PRECLINICAL PHASE
In drug development the preclinical phase (in-vitro and in-vivo) Also named preclinical
studies or nonclinical studies -> is a stage of Research that begins before clinical trials, and
during which important Feasibility, iterative testing and drug safety data are collected
most common challenges, faced by all Al start ups, in designing and running preclinical
experiments, have to do with:
1) reducing time, money and uncertainty in planning preclinical Experiments,
2) automating the selection, manipulation and analysis of cells, and
3) automating sample analysis with robotic cloud laboratory
In drug development, preclinical development, also named preclinical studies and nonclinical studies
Is a stage of research that begins before clinical trials (testing in humans) can begin, and
during which important feasibility, iterative testing and drug safety data are collected,
typically in laboratory animals
Main goals of preclinical studies are to:
1) Determine a starting, safe dose for first-in-human study and
2) Assess potential toxicity of the product, which typically include New medical devices,
prescription drugs, and diagnostics
On average, only one in every 5,000 compounds that enters drug Discovery to the stage of
preclinical development becomes an approved drug
each class of product may undergo different types of preclinical research
For instance, drugs may undergo pharmacodynamics (what the drug Does to the body (PD).
Pharmacokinetics (what the body does to the Drug) (PK), ADME, and toxicology testing
Data allows researchers to allometrically estimate a safe starting dose of the drug for clinical trials in
humans
> medical devices that do not have drug attached will not undergo
These additional tests and may go directly to good laboratory Practices (GLP) testing for safety of the
device and its components
some medical devices will also undergo biocompatibility testing which helps to show whether a
component of the device or all components are sustainable in a living model
Most preclinical studies must adhere to GLPs in ICH Guidelines to be acceptable for
submission to regulatory agencies such as the Food & Drug Administration in the United
States
typically, both in vitro and in vivo tests will be performed
studies of drug toxicity include which organs are targeted by that Drug, as well as if there
are any long-term carcinogenic effects or Toxic effects causing illness
FDA requires researchers to use good laboratory practices (GLP), defined in medical product
development regulations, for Preclinical laboratory studies
The GLP regulations are found in 21 CFR Part 58.1: Good Laboratory Practice for nonclinical
laboratory studies
These regulations set the minimum basic requirements for:
*Study conduct
*Personnel
*Facilities
*Euipment
*Written protocols
*Operating procedures
*Study reports
*And a system of quality assurance oversight for each study to help assure the safety of FDA-
regulated product
Usually, preclinical studies are not very large > however, these studies must provide detailed
information on dosing and toxicity levels
After preclinical testing, researchers review their findings and decide whether the drug
should be tested in people
In-vitro:
Refers to a medical study or experiment which is done in the lab within the confines of a test
tube or laboratory dish
In -vivo:
> refers to a medical test, experiment, or procedure that is done on (or in) a living organism, such as
a laboratory animal or human
>Clinical trials or medical studies may be performed either in viva or in Vitro
These approaches are similar in that they are both done in order to make advances in the knowledge
and treatment of illness and disease as well as understanding “wellness” and normal bodily
functions
But there are also many important differences in how in vive and in Vitro studies are
conducted, how they can be interpreted, and t Practical applications of any discoveries
which are made
Preclinical Research
Before testing a drug in people, researchers must find out whether It the potential to cause
serious harm to humans the preclinical studies are conducted on animal models under
Laboratory conditions
The two types of preclinical research are:
In-vitro: these experiments are conducted outside the animals in controlled Laboratory conditions
In-vivo: these experiments are conducted inside the animals usually, Preclinical studies are not very
large
➤ however, these studies must provide detailed information on dosing and Toxicity levels
After preclinical testing, researchers review their findings and decide Whether the drug can be
tested in people
The various experiments conducted during these studies include
• Single dose toxicity studies
• Repeated dose studies Safety pharmacology studies.
