concentrations in this study were anchored
to previous reports on environmental concentrations in different water bodies
for relevance. Expression of cyp1a was decreased in all individual treatments,
while an increase in expression occurred in the high mixture treatment.
Expression of vtg was only changed in the Tamoxifen treatments, while EROD
activity was decreased in the lower ibuprofen treatment and increased in the
high mixture treatment. While the study reports no additive toxicity, the
findings support the need to address mixture toxicity (Table 1.1). Further
studies have used transcriptomic approaches to appreciate global molecular
response from pharmaceutical pollution. Wu et al.34 exposed developing
zebrafish from 4 to 120 hpf to three antidepressants (Amitriptyline, Fluoxetine,
or Mianserin) and identified similar pathway alterations among the three
pharmaceuticals related to glycolysis and the insulin pathway, while another
study with exposure from 12 to 96 hpf to the anticonvulsant, Gabapentin,
reported alterations in genes associated with the antioxidant, immune, and
nervous systems.35 Overall, these studies are demonstrating utility of zebrafish
to link phenotypic and molecular alterations of environmental exposure to
pharmaceuticals including targeted and -omic based experimental methods.
Zebrafish studies defining pharmaceutical pollution toxicity are also
including various behavioral assays coupled to different aspects of develop-
mental toxicity. For example, the delayed toxicity of eight pharmaceuticals
(ibuprofen, Diclofenac, caffeine, Clarithromycin, Carbamazepine, Sulfameth-
oxazole, Triclosan, and Bezafibrate) and their mixtures (low, medium, and high)
observed delayed hatch for Triclosan (1 ppb) and ibuprofen (100 ppb) at 72 hpf
following exposure from 2 to 50 hpf29 (Table 1.1). Following pharmaceutical
exposure from 2 to 50 hpf, hypoactivity was observed at 100 ppb of Triclosan,
Clarithromycin, or the high mixture treatment at 118 hpf, while hyperactivity

was observed at 100 ppb Carbamazepine. Furthermore, studies have coupled
behavioral assays with biochemical and epigenetic effects of pharmaceutical
pollution. For example, the adverse exposure effects of the commonly used
pharmaceuticals paracetamol (acetaminophen) and Ciprofloxacin were
assessed.30 Concentrations were determined based on concentrations of these
pharmaceuticals in surface water and effluent in many developed countries such
as Spain, United Kingdom, and Portugal. The results of this study showed that
exposure to paracetamol produced a significant effect for all endpoints assessed,
while Ciprofloxacin only impacted behavior and biomarker endpoints
(Table 1.1). The authors concluded potential oxidative stress mechanisms of
toxicity, especially for paracetamol that were related to the observed epigenetic
modification in 5-methylcytidine. These studies are further expanding the
toolbox of methods for how the zebrafish can be used to define the health risks
and mechanisms of pharmaceutical pollution. These methods and approaches
are further detailed below.
6. Methods and approaches to assess pharmaceutical

pollutant toxicity using the zebrafish model system
6.1 Acute developmental toxicity assessments with the developing
zebrafish
Zebrafish are widely used to assess acute developmental toxicity owing to their
high fecundity, ex vivo shorter developmental periods, and transparent cho-
rions providing ease in evaluating the earliest developmental stages. Chem-
icals can be directly dissolved in fish water or in a solvent (e.g., DMSO) and
the developing zebrafish exposed via immersion in the solution. Insoluble
chemicals can also be directly injected into the developing zebrafish. Acute
toxicity studies identifying survival limits provide further understanding about
dose-dependent and/or concentration-dependent pharmaceutical toxicity and
are generally quantified as the lethal dose for which 50% of the organisms
survive (LD50) or the lethal concentration for which 50% of the organisms
survive (LC50) if tissue dose is not known.
Lethality quantification can be integrated with additional endpoints to
assess developmental toxicity including various teratogenic assessments and
provide ranking values or ranking ratios of drug toxicity. Several morpho-
logical assessments can be evaluated in the developing zebrafish, proving it as
a useful, efficient, and economic application for high-content phenotypic
screening for drug toxicity based on ex vivo embryonic development and small
size that permits use of multi-well plates (Fig. 1.3). Moreover, the zebrafish
model has several mutant and transgenic lines available with well-
characterized disease phenotypes and/or that integrate fluorescence (e.g.,
transgenic lines with eGFP) (Fig. 1.1) that are useful in chemical screening.
FIGURE 1.3 Phenotypic screening in multiwell plates permits high-content imaging in devel-
oping zebrafish. Embryos are attained from adult breeding pairs and arrayed in multiwell plates
immersed in solutions containing pharmaceutical pollutants of interest. Imaging software detects
and measures specific phenotypic outcomes to define developmental toxicity. Scoring systems can
be used to rank toxicity.
Overall, these techniques provide flexibility for researchers in experimental

