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4.5 Toxicology Studies 75
must be conducted in two mammalian species with dierent drug levels admin-
istered via the proposed therapeutic route and intravenously, if dierent. Animals
are observed for any evidence of toxic eects or changes in vital signs. All animals
are sacriced and subjected to detailed post-mortem examination. Observations on
some animals that displayed visible non-lethal eects may be continued past 14 days
to see if the eects are reversible. These studies serve to identify organs that are sub-
ject to toxic eects and provide a basis for the selection of dose levels for the repeated
dose studies that will follow.
Repeat dose studies are conducted to evaluate the long-term eects of the drug. At
least two species should be tested, one of which should be a non-rodent. The choice
should include a species that produces a response close to that anticipated in man.
Three dose levels should be chosen, representing high, intermediate and low doses.
The low level should correspond to the therapeutic range where no toxic eects are
anticipated, while the high should correspond to the range where mild toxic eects
will manifest without encountering signicant fatality. The drug will normally be
given daily by the proposed route for administration in humans. The duration of the
studies will depend on the proposed duration of the clinical trials in humans and
the specic requirements of dierent regulatory authorities. A maximum duration
of six months for rodents and nine months for non-rodents has been agreed upon
through the ICH process. General monitoring of parameters such as body weight,
clinical chemistry, haematology, and ophthalmology is carried out over the course
of the trial, and a detailed post-mortem examination of tissue and organ morphology
is conducted at the end. All results are referenced against a control group of animals.
Safety pharmacology studies may also be conducted in conjunction with the tox-
icity studies. These focus on identifying secondary pharmaceutical side eects that
may occur when the drug is administered in the therapeutic range. Emphasis is put
on identifying eects on vital systems, particularly the centralnervous, cardiovascu-
lar, and respiratory systems.
4.5.2 Genotoxicity Studies
Genotoxicity studies are required to identify compounds that can induce genetic
damage ranging from single point gene mutations to gross alterations of chromo-
somal structure. Such eects are taken as indicative of the potential to cause cancer
or heritable defects in humans. A standard battery of three types of tests is recom-
mended:
● A bacterial reverse mutation test,
● An invitro test with mammalian cells (chromosomal damage or mouse lymphoma
tk assay), and
● An in vivo test in rodents (chromosomal damage in haematopoietic cells).
The principle of the bacterial reverse mutation test is explained in Figure 4.7. The
in vitro tests maybe conducted with and withoutmetabolic activation with an extract
of liver enzymes – as noted previously drug metabolism occurs primarily in the liver
and can result in metabolites that are more hazardous than the parent drug. The
76 4 Non-clinical Studies
Bacterial reverse mutation tests, also known as Ames tests are based on using bacteria
that carry a mutation that makes them dependent on a particular ingredient (usually an
amino acid) to grow. The bacteria are inoculated onto culture plates that are decient in
the essential ingredient and exposed to dierent levels of the test substance. If the sub-
stance is mutagenic, it causes the bacteria to reverse mutate and lose their dependence
on the ingredient, enabling signicant bacterial growth. If the substance is not mutagenic,
minimal growth will be observed.
Figure 4.7 Explanation of bacterial reverse mutation tests (Ames tests).
in vitro testing will be performed before considering in vivo tests, as they are much
faster and cheaper to perform. The extent of testing may also be inuenced by con-
sideration of the structure of the substance and its similarity to known mutagenic
compounds, or the degree to which it is absorbed by the body.
4.5.3 Carcinogenicity Studies
Although positive results in genotoxic tests may be taken as indicative of human
carcinogenic potential, additional carcinogen testing may be required to identify
other agents that can induce cancer through non-genotoxic mechanisms. The
requirement for carcinogenicity studies is largely dependent on the proposed
duration of treatment in humans. Trials should be performed where a minimum
six-month treatment is anticipated. A long-term study of up to two years in a single
species, preferably rats, is now considered acceptable, with supplementary studies
in other species, where necessary. The drug should be administered at three levels
by the route proposed for humans. The high dose level should be set so as to have
relevance in humans. For drugs that display signicant toxic eects, it may be
related to the maximally tolerated dose in the toxicity tests, e.g. the dose causing
less than 10% deviation in body weight versus controls. If there is little evidence of
toxicity it may be more appropriate to base the level on a multiple (usually 25-fold)
of the maximum therapeutic dosage recommended in humans. Carcinogenicity
tests will usually not commence until preliminary clinical data is available to help
with the study design. Marketing authorisation may be granted before the studies
are completed. The requirement for such studies may be inuenced by knowledge
of how the drug is metabolised, and whether any of the metabolites are similar in
structure to known carcinogens. Carcinogenicity tests are not normally required for
protein-based drugs.
4.5.4 Reproductive Toxicology Studies
Reproductive tests are necessary to reveal any eects on mammalian reproduction.