. Genotoxicity studies
• Carcinogenicity studies
Reproductive toxicity studies
In Vitro Medical Studies
Medical studies (such as looking at the ability of a drug to treat cancer) are often first performed in
vitro-either in a test tube or laboratory dish
> an example would be growing cancer cells in a dish outside of the body to study them and
possible treatments
► studies are usually done in vitro first for ethical reasons
> In vitro studies allow a substance to be studied safely, without subjecting Humans or animals to
the possible side effects or toxicity of a new drug
> Researchers learn as much as possible about a drug before exposing humans to potential negative
effects
>If a chemotherapy drug, for example, does not work on cancer cells grown in a dish, it would be
unethical to have humans use the drug and risk the Potential toxicity
> in vitro studies are important in that they allow more rapid development of New treatments-many
drugs can be studied at one time (and they can be Studied
In a large number of samples of cells) and only those that appear to be Efficacious go on to human
studies
> an absence of biokinetics (how the body transports and metabolizestudies And toxins) is one of the
significant drawbacks of in vitro studies
> this, as well as several other factors, can make it very difficult to extrapolate the results of in vitro
tests to what might be expected when the drug is used in vivo
Sequential steps involved in design and conduct in vitro binding studies
▪ Method devpt and validation of bile acids/phosphate/Human serum albumin (HSA) or Bovine
serum albumin (BSA) using HPLC/IC/ICP-MS as per USFDA published Bioanalytical guidelines
*Kinetic binding study is conducted for test and reference formulations with bile acids/
phosphate/HSA or BSA concentrations and pH variation resembling the gastrointestinal pH
*Free bile acids/phosphate/HSA or BSA concentration is determined and bound concentration is
computed
• Saturation binding with respect to time and binding similarity between test and reference
formulation is characterized
• Equilibrium binding study for test and reference formulations with appropriate bile acids/
phosphate ranging 8 different concentrations and pH Variation resembling the Gl pH
Free bile acids/phosphate//HSA or BSA concentration is determined and Bound concentration is
computed to calculate Langmuir binding affinity (K1) and capacity (K2) constants
• Bioequivalence between test and reference formulation is determined based on the capacity
constant (K2)
In vivo Clinical Trials
in contrast to in vitro studies, in vivo studies are needed to see how tth Body as a whole will respond
to a particular substance
In some cases in vitro studies of a drug will be promising, but subsequent in vivo studies fail to show
any efficacy (or, on the other hand, find a drug to be unsave) when used within the multiple
metabolic processes that are Continually taking place in the body
An example of how in vivo studies are needed to evaluate drugs is with respect to drug absorption in
the body
a new drug may appear to work in a dish, but not in the human body
> it could be that the drug is not absorbed when it passes through the Stomach, so it has little effects
on humans
In other cases (even if a drug is given intravenously) it could be that a drug is broken down by the
body through number of reactions that occur continuously, and therefore, the drug would not be
effective when used directly in humans
It’s important to note that oftentimes in vivo studies are first done in non human animals such as
mice
> these studies allow researchers an opportunity to see how a drug works Amid other bodily
processes
➤mice and humans have important differences
> sometimes a drug that is effective in mice will not be effectivdifferences (and vice versa) due to
inherent differences in the species
Our In vivo pharmacology capabilities include:
• Testing numerous medicinal entities, including small molecules, large Molecules and antibodies
. Inducing disease by multiple techniques chemical, surgical And diet
Dosing by commonly used administration routes-P.O, I.V, S.C and I.P
Daily dosing including on weekends, at no additional cost
Performing necropsies, histopathology, gene expression and clinical pathology
Executing standard or custom protocols
Preclinical studies
Before clinical trials are undertaken for a candidate drug, vaccine, medical device, or
diagnostic assay, the product candidate is tested extensively in preclinical studies.
Such studies involve in vitro (test tube or cell culture) and in vivo (animal model)
experiments using wide-ranging doses of the study agent to obtain Preliminary efficacy:
toxicity and pharmacokinetic information
Such tests assist the developer to decide whether a drug candidate has Scientific merit for
further development as an investigational new drug
► studies that are in vivo are those in which the effects of various biological entities are
tested on whole, living organisms or cells, usually animals, including humans, and plants, as
opposed to a tissue extract or dead Organism
This is not to be confused with experiments done in vitro, i.e., in a laboratory environment
using test tubes, petri dishes, etc.