designs implementing lower to higher-throughput methodologies. For
example, low-throughput methods can attain static image phenotypes to
determine teratogenic abnormalities observed from the microscopic images,
but as these images are often captured manually, there can be more subjectivity
based on observation and fewer samples can be collected at a time. This
concern can be partially eliminated by ensuring the observer is blind to any
chemical treatments. Improvement in throughput and subjectivity can be
attained through computational methods that yield higher volumes of
morphological data. High-content imaging (HCI) strategies can be employed
to rapidly and automatically capture images in high-resolution or low-
resolution forms to assess cellular, organ-level, or whole organism. In fact,
several studies are using HCI in high-throughput toxicity screens to screen
chemical libraries for toxicity.36,37 Morphological measures are also being
used to further understand disease progression and outcomes associated with
chemical exposures. For example, studies characterized the progression and
treatment strategies for melanoma and squamous cell carcinomas.38 Several
studies also developed morphological scoring systems to rank and compare
toxicity. These scoring systems provide information about tissue-specific
teratogenicity to rank chemicals from most severe adverse outcomes to the
least.39
6.2 High-throughput screenings (HTS) for developmental toxicity

assessments
Traditionally, zebrafish were applied in low- or medium-throughput assess-
ments for developmental toxicity (Fig. 1.4). Over the past decade, there has
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Regnum confractum regis feritate
subactum
Nuper defleui, lacrimas set abinde sub spe glorie
quieui; future letatur.
Regnum purgatum probitate ducis renouatum
Amodo ridebo, nec ab eius laude tacebo.
O res laudanda, o res sine fine notanda,
Ad laudem Cristi, qui nos de carcere tristi
R. tunc custodis, quasi sit regnantis Herodis,
10 Gracius eduxit et ad inclita regna reduxit!
Nouit enim mundus, Ricardus quando secundus
Iustos deleuit proceres, quos Qualiter ad
Anglia fleuit, modum talpe,
Ipse superbire sic spirat et alcius que semper
terram
ire,
effodiens eam
Quod dedignatur proprium continue
regnumque minatur: subuertit, rex
Amplius ex more solito latitante Ricardus, vt
furore suum regnum
Seuit, et oppressit populum cui tirannice
parcere nescit. disperdat,
assiduis
Sicut humum fodit euertens talpa ymaginacionibu
que rodit, s ad populi
Vnde caret requie, sic alter nocte destruccionem
dieque, omnes suas
Vt magis euertat regnum quod cautelas
demere certat, indesinenter
Sic scelus apponit et ad hoc sua coniectat.
20 robora ponit;
Vt princeps baratri furiens regit acta theatri.
Pondera prebebat, populum quibus ipse
premebat:
Vtpote salsarum furiosa caribdis aquarum
Gurgite feruoris bibit, euomit, omnibus horis,
Sic sibi collectum facinus sub pectore tectum
Rex vomit in gentem, ve, ve! sine lege manentem.
Per prius optentum semper sibi parliamentum
Per loca conseruat, in quo mala Nota qualiter
queque reseruat; rex subtili
Est vbi persona regis residente fraude
corona, concessum sibi
optinuit, quod
Corpore presenti stat ibi vis
vbicumque
30 parliamenti: sedere vellet
Sic, vbicumque sedet presencia cum certis
regia, ledet, personis sibi
Quod nullus sciuit sceleris que assignatis, per
facta subiuit. prius inceptum
Hoc factum regis fuit abhominacio continuare
posset
legis,
parliamentum.
Quo fremuit certe populus, set
nullus aperte:
Sic tamen vt staret et tempora continuaret,
Rex sibi papales bullas habuit speciales;
Si quis in extento prius aut post parliamento
Quid contradicit, in eum sentencia vicit.
Ad scelus implendum tunc rex habet omne
timendum,
40 Excepte Cristo, qui non fuit auctor in isto;
Quicquid enim dicit clerus, populus maledicit,
Inuocat et Cristi vindictam pectore tristi.
