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In this chapter, we will discuss pre-clinical and clinical studies, pharmacovigi-
lance, pharmacogenomics, and commercialization of pharmaceutical products.
17.2 Pre-clinical Evaluations
During the beginning stage of drug discovery, the activity and specicity of the
candidate molecule were evaluated rst followed by pharmacokinetics and toxicity
studies, although many lead molecules failed at the pre-clinical evaluation due to
failure in achieving safety and efcacy of lead possibly due to failure in drug absorp-
tion, distribution, metabolism, and excretion (ADME).
Pre-clinical studies can be divided into two parts: exploratory and conrmatory.
Exploratory studies mainly involve the exploration of potential hypotheses that are
scientically driven and explore either the pathophysiology of the disease or identi-
cation of potential drug targets. Exploratory studies are often not regulated as their
sole purpose is to explore the disease progression and identify the potential target,
although computational technologies and in silico studies were developed to avoid
pre-clinical invivo exploratory studies to minimize research waste. Conrmatory
studies mainly involved conrmation of efcacy and safety of potential drug candi-
dates. These studies must be properly designed and follow different regulatory
guidelines. Data achieved from conrmatory studies must be properly analyzed,
stored, and reported to the highest regulatory authorities with transparency (Huang
etal. 2020).
With the rapid progression in the advancement of techniques such as high-
throughput screening, computational biology, and combinatorial chemistry, the syn-
thesis of different molecules has become easy and fast. Different methods to evaluate
the ADME of drug candidates increased rapidly. Many in silico methods success-
fully predicted the ADMS prole of the drug, and due to that, in silico methods
replaced invivo exploratory and ADME methods.
Fig. 17.1 Process of drug discovery
17 Pre-clinical and Clinical Studies, Pharmacovigilance, Pharmacogenomics…
426
17.2.1 In Vitro Pharmacological Studies
Over the past few years, the drug discovery approach of the new drug candidate has
been largely dependent on evaluating the compounds for their biological activity
and establishing structure-activity relationships, with assessment for better drug
efcacy, based on different experiments in moralized and immortalized cell lines as
the rst pre-clinical step which is easy and easily replicable (Perego etal. 2018;
Ziegler etal. 2000). Using monolayer culture cells which are the easiest and repli-
cable invitro models, the mechanism of action of novel drugs and their toxicity can
be easily measured (Phillips 2016). Advancements in high-throughput screening
methods and incorporating genomics and proteomics approaches are improving the
pace of pre-clinical drug evaluation from conventional to target-based approaches,
focusing on targeting gene as well as protein-level biomarkers for the sensitivity of
drugs and their resistance (Roma-Rodrigues etal. 2019). In the context of cancer
drug discovery, new insights into cellular and molecular mechanisms of different
cancers have led researchers to use different kinds of normal as well as tumor cell
models to efciently screen new potential anticancer drug molecules, including cell
lines having particular mutations, stem cells, and endothelial cells (Hamburger and
Salmon 1977; Rhim et al. 1998). Pathophysiology and molecular understanding
between complex interaction of cancer cells and the surrounding microenvironment
are not well understood. Interaction between cancer cell and microenvironment
gave rise to metastatic events. How much tumor cells inuence their microenviron-
ment is an important factor in developing new drug strategies, and for that develop-
ment of a new kind of invitro system is required (Whiteside 2008).
Two pivotal pharmacological determinants governing cellular responses encom-
pass the drug’s concentration (C) and the duration of its exposure (T). In the context
of traditional cytotoxic agents, cell proliferation and survival within two- dimensional
(2D) monolayer cultures ordinarily correlate with the product of C×T. However,
this relationship may deviate for drugs exhibiting cell-cycle phase specicity,
wherein the cellular response beyond a certain concentration threshold tends to be
directly proportional to the exposure duration. To capture temporal alterations in
response and suit the context of plate-based experiments, an array of time- dependent
measurement techniques can be employed. The attainment of consistent, pertinent,
and replicable outcomes hinges on the discerning selection of a suitable assay and
experimental framework (Kavallaris 2010; Capula etal. 2019).