▸ examples of investigations in vivo include: the pathogenesis of diseases
Phase 0
Phase 0 is a recent designation for optional exploratory trials conducted in Accordance with
the United States Food and Drug Administration’s (FDA) 2006 Guidance on Exploratory
Investigational New Drug (IND) Studies
> Phase 0 trials are also known as human microdosing studies and are Designed to speed up
the development of promising drugs or imaging Agents by establishing very early on
whether the drug or agent behaves in Human subjects as was expected from preclinical
studies
distinctive features of Phase 0 trials include the administration of single Subtherapeutic
doses of the study drug to a small number of subjects (10 to 15) to gather preliminary data
on the agent’s pharmacokinetics (what the body does to the drugs)
Phase 0 study gives no data on safety or efficacy, being by definition a dose Too low to cause
any therapeutic effect
drug development companies carry out Phase 0 studies to rank drug Candidates in order to
decide which has the best pharmacokinetic Parameters in humans to take forward into
further development
they enable go/no-go decisions to be based on relevant human models Instead of relying on
sometimes inconsistent animal data
Phase I
Phase I trials were formerly referred to as “first-in-man studies” but the field Generally
moved to the gender-neutral language phrase “first-in-humans” in the 1990s, these trials are
the first stage of testing in human subjects
they are designed to test the safety, side effects, best dose, and formulation method for the
drug
Phase I trials are not randomized, and thus are vulnerable to selection bias
normally, a small group of 20-100 healthy volunteers will be recruited
These trials are often conducted in a clinical trial clinic, where the subject cca Be observed by full-
time staff
these clinical trial clinics are often run by contract research organization (CROS) who conduct
these studies on behalf of pharmaceutical companies or other research investigators
the subject who receives the drug is usually observed until several half lives of the drug have
passed
this phase is designed to assess the safety (pharmacovigilance), tolerability
pharmacokinetics, and pharmacodynamics of a drug
Phase I trials normally include dose-ranging, also called dose escalation Studies, so that the
best and safest dose can be found and to discover the Point at which a compound is too
poisonous to administer
The tested range of doses will usually be a fraction of the dose that caused harm in animal
testing
Phase I trials most often include healthy volunteers however, there are some circumstances
when clinical patients are used, such as patients who have terminal cancer or HIV and the
treatment is Likely to make healthy individuals ill
These studies are usually conducted in tightly controlled clinics called CPUS (Central
Pharmacological Units), where participants receive 24 hour medical attention and oversight
In addition to the previously mentioned unhealthy individuals, “patients who have typically
already tried and failed to improve on the existing standard therapies” may also participate
in phase I trials
volunteers are paid a variable inconvenience fee for their time spent in The volunteer
center
before beginning a phase I trial, the sponsor must submit an Investigational New Drug
application to the FDA detailing the preliminaryData on the drug gathered from cellular
models and animal studies
Phase I trials can be further divided:
Single ascending dose (Phase la)
In single ascending dose studies, small groups of subjects are given a single dose of the drug
while they are observed and tested for a period of Time to confirm safety
typically, a small number of participants, usually three, are entered Sequentially at a
particular dose
if they do not exhibit any adverse side effects, and the pharmacokinetic Data are roughly in
line with predicted safe values, the dose is escalated, And a new group of subjects is then
given a higher dose
if unacceptable toxicity is observed in any of the three participants, an additional number of
participants, usually three, are treated at the same dose
This is continued until pre-calculated pharmacokinetic safety levels are Reached, or
intolerable side effects start showing up (at which point the Drug is said to have reached the
maximum tolerated dose (MTD)
>If an additional unacceptable toxicity is observed, then the dose escalation is terminated and that
dose, or perhaps the previous dose, isDeclared to be the maximally tolerated dose
> this particular design assumes that the maximally tolerated Cose occurs when approximately one-
third of the participants experience unacceptable toxicity
Variations of this design exist, but most are similar
Multiple ascending dose (Phase lb)
Multiple ascending dose studies investigate the pharmacokinetics and Pharmacodynamics of
multiple doses of the drug, looking at safety and tolerability
in these studies, a group of patients receives multiple low doses of the Drug, while samples
(of blood, and other fluids) are collected at various Time points and analyzed to acquire
information on how the drug is within the body
the dose is subsequently escalated for further groups, up to a Predetermined level
Food effect