Inde set oblitus rex pestifer hos sibi ritus,
Quos prius elegit, maledicto fine peregit;
Consensu, tactu, visu que ferocior actu
In regnum seuit, qui post sua crimina fleuit:
Que n o n a u d i u i t a u r i s, nec cor mala sciuit,
Tristia coniectat, populum quo perdere spectat.
Carte scribuntur et in omni parte leguntur,
Hasque sigillari iubet omnibus et Nota de primis
50 venerari: cartis, quas
Perficit hoc clerus, si debeo dicere scriptas ex
regis
verus
compulsione
Nescio, set gentes sua sunt tam clerus
exempla sequentes; quam populus
Nescia plebs legis, dum sperat formidans
premia regis, sigillauit: tali
Vt dicebatur, ad regia iussa paratur. enim subtilitate
Vrbs, ager et villa cartis posuere rex varias regni
sigilla, sui patrias
Quo magis ad plenum conspergitur spoliando
destruxit.
omne venenum:
Fallitur ex illo quisquis, cum firma sigillo
Culpa recordetur, qua proditor omnis habetur.
Cum sic quisque status sit in hiis cartis viciatus,
60 Vt veniam portet sibi soluere quicquid oportet;
Tunc exactores baratro magis auidiores
Absoluunt gentes, pacem quasi sint redimentes.
Hec set cautela nichil est nisi ficta medela,
Nam magis insanus stat morbus cotidianus;
Rex populum pressit, et ab inde quiescere nescit,
Semper turbatur, semper sua regna minatur.
Post primas cartas alias statuit magis artas,
Set de scriptura patuit non vna Nota de
figura. secundis cartis
Has eciam villis iubet affirmare que blanche-
chartres
sigillis;706 vulgariter
Qualis finis erit quisquis sub nuncupantur.
70 murmure querit;
Et sic velata facie plebs illaqueata,
Quod facit ignorat, ita dum fortuna laborat.
Accidit interea, dum terra fuit Qualiter rex
pharisea, Ricardus omnis
Est noua lis mota, quam nouerat malicie plenus
strenuissimum
Anglia tota.
dominum
Nobilis Henricus, omnis probitatis Henricum, tunc
amicus, Derbeie
Hic tunc florebat super omnes Comitem
plusque valebat; Ducisque
Vt rosa flos florum, melior fuit ille Lancastrie
bonorum, filium et
heredem, sola
Custos Anglorum, per quem lux ex inuidia, vt
fulsit eorum, ipsum perderet,
Exemplar morum que probacior ille in exilium
proborum: proiecit.
80 Ad loca bellorum leo conterit arma luporum;
Eius cognomen venerabile percipit omen,
Quod numquam victum rutilat Lancastria dictum.
Hunc patre viuente de sorte superueniente
Rex delegauit et eum sine labe fugauit;
Rex etenim nouit, ad eum quod patria vouit,
Vnde timens sortem dolet eius habere cohortem:
Inuidus hanc causam gestat sub pectore clausam,
Donec disperdat iustum sine iureque perdat.
Hic tamen ex more solito pro regis honore
90 Semper promptus erat, aliter quo premia sperat;
Sic nichil offendit, quo rex sibi dampna rependit,
Set quia cunctorum rex oderat acta proborum.
Singula non scripsi, que dux bona contulit ipsi;
Si meritum detur, tunc dux mala nulla meretur:
Exilium tortum gremio de regis abortum707
Hoc pro finali mercede datur speciali.
Purus ad omne latus sic exulat inmaculatus,
Et quem decepit rex Anglus Qualiter nobilis
Francia cepit: Henricus
Stans ibi preclarus regno fuit antedictus in
partes Francie,
vndique carus,
vt ibi tempore
Quo sibi concreuit requies, set non exilii moraretur,
100 requieuit. animo constanti
Dum genus exquirit in quo sibi iura viriliter se
requirit, transtulit.
Quem deus absoluit patri mors
omnia soluit;
Sic, patre defuncto, de consilio sibi iuncto
Est tunc querendum, melius sibi quid sit agendum:
Et sic consultus velut heres Miles adultus,
Que sua cognoscit post patrem propria poscit.
Hos per rumores adeunt ambassiatores
Regem querentes, legem super hocque petentes;
Set qui cuncta vorat non audit quod pius orat,708
110 Exheredatum set eum iubet esse fugatum.
Et sic nec regem iustum iustam neque legem
Dux probus inuenit, dum vox sibi nuncia venit.
Tunc confiscatus rapitur sine iure ducatus,
Quo se confortat dux commoda nulla reportat;
Pulli coruorum, pascit quos mater eorum,
Non ita proclamant, quin plus sibi castra reclamant