All stages ranging from pre-mating through conception, pregnancy and birth, to
growth of ospring are studied. Studies are conducted over a number of generations,
preferably in rats, with dierent dose levels administered by the proposed route in
human. Considerable numbers of animals are required, as animals will have to be
autopsied at dierent stagesin the reproductive/developmental process. Assessment
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4.7 Quality by Design (QbD) 77
of the data identies any drugs that may result in impaired male or female fertil-
ity, developmental abnormalities in the foetus (teratogens), eects on lactation, or
growth and maturation of ospring.
4.6 Chemistry, Manufacturing and Control Development
(CMC)
In addition to the safety studies, considerable work to establish the quality of drug
substance and product must be undertaken in the preclinical phase. This will usu-
ally commence with drug discovery, where initial characterisation of the substance
occurs. This characterisationshould be extendedto gain as complete an understand-
ing as possible of structure, stereochemistry, impurities, and chemical and physical
characteristics and properties of the drug substance.
Initially, the drug will have been produced at laboratory scale and may have been
extracted from a natural source. A vital element in the development process is to be
able to scale up through pilot-scale to commercial production. At a minimum, this
will require optimisation of the manufacturing steps to yield a reproducible robust
process. In many cases, this may involve developing a synthetic chemistry pathway
to produce a substance that was initially extracted from a naturally occurring source
or genetically engineering a biopharmaceutical into a suitable producer organism.
Analytical methods and specications must be established and validated so as
to dene and control the quality and purity of raw materials, intermediates and
nished products. For many standard chemical raw materials, development of spec-
ications will not be necessary as they are already published in US and European
pharmacopoeia, e.g. standards for water, organic solvents, and various excipients.
The ultimate objective of these activities is to be able to manufacture the drugs
required for clinical trials in accordance with good manufacturing practice (GMP).
4.7 Quality by Design (QbD)
Quality by Design (QbD) is a concept in existence for several decades. As its name
suggests the approach entails systematic, knowledge-based predesign of all aspects
of a product or processto ensure that the product/process will meetpre-dened qual-
ity characteristics every time.
Initially championed by the FDA, QbD principles are now routinely applied
internationally during the discovery, development and ongoing manufacture of
medicines. In the context of drug development, QbD principles focus upon, for
example, predening the product’s essential quality attributes or characteristics
(e.g. its exact molecular structure, structure–function relationship, and purity
required) and designing a production process capable of ensuring the consistent
manufacture of nished product meeting those essential attributes. From a regula-
tory perspective, QbD principles are incorporated into a wide variety of regulatory
guidelines, most notably ICH quality guidelines Q8–Q11 (see Figure 4.3).
78 4 Non-clinical Studies
4.8 Quality of Biotech Products
Specic additional tests are required to assure the quality and safety of biophar-
maceutical products derived from human or animal cell lines. There is always the
possibility that harmful viruses such as herpes virus may be present in the original
cell line or could be introduced as adventitious agents in culture media during the
growth of such cells. Thus, appropriate assaysmust be applied to detect such viruses
and demonstrate that there is eective elimination or inactivation of any infective
agents during the subsequent purication and processing steps. Considerable
studies are also required to assess the stability of expression of products obtained
from genetically modied organisms. Potential variation could arise because of
susceptibility to mutation of the primary DNA sequence, or alteration in the level
of post-translational modication experienced under dierent culture conditions,
particularly as a process is scaled-up. Specic guidance is provided in ICH Q5
guidelines.
4.8.1 Stability Studies
Data to demonstrate the stability characteristics of both the drug substance and
the drug product must be collected. Studies using three dierent batches of both
substance and product in their respective containers/packaging must be conducted.
Real-time data should be collected under conditions of temperature and relative
humidity in line with the recommended storage. Conditions in dierent world
climatic zones must be taken into consideration for cases where normal environ-
mental storage conditions will be used. Standard conditions to simulate normal
storage are temperatures of 25 or 30
∘
C and relative humidity of 60% or 65%. A
minimum of 12 months real-time data are required for marketing authorisation
application. Real-time data should be supported by accelerated and intermediate
stress testing at higher temperature/relative humidity. Photostability should be
veried using a single batch.
4.9 Good Laboratory Practice (GLP)
All nonclinical studies used to demonstrate the safety of the drug must be con-
ducted in accordance with the principles of good laboratory practice (GLP). GLP
is a quality system concerned with the organisational process and the conditions,
under which nonclinical health and environmental safety studies are planned,
performed, monitored, recorded, archived, and reported. The principles of GLP
were developed under the auspices of the Organisation for Economic Coopera-
tion and Development (OECD) so that national regulators could mutually recognize
and have condence in safety studies that are conducted outside their jurisdiction.
