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10.2.1 Chemistry, Manufacturing, and Control Information
Information related to the composition, manufacturing, stability, packaging, and
controls used to produce the drug substance and the drug product is detailed in the
CMC section of the application. Documentation in the CMC section ensures
consistent batches of the drug candidate for production and packaging.
10.2.2 Animal Pharmacology and Toxicology Studies
Preclinical data assessing whether the product is likely to be efficacious and most
importantly safe for testing in humans is the primary focus of this section.
10.2.3 Clinical Protocols and Investigator Information
Protocols for the proposed clinical studies to insure subjects are not exposed to
unnecessary risks, and information regarding the qualification of the clinical investigators who will oversee the administration of the experimental drug are described
in these sections.
10.2.3.1 Clinical Trials Phase 1–3 [2] In addition to Phase 1–3, there are also
Phase 0 (exploratory) and Phase 4 (post marketing) clinical trials; both not discussed
further in this chapter.
10.2.3.2 Phase 1 In a Phase 1 trial, a small (20–80) number of healthy volunteers
are dosed to assess the safety, tolerability, pharmacokinetics, and pharmacodynamics
of a drug. It is rare that any indication of the efficacy of the drug candidate can be
assessed in a Phase 1 trial. A typical Phase 1 study design is a single ascending dose
(SAD) in which a single dose is administered, and if no adverse effects are observed,
a higher single dose is given. Dosing continues until either the plasma drug levels
reach those estimated to be safe from the preclinical studies or side effects are noticed
(maximum tolerated dose (MTD)). In a multiple ascending dose (MAD) study,
a group of healthy volunteers are administered multiple doses of the drug. If no side
effects are noticed, a second group is given a higher dose, and the process is continued
until the dose calculated to be safe from the preclinical studies are reached, or side
effects are noticed. Administering the drug under fasting and fed conditions and
determining the effects of food on drug concentration in blood levels is also
investigated.
The two main endpoints of a Phase 1 trial are determining the blood levels of the
drug versus time after dosing (pharmacokinetics) and establishing the MTD
10.2.3.3 Phase 2 Phase 2 trials are designed with a larger group of volunteers
(20–300 patients) to continue to establish safety and to begin to assess the efficacy of
a drug candidate. Doses selected are believed to be safe, either based upon the MTD
found in Phase 1 or the Phase 1 pharmacokinetics in conjunction with the preclinical
pharmacokinetic and toxicological studies. In addition to being safe, the selected
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doses administered to patients are also predicted to be efficacious based upon the
efficacy established in animal and human cell or tissue studies during the discovery
phase of the project.
10.2.3.4 Phase 3 Phase 3 trials involve several clinical centers with a large
number of patients (300–3000 or more depending upon the disease/medical condition
studied). The aim is to assess the safety and efficacy of the drug candidate compared
to the standard of care treatment for the disease indication. The length of the studies
will vary depending on the disease indication. For example, according to the FDA
guidelines [4], in an obesity trial, patients are dosed for 12 months which provides
a complex and costly study to conduct.
A new drug application (NDA) can be submitted to the FDA based upon the data
obtained from one or several Phase 3 studies. Upon FDA approval, the drug can be
marketed.
10.3 STRATEGIC GOALS FOR THE PRECLINICAL DEVELOPMENT
The attrition rate of dr ug discovery and development candidates (Figure 10.2) is key
to the strategic goals for selecting a preclinical candidate. There are two major
decreases in the number of compounds advancing from one stage to the next. The
first and largest decline is the number of drug candidates in discovery advancing to
the clinic. Based on the data set, only 1 out of every 150 d rug candidates in discovery
enters clinical development (Figure 10.2). Once a drug candidat e has been shown to
be sufficiently efficacious in a nimal models of the disease, and safe in preclinical
development for the FDA to approve the IND application, the average likelihood for
the drug candidate to reach market is about 1 in 10. For a pharmaceutical company,
Figure 10.2 Attrition rate of drug candidates. The number of compounds prepared versus
the phase of development is plotted. For 1500 compounds in discovery, only 9 enter a Phase 1
clinical trial. Data from Ref. 3.