Regis fautores, terras que ducatus honores:
Rex bona dispergit, qui non sine crimine pergit,
Distribuens sortes, ditescat vt inde cohortes.
120 Quod sic decreuit rex fama perambula creuit,
Per mundum totum scelus hoc erit amodo notum.
O quam plura sinit deus, et cum Nota qualiter
tempora finit, post obitum
Omnia tunc certe que sunt patris sui ducis
Lancastrie
demonstrat aperte!
nobilissimus
Dux inspiratus tandem, quasi sit filius suus
renouatus, Comes
Singula compensat perfecto antedictus, tunc
cordeque pensat: de iure dux, vt
Tortorem regem tortam ipse
creuisseque legem hereditatem
suam
Cernit, et errores in vtroque statu vendicaret, de
grauiores: partibus
Signans se Cristo quesiuit opem Francie prouiso
super isto, sapienter
Qui, bene dum sperat, iubet vt sua itinere Calisias
propria querat. adiit, vbi cum
Ex subito more, saluo sibi semper domino Thoma
Cantuariensi
130 honore, Archiepiscopo,
Partes subtiles Francorum dux necnon Thoma
quasi miles 709 filio et herede
Cum paucis transit, nec ibi Ricardi Comitis
tardando remansit: Arundellie, vt
prefertur,
Calisias iuit, vbi propria regna defuncti, vt in
petiuit; Angliam
Cum modica classe sic transfretaret,
magnanimum remeasse Cristo se
commendans
Constat, et in naui dux ducitur inde nauem
suaui. ascendit.
Primas Anglorum, tunc exul fraude malorum,
Thomas deuote stat ibi, comitante nepote:
Hos dux regalis, veluti gallina sub alis,
Secum votiua saluos duxit comitiua.
140 Dux, Comes, Antistes, pariter solamina tristes
Querunt sperantes, vbi venti sunt Qualiter nobilis
agitantes: Henricus, tunc
Vela petunt portum quem sors Dux Lancastrie,
per mare
prope contulit ortum;
nauigando
Vt dux concepit, Aquilonica littora portum querens
cepit. tandem prope
Tunc magis audaci vultu cum plebe Grymmesby,
sequaci Cristo
Exultans dicit, quod in hoc quasi mediante,
prelia vicit. littora pacifica
sortitus est.
Ex animo forti dederat bona corda
cohorti,710
Quod bene sperarent, quicquid sibi fata pararent.
Sic congaudentes sub spe que nichil metuentes,
Quo melius querunt, naues simul applicuerunt:
150 Dux prius egressus disponit humo sibi gressus,
Primitus exorat que deum genuflexus adorat
Votis sincere mentis, quod possit habere
Victoris palmas, extendit ad ethera palmas;
Vtque scelus guerre superet, dedit oscula terre,
Pluraque deuota dux fecit ibi pia vota.
De prece surrexit, surgendoque se cruce texit,
Et tunc quam letas incepit adire Qualiter ad
dietas: seruicium
Patria cum sciret quod saluus dux nobilis ducis
quasi vniuersa
reueniret,
terra gratanter
Totus ei mundus occurrit vbique se optulit.
iocundus.
160 Tunc rex Ricardus lepus est et non leopardus;
Quem timor astrinxit, alibi sua robora finxit:
Hic ducis aduentum presciuit ab Qualiter rex
ore scientum, Ricardus
Quo celer exiuit et Hibernica regna tempore quo
nobilis dux
petiuit.
Henricus
Sepe silens plangit, quem tunc applicuit, in
vecordia tangit, partibus
Ex quo singultus plures rex cepit Hibernie
adultus. invtiles dies ad
Sic redit absente dux noster rege sui
timente, confusionem
infortunate
Nec quid presumit, sua propria consumpsit.
dumque resumit.
Dux probus audaci vultu cum plebe sequaci
Regnum scrutatur, si proditor Qualiter apud
inueniatur; Bristolliam capti
Sic tres exosos magis omnibus et decapitati
fuerunt tres
170 ambiciosos
precipue regis
Regni tortores inuenerat ipse fautores, qui in
priores: mortis articulo
Ense repercussi periunt Scrop, dicti regis
Grene que Bussy;711 condiciones
Hii quasi regales fuerant cum rege multipliciter
accusarunt.
sodales.
Scrop Comes et Miles, eius Bristollia viles
Actus declarat, quo mors sua fata pararat;
Greneque sorte pari statuit dux decapitari,
Bussy conuictus similes quoque sustinet ictus:
Vnanimes mente pariter mors vna repente
Hos tres prostrauit, gladius quos fine vorauit,
180 Sicut et egerunt aliis, sic hii ceciderunt;
Quo dux laudatur regnumque per omne iocatur.
Sunt tamen Henrici quamplures tunc inimici,