The GLP requirements are set out in EU legislation under Directive 2004/10/EC
and in US regulations under 21 CFR Part 58. The main headings of both regulations
are outlined in Table 4.1. Although the headings are slightly dierent, the topics
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4.9 Good Laboratory Practice (GLP) 79
Table 4.1 Comparison of the content of EU and US GLP regulations.
EU Directive 2004/10/EC US 21 CFR Part 58
SECTION I
INTRODUCTION
Preface
1. Scope
2. Denitions of terms
SECTION II
GOOD LABORATORY PRACTICE
PRINCIPLES
1. Test facility organisation and
personnel
1.1 Test facility management’s responsibilities
1.2 Study director’s responsibilities
1.3 Principal investigator’s responsibilities
1.4 Study personnel’s responsibilities
2. Quality assurance programme
2.1 General
2.2 Responsibilities of the quality assurance
personnel
3. Facilities
3.1 General
3.2 Test system facilities
3.3 Facilities for handling test and reference
items
3.4 Archive facilities
3.5 Waste disposal
4. Apparatus, material, and reagents
5. Test systems
5.1 Physical/chemical
5.2 Biological
6. Test and reference items
6.1 Receipt, handling, sampling, and storage
6.2 Characterisation
7. Standard operating procedures
8. Performance of the study
8.1 Study plan
8.2 Content of the study plan
8.3 Conduct of the study
9. Reporting of study results
9.1 General
9.2 Content of the nal report
10. Storage and retention of records and
materials
Subpart A—General Provisions
Sec. 58.1 Scope
Sec. 58.3 Denitions
Sec. 58.10 Applicability to studies performed
under grants and contracts
Sec. 58.15 Inspection of a testing facility
Subpart B—Organization and Personnel
Sec. 58.29 Personnel
Sec. 58.31 Testing facility management
Sec. 58.33 Study director
Sec. 58.35 Quality assurance unit
Subpart C—Facilities
Sec. 58.41 General
Sec. 58.43 Animal care facilities
Sec. 58.45 Animal supply facilities
Sec. 58.47 Facilities for handling test and con-
trol articles
Sec. 58.49 Laboratory operation areas. Sec.
58.51 Specimen and data storage facilities
Subpart D—Equipment
Sec. 58.61 Equipment design
Sec. 58.63 Maintenance and calibration of
equipment
Subpart E—Testing Facilities Operation
Sec. 58.81 Standard operating procedures
Sec. 58.83 Reagents and solutions
Sec. 58.90 Animal care
Subpart F—Test and Control Articles
Sec. 58.105 Test and control article character-
ization
Sec. 58.107 Test and control article handling
Sec. 58.113 Mixtures of articles with carriers
Subpart G—Protocol for and Conduct of
a Nonclinical Laboratory Study
Sec. 58.120 Protocol
Sec. 58.130 Conduct of a nonclinical labora-
tory study
Subpart J—Records and Reports
Sec. 58.185 Reporting of nonclinical labora-
tory study results
Sec. 58.190 Storage and retrieval of records
and data
Sec. 58.195 Retention of records
80 4 Non-clinical Studies
Table 4.1 (Continued)
EU Directive 2004/10/EC US 21 CFR Part 58
Subpart K—Disqualication of Testing
Facilities
Sec. 58.200 Purpose
Sec. 58.202 Grounds for disqualication
Sec. 58.204 Notice of and opportunity for
hearing on proposed disqualication
Sec. 58.206 Final order on disqualication
Sec. 58.210 Actions upon disqualication
Sec. 58.213 Public disclosure of information
regarding disqualication
Sec. 58.215 Alternative or additional actions
to disqualication
Sec. 58.217 Suspension or termination of a
testing facility by a sponsor
Sec. 58.219 Reinstatement of a disqualied
testing facility
covered by the regulations are identical. It should be noted that the EU directive is
also intended to cover safety testing of general chemicals and eld studies.
As with all quality systems the regulations address basic aspects such as person-
nel, documentation, facilities, and equipment. However, there are some particular
requirements that are worthyof note. AStudy Director must be appointed with over-
all responsibility for the conduct and reporting of each study or group of related
studies. A written protocol or study plan must be prepared to outline the purpose
and detail of how the study will be conducted, with justication for approach taken
where appropriate (length, animals, etc.). An independent quality assurance group,
in addition to the study director, must approve the study. The QA group is also
responsible for oversight of the study and must certify the nal report with respect
to GLP compliance. Samples of materials and tissues must be retained in addition
to all documentary records. The retention times for samples and documents will
depend on the stage of the authorisation process that the study supports and the
requirements of the particular regulatory authority. Facilities must be inspected and
accredited by external agencies; notied bodies in the EU, or directly by the FDA in
the US.