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there is increased motivation to support the project and drive the drug candidate
forward toward the market. For a smaller company, compounds in the clinic will
significantly increase the chance of attracting investors willing to provide the
capital required to continue developmen t, close an attractive licensing deal, or sell
the asset or company.
In the latter case, a business plan may incorporate preclinical studies early in the
development plan to initiate a Phase 1 clinical study even if these preclinical studies
need to be supplemented to initiate Phase 2 and 3 studies. For most indications, the
efficacy endpoints of a clinical study (Phase 2 and 3) will involve dosing for months
to years. The preclinical toxicological studies have to support this length of human
dosing, which requires long and costly preclinical toxicological studies. If the
business strategy is to place a compound into the clinic and conduct a Phase 1 study
early in the development plan, 1 month toxicological studies are all that is required.
These preclinical studies are substantially less time and money consuming than a
preclinical package that may be required to support Phase 2 and 3 clinical trials. Thus
the clinical development plan must account for the risk for conducting a Phase 1 trial
before the entire preclinical data package has been completed.
10.4 SELECTION OF PRECLINICAL DEVELOPMENT CANDIDATE
The cost and the precise IND enabling preclinical toxicology and pharmacology
studies will vary depending on the disease indication. Typically, one should budget
$1.5 million with a timeline of 9–12 months to complete the studies required for an
IND submission. It is therefore unrealistic to conduc t IND enabling studies for several
candidates and strategies to select one candidate are necessary.
The reasons for drug candidates to fail in development have evolved over time. In
1991, the major reasons for failure where attributed to issues with PK and bioavailability. Since then, assays to assess these parameters prior to selecting a preclinical
candidate have been developed. In 2000, the number of clinical failures due to poor
PK or bioavailability was less than 10%; poor efficacy, problems with toxicology or
safety, and commercial reasons attributed to the majority of failures [5]. It is clear that
assessment of PK, efficacy, and safety and toxicology should be evaluated prior to
selecting a candidate for preclinical development (commercial reasons are beyond
the scope of this chapter).
10.4.1 Efficacy
The efficacy of the drug candidate is measured in the discovery phase (Figure 10.1).
As the compound advances to preclinical and Phase 1 clinical development, the drug
candidate is evaluated for safety as opposed to efficacy.
Based upon in vitro efficacy, the most potent molecules are the preferred
candidates for development. However, molecules that are potent in vitro may not
be the most potent and efficacious in vivo. A number of parameters such as cell
permeability, stability in plasma, plasma protein binding, and clearance from plasma
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will determine the concentration of the drug candidate at the molecular target in vivo.
These parameters are also considered when selecting leads to be tested for efficacy
in vivo. The most potent and efficacious lead in vivo are selected as the preclinical
candidates. The more potent the drug candidate, the lower the dose will be, which
presumably reduces the risk for off-target side effects.
A clear relationship between the pharmacokinetics (plasma/tissue levels of the
drug or active metabolites versus time after dosing) and pharmacodynamics (efficacy) of the drug candidate should be known. If a drug candidate interacts with a
molecular target with an EC
50
of 1 nM, in principle, the free fraction (not bound to
plasma proteins) of the drug candidate needs to be in the low nanomolar range for as
long after dosing as the drug candidate shows efficacy. Alternatively, the efficacy of
a drug candidate may depend on a more or less complete inhibition of a target and
thus the drug concentrations for that target need to be greater. Lastly, the efficacy
may involve activating a target that leads to a cascade of events that is sustained
even after the drug levels are below target activation. Regardless of the mechanism
for efficacy, the PK/PD relationship needs to be described for the preclinical
candidate.