Tales qui querunt obsistere, nec potuerunt:
Sepius effantur et eum post terga minantur,
Set non audebant, faciem cum respiciebant.
Tempore sic stante stat rex vbi stabat ab ante,
Donec commota tremit eius concio tota:
Sic magis ignari sceleres fiunt Qualiter
quasi rari,712 Ricardus rex de
partibus
Omnes sorte pari dubitant qua Hibernie
parte iuuari. rediens Wallie
Tunc fortuna rotam diuertit ab inde littora cepit.
190 remotam,
Cecaque permansit, dum rex super equora transit.
Quos laqueos fecit in eos, sua culpa reiecit,
Qui laqueatus erit, patrie dum littora querit.
Hoc non obstante, vento tamen exagitante,
Portum fatalem sors reddit ei specialem;
Inque suas claues cepit fera Wallia naues,
Quas cito dissoluit, regis cum facta reuoluit.713
Rex mittens sortes mandauit habere cohortes,
Set nichil inuenit, vbi gracia nulla reuenit.
Hoc ita cumque vident, quidam sub murmure
200 rident,
Et quidam flentes fuerant de corde dolentes:
Prospera que nescit, tunc regia pompa recessit,
Quisque viam vertit subito, nec ad arma reuertit.
Tunc rex, vt dicit, sua fata dolens maledicit,
Nec timet hinc Cristum, mundum nec abhorruit
istum,
Non est contritus, nec vult dimittere ritus,
Vt prius errauit, sic semper continuauit;
Sic furit ipse malis semper sine lege feralis,
Principio qualis steterat, stat fineque talis.
210 Cautus vt inuadit agnos, quos ledere vadit,
Vulpis, in occulto sic rex a tempore multo,
Pectore subtili iuuenis sub fraude senili,
Omne scelus poscit, regnum quo perdere possit:
Tunc super omne tamen conspirat habere
leuamen,
Vnde ducis sortem fallat fugiatque cohortem.
Hinc perscrutatur dolus et fraus continuatur,
Si quid prodesse poterit cogente necesse:
Est ibi vis nulla, velut os perit absque medulla,
Rex qui posse caret pro tunc sine viribus aret:714
220 Per loca, per castra fugit, et si tunc super astra
Scandere sciuisset, transcendere tunc voluisset.
Sic tumor elatus nuper tam magnificatus
Est timor effectus, latitans quasi talpa reiectus.
Quem non preseruat Cristus, se non homo seruat,
Et quamuis tarde de te loquor ista, Ricarde.
Peruigil a sompnis quod dicitur Qualiter rex
audiat omnis, Ricardus cum
Et quod dicetur regnis suis fautoribus
nobili duci
exemplificetur.
Henrico eisdem
Est rota fortune quodamodo regula in Wallia
lune, occurrenti se
Que prius albescit de nocte que reddiderunt.
post tenebrescit;
230 Sic de quo scripsi Ricardo contigit ipsi:
Dum stetit ad plenum, steterat sibi tempus
amenum,
Set cum decrescit, lucem tunc nebula nescit;
Cum se peruertit, sua spera retrograda vertit.
Nil sibi de bellis, quia stat sibi terra rebellis,
Nec mare succurrit, fugiens quia nauta recurrit;
Spes sibi collata non est, set et vndique fata
Ipsum torquebant, et ad ima repente ruebant:
Non ita secreta loca sunt neque castra quieta,
Que tunc secura fuerant pro sorte futura.715
240 Finis adest actus, capitur rex fitque subactus,
Et reliqui tales, sibi sunt qui collaterales,
Caute ducuntur capti, qui fata sequntur:
Sic rex preuentus ducis est virtute retentus.
Augusti mensis dedit hoc, quo Londoniensis
Vrbs congaudebat, que ducem cum Qualiter nobilis
laude canebat. Henricus vna
Sicut arena maris, occursus adest cum rege
Ricardo et aliis
popularis,
Londonias
Tanti victoris benedicens gesta veniunt, vbi
vigoris.
In Turrim transit R., sub custode dictus rex in
remansit; turrim positus
Sic caput Anglorum minimus iacet per aliquod
tempus sub
ipse minorum. custodia
Vt sit opus planum, nichil et de remansit.
250 pondere vanum,
Apponendo manum dux purgat ad Qualiter nobilis
horrea granum; dux Henricus
Iustos laudauit, iniustos vituperauit, proceres
Hos confirmauit, hos deprimit, hos quoscumque
per regem
releuauit. Ricardum in
Regni primatem, crudelem per exilium
feritatem delegatos ad
Quem rex explantat, dux ex pietate propria
replantat: mitissime
Humfredum natum patre defuncto reuocauit.
spoliatum,
Quem rex transduxit, hunc dux probitate reduxit.