17.2.2 In Vivo Toxicity Studies
The main goals of initial invivo preclinical toxicity assessments for drug candidates
are to identify possible untoward outcomes and provide preliminary safety margin
assessments. These evaluations guide the suitability of a compound for human
application or the necessity to exclude it from subsequent developmental stages.
Furthermore, additional investigations can be carried out to nd possible molecular
mechanisms behind observed toxic effects, thereby changing the strategies in the
M. Joshi and B. M. Patel
427
preclinical drug development phase and evaluating possibilities for human hazard
evaluation (Gross and Kramer 2003; Greaves etal. 2004). Based on the industry
inputs, toxicity data from rodents anticipate only 43% of human toxic reactions,
whereas non-rodent inquiries foresee 63%. Interestingly, the synergy arising from
considering both these species escalates the predictability to approximately 71% for
human toxicities. Interpretation of toxicity data is a straightforward task, predicting
human response based on these data remains controversial (Olson etal. 2000). This
difference in interpretation arises mainly due to biological as well as pathological
differences between species along with intrinsic variations in pharmacokinetic
aspects like absorption, distribution, metabolism, and excretion properties. Many
studies have documented variations in toxicity proles where toxicity was present
exclusively in rodents in drug metabolism (Mutlib etal. 2000), thyroid hormone
regulation (Wu and Farrelly 2006), or the formation of bladder tumors (Cohen and
Lawson 1995). Enhancing human risk assessment and the judicious selection of
viable drug candidates for developmental pursuits goes beyond simply advancing
regulatory toxicity investigations during the early stages of discovery or conducting
tests across a wider array of models. Instead, it hinges upon cultivating a deeper
understanding of the intricacies of toxicity mechanisms. This proactive investment
should yield substantial returns by facilitating a more astute utilization and interpre-
tation of both invivo and invitro models (Fielden and Kolaja 2008).
As of now, the Organization for Economic Co-operation and Development
(OECD) provides guidelines to conduct toxicity studies of different drug candi-
dates. It includes Acute Oral Toxicity, Acute Dermal Toxicity, Acute Inhalation
Toxicity, Acute Dermal Irritation/Corrosion, Acute Eye Irritation/Corrosion, Skin
Sensitization, Repeated Dose 28-day Oral Toxicity Study in Rodents, Repeated
Dose 90-day Oral Toxicity Study in Rodents, Repeated Dose 90-Day Oral Toxicity
Study in Non-Rodents, Repeated Dose Dermal Toxicity: 21/28-day Study,
Subchronic Dermal Toxicity: 90-day Study, Subacute Inhalation Toxicity: 28-Day
Study, Subchronic Inhalation Toxicity: 90-day Study, Prenatal Development
Toxicity Study, One-Generation Reproduction Toxicity Study, Two-Generation
Reproduction Toxicity, Toxicokinetics, Acute Oral Toxicity: Up-and-Down
Procedure, and many more (Buschmann 2013).
17.2.3 In Vivo Efficacy Studies
In vivo, efcacy studies mainly involve the assessment of pharmaceutical drugs in
biological systems except humans. In vivo, efcacy studies mainly involve the
induction of a particular disease into the animals and then treating the disease with
investigational drugs. As of now, animals such as mice, rats, rabbits, dogs, zebrash,
and fruit ies are the most commonly used animals to evaluate toxicity and effec-
tiveness of drugs due to their somewhat resemblance to human anatomy and physi-
ology. However, it remains controversial regarding the translation of animal data
into human data. Regarding conducting the efcacy studies, the Institutional Animal
Ethics Committee (IAEC) is formed which provides approval and guidelines for
17 Pre-clinical and Clinical Studies, Pharmacovigilance, Pharmacogenomics…
428
public participation in the regulation of animal research. IAEC decides whether the
study requires invivo testing or not and if required then how many numbers of ani-
mals are required. The three Rs (3Rs) are another ethical principle coined by
W.M.S. Russell and R.L.Burch for the utilization of animals in experiments. The
3Rs are replacement, development of alternative procedures to replace the use of
animals in experiments; reduction, use of protocols that allow the generation of
more meaningful data with the use of fewer animals; and renement, use of proto-
cols that alleviate or minimize distress, pain, and discomfort to animals while
enhancing the comfort and welfare of animals used in experiments.