The FDA will not approve a drug that is inferior to a drug already marketed thus
establishing the goal for efficacy. For an indication that does not have any marketed
drugs, efficacy may be based upon estimates for what can be successfully marketed.
At project initiation, a target profile should be established to favorably compare a
preclinical candidate to marketed benchmarks for the desired indication. All lead
compounds for preclinical development should be at least as efficacious as the target
profile dictates.
If a drug candidate has the same target as an already marketed drug, the drug
candidate must be at least as efficacious in pharmacological models of the disease,
more potent to the target and possess a greater safety margin. Statins are an excellent
example of a class of drugs in which different compounds have been successfully
developed and commercialized. Bayer’s Baycol was initially marketed until severe
side effects removed the drug from patients [6]. Merck’s Zocor is on the market and
is metabolized by a single Cyp(3A4) [7], which increases the risk of drug–drug
interactions. Astra Zeneca’s Crestor is more potent than Zocor, and is not metabolized [8]. These improvements over previously marketed drugs have allowed Crestor
to gain a substantial market share.
Another example of efficacy guiding the selection of a preclinical candidate is
OSI Prosidion’s PSN602, a monoamine reuptake inhibi tor with 5-HT1A agonism [9]. In this case Abbott’s Meridia (Sibutramine) [10] is the benchmark.
Sibutramine is a dual serotonin (5-HT)/norepinephrine (NE) reuptake inhibitor
approved for the treatment of obesity. The drug is associated with elevations in blood
pressure and heart rate in some patients limiting the dose that can be used. By
designing a 5-HT1A agonist in conjunction with monoamine reuptake inhibition,
OSI Prosidion PSN602 was as efficacious as sibutramine at reducing body weight in
a rodent model of obesity, but exhibits a more favorable cardiovascular safety profile
after single doses.
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It is more difficult to set the efficacy criteria if the molecule under development is
first-in-class. Metabolex MBX-2982 [11] targets the G-protein coupled receptor 119
(GPR119) as a therapy for Type-2 diabetes [12] and is currently in Phase 1 clinical
studies. Most likely, Metabolex aims to advance MBX-2982 to Phase 2 as soon as
possible to generate proof-of-concept efficacy data in terms in patients. Concurrently,
Metabolex is developing second-generation compounds as an improvement to the
properties of MBX-2982. If the human efficacy data indicates that MBX-2982 is a
validated target for Type-2 diabetes, Metabolex may terminate the development of
MBX-2982 and initiate the clinical development with an improved second-generation
molecule.
10.4.2 Safety/Tolerance
In addition to efficacy (discussed in Section 10.4.1), safety is another parameter to be
thoroughly assessed in guiding the selection of a preclinical candidate. There are a
number of assays that are both inexpensive and may be used under non-GLP
conditions to generate data on several repr esentatives from a chemical lead series
to help selection of a preclinical candidate with a strong probability of passing the
preclinical filter in terms of safety parameters (Table 10.1)
10.4.2.1 HGPRT Forward Mutation, Ames Test, and Herg Channel Inhibition
For most disease indications a positive HGPRT forward mutation, Ames, or Herg
channel inhibition test is a “show stopper” for a drug candidate and the compound
should not be considered for future development. However, there are exceptions such
as the cancer drug Sutent. Sutent inhibits the Herg channel [13], and yet is approved
and marketed. Nevertheless, it is advisable to generate data on these assays mentioned
above at an early stage to be able to screen compounds that do not warrant any further
testing.
10.4.2.2 Receptor Binding and Kinase Panels To assess the possibility for offtarget side effects, the drug candidate is screened against a panel of receptors and
kinases. If the drug candidate does interact with some of the receptors in the panel,
the risk for off-target effects is greater than if there were no interactions with the
TABLE 10.1 Non-GLP Assays to Assess Safety of a Chemical Lead
In Vitro In Vivo
HGPRT forward mutation Pharmacokinetics
Ames test Non-GLP toxicological study
Herg channel
Receptor binding and kinase panels
Cyp inhibition, induction, and metabolism
Protein binding
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receptors. A drug candidate’s interaction with receptors does not necessarily “kill” the
compound since there still may be a range for on-target efficacy. However, positive
results in any nontarget assay raises concerns about the compound and the data may
assist the selection of a candidate among a series of molecules. For example, assessing
the effects on as many kinases as possible is valuable to develop a selective kinase
inhibitor per-clinical candidate.