Nil tibi desperes, Arundell profugus heres,716
Prospera namque ducis fatis tua fata reducis.
260 Warwici Comitem, cuius sine crimine litem
Dux pius agnouit, saluum de carcere mouit:
Cobham sorte pari dux fecit et hunc reuocari;
Exilio demptus iustus redit ille redemptus
Nec prece nec dono, Cristo mediante patrono.
Tanta tulit gratis primordia dux bonitatis:
Vt bona tam grata super hoc sint continuata,
Cristus adhuc mentem ducis efficit esse
manentem.
Londoniis festo Michaelis tunc Qualiter
manifesto, assignatum fuit
Stent vt ibi tuta, sunt parliamenta parliamentum
tenendum apud
statuta:
Westmonasteri
Quilibet attendit que sors sibi fata um ad festum
270 rependit, Sancti
Semper et in gente fit murmur rege Michaelis tunc
regente. proximi. Et
Interea transit moriens nec in interim
orbe remansit Humfredus
filius et heres
Humfredus dictus, r e d i t i l l e
ducis Glouernie
d e o b e n e d i c t u s: vna cum matre
Defuncto nato, cito post de fine sua corporis
beato infirmitate
Mater transiuit, nati dum funera mortui sunt.
sciuit:
Primo decessit Cignus, dolor vnde repressit
Matrem cum pullo, sibi mors nec parcit in vllo.
Est apud antiquos dictum, ‘Defunctus amicos
Vix habet,’ a tergo caueat sibi quilibet ergo:
280 Quisque suum pectus tangat viuens homo rectus,
Nec sic gaudebit, quia singula vana videbit.
Scribere iam restat, que mundus adhuc
manifestat,
Vt sit opus tale cunctis speculum generale.
Tunc prius incepta sunt parliamenta recepta,
De quibus abstractus Ricardi Qualiter primo
desinit actus. die parliamenti
Ecce dies Martis, nec adest rex Ricardus
personaliter
presentia partis,
non comparuit,
Non sedet in sede, quem culpa set alibi
repellit ab ede; existens titulo
Denegat in scanno loca tunc corone sue sub
fortuna tiranno, forma magis
A visu gentis quem terruit accio auctentica
mentis. penitus
renunciauit;
R. non comparet, alibi set super quo
290 dummodo staret, nobilis
Causas assignat, quibus H. sua Henricus,
sceptra resignat: 717 vniuerso
Substituit aliquos proceres tunc populo in eius
laudem
iuris amicos,
conclamante vt
Ad quos confessus proprio fuit ore rex efficiatur,
repressus. electus est.
Hiis circumspectis aliisque sub ordine lectis,
R., qui deliquit, hunc curia tota reliquit;
Hunc deponebant, plenum quem labe sciebant,
Nec quis eum purgat, iterum ne forte resurgat:
Tunc decus Anglorum, set et optimus ille bonorum,
H. fuit electus regno, magis est quia rectus.
300 Sola dies tentum tulit istud parliamentum,
Nec magis expressit pro tunc, set ab inde recessit:
H. tamen extenti noua tempora parliamenti
Proxima decreuit, quo regni gloria creuit.
Quando coronatus foret et de fine leuatus,
Tunc processus erit super hoc quod curia querit;
Interea gentes viuunt sub spe recolentes,
Quod nouus errores rex conteret anteriores.
Sexta dies stabat Octobris, Qualiter
quando parabat parliamentum
Rex nouus optata sua parliamenta continuatur718
nouata: vsque post
310 Curia verbalis fuit et non iudicialis, coronacionem.
Ad tempus restat nichil et de pondere prestat:
Dicitur expletum quod nil valet esse quietum,
Donec persona regis sit operta corona;
Sicque coronari, quem Cristus vult venerari,
Corditer exultat plebs omnis et inde resultat.
Qui res disponit et eisdem Qualiter in die
tempora ponit, solempni
Ille diem fixit, Henricum quo nobilis
Henricus in
benedixit:
Solium regie
Predestinauit deus illum quem maiestatis719
titulauit, sublimatus cum
Vt rex regnaret sua regnaque omni gaudio
iustificaret. coronatur.
Quem deus elegit, regali laude
320 peregit,
Vnde coronatur in honoreque Nota, qualiter
magnificatur: iura corone
serenissimo
Tempore felici poterunt sollempnia iam regi nostro
dici, Henrico quarto
tribus modis
Que tam sacratis horis patuere
accrescunt:
beatis;720 Primo
Edwardi festa Confessoris Successione:
manifesta Secundo
Henrici festum Regis testantur eleccione:
honestum. Tercio
conquestu sine
Plebs canit in mente que resultat in sanguinis