The 3Rs have a broad value and meaning to improve animal care and quality of
research and encourage researchers to use alternatives to avoid excessive use of
animals. In many countries, these 3Rs are now introduced into the governing body
for animal use (Lewis 2019).
Many different animal models have been developed over the years for almost all
diseases. With the progression of genetic and molecular insights into each disease,
different gene knock-in and knock-down models were developed to understand the
pathophysiology in a better way (Robinson etal. 2019).
17.3 Clinical Evaluations
Clinical research is a study of investigational drugs. In some cases, it includes both
healthy volunteers and patients. Types of clinical trial can broadly divide to obser-
vational trial and clinical trials.
Observational inquiries meticulously observe individuals within their natural cir-
cumstances. Investigators gather insights from individuals and scrutinize shifts that
unfold over time. To illustrate, a cohort of seniors might be queried about their
physical activity routines, followed by a year of monthly memory assessments. This
endeavor aims to unveil the correlation between physical engagement and cognitive
well-being. While observational studies abstain from testing medical interventions
like pharmaceuticals or devices, their outcomes can potentially unearth innovative
treatments or prevention tactics warranting assessment in clinical trials.
17.3.1 Clinical Trial Phases
17.3.1.1 Phase 0
The main difference between phase 0 and another phase trial in the regard of tradi-
tional Investigational New Drug (IND) application lies in the absence of therapeutic
intent within phase 0 trials. Both types of subjects, healthy individuals and patients,
are involved in the study and exposed to doses of an intervention that are subthera-
peutic yet still pharmacologically active. The primary objective of these trials is not
to evaluate the toxicity of drug, unlike other traditional trials. In phase 0 trials, the
focus remains on administering doses that are below the level of expected therapeu-
tic effects. While the exposure of the drug remains limited to subjects, exibility
M. Joshi and B. M. Patel
429
exists for dose escalation. The anticipation of signicant adverse events related to
the drug is low due to the use of lower doses. This specic advantage was recog-
nized by many regulatory bodies, allowing for a more regulated approach to pre-
clinical toxicology studies. Instead of necessitating extensive dose-limiting toxicity
studies, phase 0 trials remain dependent on single dose or short-term treatment or
short-term toxicity assessment. Moreover, the reduced quantity of the investiga-
tional drug required for phase 0 trials does not require the need for full-scale pro-
duction and clinical-grade manufacturing that adheres to good manufacturing
practices (GMP) before the commencement of the trial. Consequently, this advan-
tage allows the initiation of phase 0 trials at a much earlier stage compared to the
traditional phase I studies. This early initiation allows researchers to understand the
pharmacokinetics (PK) and the effects on drug targets in human participants at an
earlier developmental stage of the therapeutic agent. In summary, the requirement
of phase 0 trials arises from their non-therapeutic intent, employing subtherapeutic
yet pharmacologically active doses in a limited exposure setting, accompanied by
the graded dose escalation. Lower chances of signicant adverse events allow the
utilization of condensed pre-clinical toxicology studies. Furthermore, the minimal
quantity of study drugs needed for these trials allows for their initiation ahead of
traditional phase I studies. This, in turn, grants researchers an invaluable opportu-
nity to delve into the pharmacokinetic and drug target effects in human subjects
much earlier in the trajectory of clinical development (Kummar etal. 2008).