4.10 Chapter Review
This chapter examined the various nonclinical studies that are at the core of the
pre-clinical phase of drug development. These studies are intended to gain a basic
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4.10 Chapter Review 81
understanding of the pharmacology of the drug and address safety and quality con-
cerns prior to engaging in trials with human subjects. The studies must provide
detailed knowledge of pharmacodynamics and pharmacokinetics in a combination
of in vitro and animal models, and permit adequate assessment of the toxicologi-
cal properties of the drug. The drug substance and product must be characterized
in terms of its chemical/physical characteristics, manufacture, control, and stabil-
ity. All safety studies must be conducted according to the principles of GLP. Finally,
an overview of non-clinical studies reported in respect of a sample-approved drug
(Eliquis) is presented as a case study (Box 4.1).
Box 4.1 Non-clinical Studies of Eliquis
Eliquis is the tradename given to one of the most globally lucrative drugs on the
pharmaceutical market. The chemical structure of its active ingredient (apixa-
ban) is shown below (a). Marketed in tablet form, apixaban binds to and inhibits
blood coagulation factor Xa (FXa, also known as Stuar t factor); see (b) below.
Inhibition of factor Xa prevents blood clot formation. As such, the product is
used to prevent conditions such as stroke, venous thromboembolism and pul-
monary embolism. Eliquis was first approved for medical use in the EU in 2011
and subsequently in the US in 2012.
An overview summary of the main data/findings provided to the regulatory
authorities by a company seeking marketing approval is generally published
on the regulatory authority’s website, for purposes of transparency in the reg-
ulatory approval process. For example, upon drug approval, the EMA publishes
a drug EPAR (European Public Assessment Report), summarizing non-clinical,
clinical and quality data/findings which underpinned the agency’s decision to
approve the drug for general medical use. The EPAR published in respect of
Eliquis indicates that nonclinical studies/findings included:
● Apixaban was shown to directly and specifically inhibit human factor Xa and
its molecular mechanism of action is understood.
● The major breakdown metabolite of apixaban (O-dimethyl apixaban sulfate)
does not significantly inhibit human factor Xa in vitro.
● Apixaban demonstrated antithrombotic efficacy in a number of animal models
(mainly rabbits, rats and dogs).
● Various safety studies were performed to assess any potential effects of
apixaban on the cardiovascular, respiratory, and central nervous systems in
particular. These safety studies included both short and long-term toxicity
studies (mainly in dogs) and concluded that, at doses exceeding the human
(Continued)
82 4 Non-clinical Studies
Box 4.1 (Continued)
recommended dose, apixaban did not induce any respiratory or central
nervous system toxicity.
● In vitro pharmacokinetic studies relating to drug absorption focused upon drug
absorption across excised portions of the rat digestive tract, as well as a vari-
ety of different cultured cell types.
● Pharmacokinetic studies were also undertaken in rats, dogs, and chimpanzees,
and focusing upon, e.g. establishing drug bioavailability, distribution, and
elimination. For example, 2 single radiolabelled distribution studies were
conducted in rats.
● Single-dose oral toxicity studies were undertaken in mice, rats, dogs, and
cynomolgus monkeys, while repeat-dose toxicity studies were undertaken in
rats (six months) and dogs (one year), with any effects, noted generally relat-
ing directly to the drug’s mode of action.
● Genotoxicity studies entailed application of four different assay approaches
(bacterial mutation tests, in vitro chromosomal aberrations in mammalian
cells, and in vivo assays in rat bone marrowand peripheral bloodlymphocytes),
with no evidence of genotoxicity recorded.
● Two two-year carcinogenicity studies were undertaken, in rats and mice.
● Reproductive toxicity studies wereundertaken in rats, mice, and rabbits, focus-
ing upon fertility and embryo–foetal development.
● Ecotoxicity and environmental risk assessment studies were also undertaken.
Such non-clinical studies would have been informed by the various regulatory
guidelines cited within the body of this chapter.
1. chemical structure of apixaban
H
2
N
H
3
CO
N
N
N
O
O
O
N
2. Structure of human factor Xa complexed with apixaban (latter highlighted by
broken line).
Source: Available at www.rcsb.org. Alexander, R. 2007/RCSB PDB/Licensed under CC0 1.0.
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4.11 Further Reading 83
4.11 Further Reading
ICH Harmonized Guidance Documents
www.ich.org
European Medicines Agency nonclinical guidelines
https://www.ema.europa.eu/en/human-regulatory/research-development/scientic-
guidelines/non-clinical-guidelines
FDA Non-harmonized Guidance Documents
https://www.fda.gov/regulatory-information/search-fda-guidance-documents#
guidancesearch
GLP Directive 2004/10/EC
https://eur-lex.europa.eu/homepage.html
US GLP Regulations
Code of Federal Regulations, Title 21, Part 58
www.fda.gov
Yu, L. et al. (2014) Understanding pharmaceutical quality by design. AAPS Journal, 16
(4), 771–783.
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