ore loquente, effusione.
Quisque colit Christum, quia regem
suscitat istum;
Vix homo pensare poterit seu dinumerare,
Que tunc fulserunt sollempnia quanta fuerunt:
330 Omnis terra deum laudat que canit iubileum,
Henricum iustum que pium que ferum que
robustum.
Vnde coronatur trino de iure probatur,
Regnum conquestat, que per hoc sibi ius
manifestat;
Regno succedit heres, nec ab inde recedit;
Insuper eligitur a plebe que sic stabilitur:
Vt sit compactum, iuris nil defuit actum;
Singula respondent Henrici iuraque spondent.
Fama volans creuit, que climata Qualiter
cuncta repleuit, parliamentum
Quo laus vexilli super omnes adhuc fuit
continuatum.
prefuit illi:
340 Sic regnat magnus reprobis leo, mitibus agnus,721
Hostes antiquos qui terret et augit amicos.
Luna diem donat, qua Regem terra coronat,
Marsque sequens terre dat parliamenta referre:
Rex sedet et cuncti proceres resident sibi iuncti,
Stant et presentes communes plus sapientes;
Tempus erat tale communeque iudiciale.
Quod bene prouisum nichil est a iure
res c i sum;722
Est quia protectus, letatur sic homo rectus,
Et metuunt reliqui sua dampna dolenter iniqui.
Set quia plus dignum prius est Qualiter
350 recitare benignum, Henricus, Regis
Que sunt maiora scribens recitabo tunc Henrici
primogenitus,
priora:
statum que
Henrici natus Henricus, honore nomen
beatus, Principis de
Est confirmatus heres Princepsque consensu
vocatus: omnium
Sic pars abscisa, summo de iudice gloriose
visa, adeptus est.
Arboris est vncta veteri stipitique reiuncta.
Istud fatatum fuit a sanctisque relatum,
Quod tunc compleuit deus, ex quo terra quieuit:
Hoc facto leta stupet Anglia laude repleta,
Cordeque letatur, quia stirps de stirpe leuatur.
Tunc de consensu Regis procerum quoque
360 sensu,
Plebe reclamante, stant Qualiter ea que
parliamenta per ante; nuper in
Sic procedebant super hiis que parliamento
tempore Ricardi
gesta videbant
per ducem
Ad commune bonum, recolentes Glouernie et
gesta baronum. socios suos
Que prius Vrsus, Equs et Olor, qui gesta fuerunt,
dicitur equs, presens
Nuper fecerunt, firmissima parliamentum
constituerunt; confirmauit; et
ea que
Et que pomposa peruersaque Ricardus in
fraude dolosa vltimo suo
Ricardus fecit, hec curia tota parliamento
reiecit. constituit,
Et tunc tractatum fuit illud opus presens eciam
sceleratum, parliamentum
Quo dudum Cignus periit sine i u r e penitus
cassauit.
benignus;723
370 Iusticie vere vindictam clamat habere
Omnis ob hoc funus populus, quasi vir foret vnus:
Sic communis amor popularis et vndique clamor
Extitit acceptus a Rege que lege receptus.
Infortunatus Ricardus, plus sceleratus,
Omnibus ingratus, fuit vndique tunc Qualiter
maculatus; Ricardo suis ex
Sic quasi dampnatus abiit pre labe demeritis
iudicialiter
reatus,
condempnato,
Quo stetit elatus sub carcere ceteri qui cum
magnificatus. eo accusati
Eius fautores, qui sunt de sorte erant,
priores, tantummodo ex
Tunc accusati sunt ad responsa mera regis
vocati: pietate quieti
permanserunt.
Hi responsales submittunt se
380 speciales
Iudicio Regis, per quem silet vlcio legis.
Regia nam pietas sic temperat vndique metas,
Quod nil mortale datur illis iudiciale;
Est tamen ablatum, quod eis fuit ante beatum,
Vocibus Anglorum venerabile nomen eorum;
Corpora stant tuta, cecidit set fama minuta,
Dux redit in Comitem, quatit et sic curia litem,
Labitur exosus Bagot, quem rex pietosus
Erigit, et mite prolongat tempora vite.
390 Sic pius Henricus, inimico non inimicus,
Gracius, vt debet, pro dampno commoda prebet;
Ipse pium frenum laxat, quia tempus amenum
Appetit, et Cristo placuisse putauit in isto.
Non tamen in gente placet hoc, set in ore loquente
Publica vox dicit, leges quod mammona vicit;
Iusticiam queri plebs vult, rex vult misereri,
Et sic fortuna pro tempore non fuit vna:
Rex excusatur, nam dicunt quod variatur
Consilio tali, quo res latet in speciali.
Quatuor auctores sceleris, Iuda nequiores,