17.3.1.2 Phase I
Phase I clinical trial is carried out primarily to identify the recommended dose for
phase II.It is commonly carried out by dose escalation and achieves the maximum
tolerated dose (MTD) using different methods such as 3+3 algorithms and continu-
ous evaluations. Phase I trials are very small, and a maximum of around ten patients
are treated with MTD.To gather more data on the toxicity and efcacy of the inter-
vention, phase I trials consider the inclusion of “expansion cohorts” that allow for
the treatment of additional participants at the MTD.In terms of efcacy, meaning
undesirable clinical effects, several factors are taken into consideration after admin-
istration of the drug. The conventional approach in the phase I trial is to achieve a
minimum recommended dose (P2RD) by focusing only on toxicity outcomes from
a limited sample size, while not focusing on the integration of efcacy within the
dose-nding algorithm, leading to certain unfavorable ramications. In a recent
advancement, the number of patients in expansion cohorts has dramatically
increased with 100–200in some trials. A large “phase I expansion cohort” can be
considered a phase II study without any particular design and comparison with a
placebo or any other intervention. This approach can eliminate the problems associ-
ated with small cohorts. Phase I can be considered as a phase II trial, but carried out
without having any particular plan and design other than a xed sample size. This
method mitigates the problem associated with small expansion cohorts. The MTD
easily may turn out to be either toxic or ineffective.
The main focus of this clinical trial and drug discovery is to select the right dose
for the large population. To achieve this, modications are required from time to
17 Pre-clinical and Clinical Studies, Pharmacovigilance, Pharmacogenomics…
430
time in terms of patient selection, trial design, cautiously selected objectives, end-
points, and inclusion and exclusion criteria. Future efforts should be made in the
direction of improving patient selection, along with identifying the mechanism of
action of intervention and minimizing the adverse effects of the drug (Muglia and
Digiovanna 1998).
17.3.1.3 Phase II
The objective of the phase II clinical trial is to identify the efcacy of the interven-
tion that can then proceed to further scrutiny in the conclusive phase III trials.
Randomized arrangements in phase II trials are classied within three distinct
frameworks:
Parallel noncomparative exploration: This category involves the random assign-
ment of participants to separate experimental regimens, each with its indepen-
dent decision-making criteria.
Randomized selection: Within this design, various experimental regimens are pre-
sented, and the selection of the most auspicious among them is randomized.
Randomized screening design: This type facilitates the comparison between an
experimental regimen and the established standard of care through randomized
allocation.
In a current scenario with limited availability of patients and resources with sev-
eral potential therapies for various diseases, the utilization of randomized phase II
designs is surging in popularity. This mainly has a role in the eld of cancer drug
development, where the abundance of prospects calls for astute clinical trial strate-
gies. By employing randomized phase II designs in a manner consistent with their
foundational inference principles, the judicious allocation of phase III nancial and
patient resources can be ensured (Van Norman 2019).
17.3.1.4 Phase III
Based on the previous ndings that have demonstrated the safety and efcacy of the
intervention, a phase III trial is sometimes referred to as a “therapeutic conrma-
tory,” “comparative efcacy,” or “pivotal” trial carried out. This particular phase of
drug evaluation occurs within a broader and often more diverse population. Its pri-
mary objectives are to validate safety and effectiveness, as well as the determination
and estimation of possible adverse reactions.
It is noteworthy that phase III trials while encompassing a range of 300–5000
participants have more statistical power compared to phase II studies. This statisti-
cal power allows for the establishment of adverse event rates not exceeding 1in 100
individuals, in alignment with Hanley’s “Rule of 3.” This prominently proves the
importance of phase IV trials in the detection of infrequent adverse drug reactions
(Eypasch etal. 1995). Consequently, it’s apparent why the FDA commonly requires
multiple phase III trials as a prerequisite for the establishment of both the safety and
efcacy of a drug.
M. Joshi and B. M. Patel
431
The different types and designs of phase III trials, often recognized as “superior-
ity” or “placebo-controlled trials,” focused on comparative efcacy outcomes.
These trials compare the new intervention of interest with either an established stan-
dard therapy or a placebo. A distinctive feature of the phase III trial framework lies
in the balance achieved in the allocation of treatments through randomization to
evaluate the treatment effectiveness unbiasedly (Cahana and Romagnioli 2007;
Foddy 2009; Wilcox 2008).
The preferred method to evaluate the phase III trials is the intention-to-treat anal-
ysis. This analytical approach evaluates participants based on the treatment arm to
which they were initially randomized, regardless of the actual treatment they even-
tually received. This principle, often referred to as the “analyzed as randomized”
rule, serves to uphold the integrity of randomization and eradicate the inuence of
selection bias. Consequently, any observed disparities in outcomes can be directly
correlated to the treatment itself rather than any confounding factor.