short trial designed to investigate any differences in absorption of the drug by the body,
caused by eating before the drug is given
> these studies are usually run as a crossover study, with volunteers being given two
identical doses of the drug while fasted, and after being fed
Phase II
once a dose or range of doses is determined, the next goal is to evaluate whether the drug
has any biological activity or effect
Phase II trials are performed on larger groups (50-300) and are designed to assess how well
the drug works, as well as to continue Phase I safety assessments in a larger group of
volunteers and patients
Genetic testing is common, particularly when there is evidence of Variation in metabolic
rate
>when the development process for a new drug fails, this usually occurs during Phase II trials when
the drug is discovered not to work as planned, or to have toxic effects
Phase II studies are sometimes divided into Phase lla and Phase llb
there is no formal definition for these two sub-categories, but generally:
Phase Ila studies are usually pilot studies designed to demonstrate clinical Efficacy or
biological activity (‘proof of concept studies)
Phase IIb studies determine the optimal dose at which the drug shows Biological activity
with minimal side-effects (‘definite dose-finding’ studies)
Trial design
Some Phase II trials are designed as case series, demonstrating a drug’s safety and activity in a
selected group of participants
Other Phase II trials are designed as randomized controlled trials, where Some patients receive the
drug/device and others receive placebo/standard Treatment
Randomized Phase II trials have far fewer patients than randomized Phase III trials
>example: cancer design
In the first stage, the investigator attempts to rule out drugs that have no or little biologic activity >
for example, the researcher may specify that a drug must have some Minimal level of activity, say, in
20% of participants
If the estimated activity level is less than 20%, the researcher chooses not To consider this drug
further, at least not at that maximally tolerated dose
if the estimated activity level exceeds 20%, the researcher will add more participants to get a better
estimate of the response rate
typical study for ruling out a 20% or lower response rate enters 14 Participants
If no response is observed in the first 14 participants, the drug is Considered not likely to
have a 20% or higher activity level
The number of additional participants added depends on the degree of Precision desired,
but ranges from 10 to 20
Thus, a typical cancer phase II study might include fewer than 30 people to Estimate the
response rate
Efficacy vs effectiveness
▸ when a study assesses efficacy, it is looking at whether the drug given in the specific manner
described in the study is able to influence an outcome of interest (e.g. tumor size) in chosen
population (e.g. cancer patients With no other ongoing diseases)
when a study is assessing effectiveness, it is determining whether aTreatment will influence the
disease in an effectiveness study, it is essential that participants are treated as they Would be when
the treatment is prescribed in actual practice
that would mean that there should be no aspects of the study designed to increase compliance
above those that would occur in routine clinical Practice
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The outcomes in effectiveness studies are also more generally applicable than in most
efficacy studies (for example does the patient feel better, come to the hospital less or live
longer in effectiveness studies as opposed to Better test scores or lower cell counts in
efficacy studies)
> there is usually less rigid control of the type of participant to be included in effectiveness studies
than in efficacy studies, as the researchers are interested in whether the drug will have a broad
effect in the population of Patients with the disease.
Success rate
Phase II clinical programs historically have experienced the lowest success rate of the four
development phases
in 2010, the percentage of Phase II trials that proceeded to Phase III was 18% and only 31% of
developmental candidates advanced from Phase II to Phase III, in a large study of trials conducted
over 2006-2015
Phase III
This phase is designed to assess the effectiveness of the new intervention And, thereby, its
value in clinical practice
> Phase III studies are randomized controlled multicenter trials on large patient groups (300-3,000
or more depending upon the disease/medical condition Studied) and are aimed at being the
definitive assessment of how effective.
The drug is, in comparison with current ‘gold standard’ treatment
Because of their size and comparatively long duration, Phase III trials are the Most
expensive, time-consuming and difficult trials to design and run, especially in therapies for
chronic medical conditions
> Phase III trials of chronic conditions or diseases often have a short follow-up period for evaluation,
relative to the period of time the intervention might be Used in practice
This is sometimes called the “pre-marketing phase” because it actually Measures consumer
response to the drug it is common practice that Certain Phase III trials will continue while
the regulatory submission is Pending at the appropriate regulatory agency