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Contemporary Chemical Approaches

for Green and Sustainable Drugs 1st

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1. Using the zebrafish model system to identify the

2. Analysis of pharmaceuticals in the environment

3. Analytical methods for trace level analysis of water samples 32

3. Leaking of antibiotics in the aquatic environment

4. Advances in drug development with the

5. Virtual screening techniques in pharmaceutical

6. In silico modeling of environmental toxicity

7. Sustainable separations in pharmaceutical

8. Green synthetic methods in drug discovery and

9. Characterizing the environmentally benign nature of

10. Green chemistry approaches to drugs that treat

11. Dynamic effects of organic molecules for drug

3. Molecular drug delivery by organized self-assemblies 337

12. Antibody-drug conjugates for targeted delivery

13. Toward the green synthesis of peptides and

2.4 Development of coupling agents and coupling

14. The multitarget approach as a green tool in

15. Directed evolution: a new powerful tool in drug

16. Conventional and advanced treatment methods

Anderson Alles de Jesus, Coordination of the Interdisciplinary Bachelor of Science

Levente Kárpáti, Department of Organic Chemistry, Faculty of Pharmacy, Semmel-

Using the zebrafish model

Contemporary Chemical Approaches for Green and Sustainable Drugs

2. The zebrafish model system

3. Use of the zebrafish model system in drug discovery

Although current methods work in preventing the toxic compounds from

FIGURE 1.2 The generalized

development was accomplished by expanding the use of animal models

4. Significance of pharmaceutical pollution

groundwater, and wastewater.1,20 It is known that most often human phar-

5. Use of the zebrafish model system to assess

Pharmaceuticals Exposure parameters Results Implications References

8 Contemporary Chemical Approaches for Green and Sustainable Drugs

Using the zebrafish model system to identify Chapter | 1

products in singular or mixture exposures of acetaminophen, Carbamazepine,

releasing hormone 3 (gnrh3) were observed.27 Specifically, Carbamazepine

Clarithromycin, or the high mixture treatment at 118 hpf, while hyperactivity

6. Methods and approaches to assess pharmaceutical

Overall, these techniques provide flexibility for researchers in experimental

6.2 High-throughput screenings (HTS) for developmental toxicity

Hic in tercia parte Cronice finaliter scribit

Tristia post leta, post tristia Hic in tercia

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