Another type of analysis is termed “as-treated” or “per-protocol.” Here, partici-
pants are assessed according to the treatment they received, irrespective of their
initial randomization. This approach, while offering valuable insights, diverges
from the intent of randomization and can, in essence, approximate the conditions of
an interventional cohort study, which introduces the potential for treatment selec-
tion biases (Tsiatis 1990).
To further minimize the intended biases, the phase III trial can be designed and
designated as “blinded” (or masked) where the investigator or patient does not know
about the intervention arm. Specic blinding strategies such as “single blinding”
(subject only), “double blinding” (both subject and investigator), or “triple blind-
ing” (data analyst, subject, and investigator) can be applied based on the study
requirement and study design. Unfortunately, it is not possible to blind all the trials.
To design a phase III trial, phase III study, the Consolidated Standards of Reporting
Trials (CONSORT) guideline was created to improve the design of the trial while
minimizing intended and unintended biases (Umscheid etal. 2011) (Fig.17.2).
17.4 Pharmacovigilance
The World Health Organization (WHO) has dened pharmacovigilance as the “sci-
ence and activities relating to the detection assessment, understanding, and preven-
tion of adverse effects or any other possible drug-related problems.” The
responsibility to establish proper guidelines was taken up by the Council for
International Organization of Medical Sciences (CIOMS). Initially founded in 1949
as an independent body for the worldwide communication of medical science
knowledge, CIOMS underwent reconguration in 1966 to create fundamental con-
cepts, regulations, and protocols concerning the safe utilization of pharmaceutical
products (medications and vaccines). Employing a collaborative network involving
a worldwide working group comprised of medical and paramedical professionals
from governmental, academic, and industry, CIOMS undertook the resolution of
expanding the scope of pharmacovigilance. Pharmacovigilance mainly includes
17 Pre-clinical and Clinical Studies, Pharmacovigilance, Pharmacogenomics…
432
adverse drug reactions, intermittent reporting, risk-benet evaluations, safety docu-
mentation during clinical trials, signal recognition, and risk mitigation (Tsintis and
La Mache 2004).
Thus, the three main objectives of PV are case management, signal management,
and benet management.
Activities and task currently included in the PV domain are monitoring clinical
trials to ensure the safety of the patient, identifying the safety prole of the drug in
regard to selecting rst safe dose for humans, communication regarding safety and
progress in clinical trials to stakeholders, determining the casual relationship
between drug and adverse effects, monitoring issues related to drug safety and qual-
ity of manufactured drugs, and preparing and carrying out inspection in various
departments.
It is important to keep in mind that pharmacovigilance has broadened the data
and knowledge of pharmacological drugs. It is an important addition to the lack of
knowledge of adverse drug reactions during clinical trials. Reporting and analyzing
those adverse drug reactions increase our understanding of the mechanism of drugs,
their efcacy on different populations, and their impact on genetics.
Pharmacovigilance remains in a state of constant modications and is adapted nely
in the context of drug safety and also in personalized medicine.
Pharmacovigilance mainly deals with detecting and reporting adverse drug reac-
tions (ADRs). Adverse drug reactions can be dened as unintended or obnoxious
drug responses that take place at the normal dose levels intended to be used as
therapy, diagnosis, or prophylaxis.
ADR can be divided into six different categories:
Type A: Dose-dependent effect mainly due to exaggerated pharmacological response
Type B: Not dose-dependent on dose and occurs in a small number of populations
Fig. 17.2 Different phases of clinical trials
M. Joshi and B. M. Patel
433
Type C: Chronic effect associated with the development of spontaneous disease
Type D: Carcinogenicity, delayed effect, and teratogenicity
Type E: ADR reported after the end of treatment
Type F: Failure of treatment (Zazzara etal. 2021)
17.4.1 Role ofPV intheDrug Development Process
Direct involvement of PV professionals in the clinical trials generally fell out scope
of PV activities although based on their eld experience, they could be strategically
positioned to assume a vital role in comprehending the broader context and ensuring
effective communication with all stakeholders regarding ongoing advancements.