10.4.2.3 Cyp Inhibition, Induction, and Metabolism A drug candidate that
affects CYP inhibition, CYP induction, or is metabolized by a single CYP should
be “flagged” as a potential issue. As an example, the drug candidate inhibits an
isoform of CYP metabolizing other drug substances. Thus, upon administration of the
drug candidate, the metabolism of any compound which is a substra te for this CYP
will be reduced, presumably leading to higher drug levels, and prolonged exposure of
these compounds. This drug–drug interaction can then effectively lead to an overdose
of the compounds metabolized by the particular CYP. Inhibition of the major human
drug metabolizing CYPs [14] 3A4, 2D6, 2C19, 1A2, 2A6, 2B6, 2C8, 2C9, and 2E1
should be examined. A positive result may not “kill” the compound but should be
carefully reviewed before advancing the candidate further.
A compound that induces a CYP has the potential to have an opposite effect. If a
drug candidate induces a CYP, any drugs the patient is taking that is metabolized by
this CYP will presumably be eliminated faster, and effectively be under dosed.
If a compound is metabolized solely by one CYP, there will be a greater risk for
drug–drug interactions. Examples on the market displaying this phenomenon include
Zocor which is metabolized solely by CYP 3A4. In this case, in addition to the risk for
drug–drug interactions, the drug should not be taken with grape fruit juice. Drinking
grape fruit juice will inhibit CYP3A4 and cause a substantial increase in the plasma
concentrations of Zocor [7] as well as other marketed drugs metabolized by CYP3A4.
Drugs metabolized by one CYP can also be strongly affected by CYP polymorphisms
in the population. These variations include different expression levels or activities of
CYPs and can lead to variations in drug concentration levels.
10.4.2.4 Protein Binding Binding of a drug candidate to plasma proteins will
lower the free fraction of the compound available to a target. A compound possessing
strong protein binding typically requires an increase in dose to affect the target. Also,
strong binding to plasma proteins poses a risk for drug–d rug interactions. If a large
fraction of a molecule is bound to plasma proteins and a second molecule is introduced
binding to the same site on the plasma proteins, the compet ition for binding can result
in a significant increase in the free-drug concentration. For example, if 99% of a
molecule is protein bound and competition by a second molecule reduces protein
binding to 90%, there will be a 10-fold increase in the free-drug concentration of the
first molecule.
Conversely, binding to plasma proteins is not necessarily an undesired property.
Novo Nordisk GLP-1 analog Liraglutide is designed to bind to albumin. Bound to
albumin, Liraglutide is protected from proteolysis by dipeptidyl peptidase-4 that
results in prolonging the plasma half-life of the drug candidate [15]
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10.4.3 PK
Determination of the bioavailability, half-life, and clearance of a drug candidate and
the ability to correlate the plasma concentration of the drug candidate with potency
on the target are important to select a lead compound. With several potential lead
candidates, these parameters are used to prioritize compounds with the bioavailability
and clearance rates that support the desired dosing regimen. To obtain these PK
parameters, typical studies include a single dose administered by intravenous and the
expected route of administration (i.e., oral).
In addition to single-dose PK studies, a multiple dose PK study will determine the
dose proportionality of the drug candidate. Plasma levels of the drug candidate should
increase linearly with dose. If the plasma concentrations of the drug do not increase
above a certain dose, presumably the absorption of the drug limits the exposure.