The process of gathering essential data to establish and uphold drug safety pro-
les throughout a product’s life cycle is generally consistent on a global scale. The
uncertainties inherent in post-marketing encounters serve as cues that underscore
the overarching realization that safety issues linked to drugs can manifest unexpect-
edly and from diverse sources. PV professionals are tasked with upholding a state
of constant “risk-awareness” concerning both the intended and unintended impacts
of their drugs.
The procedural aspects associated with these documents are typically, though not
universally, overseen by a company’s regulatory affairs department. However, the
accountability for the substance of these documents lies with the departments
responsible for pre-clinical animal studies, clinical indications, adverse events, and
product packaging. Within this framework, PV assumes a proactive role in suggest-
ing modications to the product’s safety prole. This necessitates the establishment
of robust governance structures and processes to effectively advocate for changes in
the content, format, and placement of such proposals within the relevant user
documents.
The contrasting regulatory strategies employed by the FDA and the EMA neces-
sitate PV professionals to be exceptionally vigilant and perceptive in the face of
emerging signals. Each new signal’s implications may diverge depending on the
regulatory jurisdiction, demanding distinct approaches to management
(Beninger 2018).
17.4.2 Clinical Trial Designs
During the era of evidence-grounded medicine, randomized controlled clinical tri-
als hold a paramount status in enlightening clinicians regarding the relative effec-
tiveness and safety of treatments. The insights emanating from a clinical trial hold
merit only when the trial itself is meticulously crafted. This entails an explication of
the research queries and a precise delineation of tting criteria for participants, out-
comes, and study frameworks. These measures serve to mitigate distortions and
extraneous inuences. It’s important to note that no solitary trial blueprint reigns
supreme across all conceivable scenarios. Hence, a robust comprehension of the
17 Pre-clinical and Clinical Studies, Pharmacovigilance, Pharmacogenomics…
434
merits and demerits intrinsic to diverse clinical trial architectures becomes
imperative.
17.4.3 Randomized Controlled Trials
Randomized controlled trials (RCTs) are type of a study design that evaluates the
safety and efcacy of novel interventions or treatments. While individual studies
might not independently establish causation, randomization of subjects in the trial
can effectively mitigate biases, offering a robust mechanism to scrutinize the causal
links between interventions and outcomes. This arises from the fact that the process
of randomization can divide the characteristics of subjects in a balanced way among
different groups.
During the process of designing the RCTs, the type of subjects, different inter-
ventions, and desired outcomes should be taken into consideration carefully. After
considering these factors, the next step should be a selection of the total number of
required participants which can robustly establish the presence of any potential con-
nections (conducted through power calculation). Subsequently, participants are
enlisted and arbitrarily divided into either the intervention cohort or the comparator
group (Torres-Saavedra and Winter 2022).
17.4.3.1 Parallel Arm Design
In the parallel arm design, participants are assigned randomly to one or more study
arms, with each branch being linked to a unique intervention. After the randomiza-
tion procedure, each participant remains exclusively associated with their desig-
nated treatment arm throughout the entire study period. This parallel group design
can be used in any disease condition, and many studies can be carried out simultane-
ously. This study design requires strong vigilance to avoid unintended co-
interventions or cross-overs.
17.4.3.2 Cross-Over Design
In the cross-over design, each subject receives both treatments at different time
intervals. For example, subjects receiving treatment A will be receiving treatment B
after the washout period, and subjects receiving treatment B will be receiving treat-
ment A after the washout period. A washout period must be present to eliminate any
effect of the rst intervention and avoid synergic and antagonist interaction between
the two interventions. In this method, the outcome can be compared in the same
subject as well as between the groups. One of the important requirements to design
cross-over RCT is that the disease must be chronic (e.g., diabetes, hypertension) and
the effect of the drug should be washed out before starting the next intervention.
17.4.3.3 Randomized Withdrawal Design
In this design, all the subjects will receive the intervention, and no randomization
was carried out. Subjects who responded to therapy will be considered in the study,
while non-responders were left out of the study. Responders were randomized in
M. Joshi and B. M. Patel