The highest dose producing an increase in plasma concentration of the drug will be the
highest dose that can be used in toxicological studies in this species. Since doses
proposed to be tested in humans need to be supported by toxicological studies in
animals, this will limit the doses than can be tested in the clinic. Ideally, repeat dosing
of a drug candidate should not change the PK parameters. If there is an increase in drug
concentration in plasma due to repeat dosing, this in conjunction with protein binding
is correlated with the efficacy of the compound (PK/PD relationship).
The cost of non-GLP repeat dose PK studies with a few select drug candidates is
rather nominal com pared to the IND enabling studies.
10.4.4 Non-GLP Toxicological Study
The in vivo efficacy studies in discovery should generate data at varied doses to
identify a minimum efficacious dose (MED) and an MTD. The difference between
an MED and an MTD should be as large as possible to decrease the likelihood of side
effects at efficacious doses. The assessment of an MTD varies with the disease
indication and target. In some cases, an MTD is defined by off-target effects and an
assessment is made based upon measures of gross toxicology; change in organ
weights, animals behavior, morbidity, or mortality. Alternatively, an MTD may be
driven by the efficacy on the target. For example, a molecule increasing the secretion
of insulin may lower blood glucose levels below physiological levels causing
hypoglycemia.
In addition to these fundamental assessments of toxicology, prior to selecting a
final preclinical candidate, conducting a 14-day non-GLP formal toxicological study
in rats at several doses will identify any potential issues associated with the drug
candidate before costly GLP studies are initiated.
From the analysis of the toxicological screening data, drug candidates may be
ranked. There are no generic rules to rank compounds based on this data since the
disease indication will dictate the safety or allowable side effect. Oncology drugs
often are associated with rather severe side effects. Alternatively, a recent FDA
guidance document requires data demonstrating that antidiabetic drugs will not result
in an unacceptable increase in cardiovascular risk [16].
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10.5 CMC
There are several factors in a CMC sect ion that may be assessed under non-GLP
conditions at a modest cost in time and money to help select a successful preclinical
candidate.
10.5.1 Solubility
Typically compounds with low solubility are more difficult to develop since this
impacts testing the activity of the compound in vitro and in vivo for activity and safety.
Thus for compounds with equal other properties, it is advisable to select a candidate
with high solubility.
There are examples of drugs with very low solubility that were commercialized.
The COX-2 inhibitor Vioxx (rofecoxib) (removed from the market due to cardiovascular issues) had high bioavailability (>90%) for a dose of <50 mg [17].
10.5.2 Solutions Stability
A drug candidate needs to be stable in solution. Typically, a high- performance liquid
chromatography (HPLC) method analyzes the concentration of a compound at
varying pH values to assess its stability. An acid-labile compound planned to be
dosed orally might need to be formulated in a capsule to prevent degradation in the
stomach, while a compound which is not very stable at any pH is potentially
problematic.
10.5.3 Synthetic Feasibility, Solid-State Stability, and Hygroscopicity
The large-scale synthesis required for GMP production is typically different from the
laboratory-scale synthetic route. In some instances, it is difficult to evaluate the final
synthetic route at an early stage; however, understanding the cost of goods is essential
for selecting a drug candidate.
Large-scale synthesis can produce material that is different in the solid state
compared to laboratory-scale material. These polymorphs of a compound can behave
differently chemically and biologically. Solid-state stability may be assessed with
micro calorimetry and an accelerated stability program will provide data that may
assist in selecting a preclinical candidate. A hygroscopic compound is more difficult
to manufacture and formulate and should not be selected as a lead compound.
10.5.4 Patent Position
Although patents are not part of the IND application per se, the intellectual property
of the invention is a commercially important factor and should be addressed in the
hit-to-lead stage. Typically, claiming the composition of matter for a drug candidate
is the most critical claim. Ironically, there are examples in which a development
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candidate without a strong patent position was advanced to the clinic. Biostratum had
completed Phase 2 clinical trials with Pyridorin with promising efficacy when it
became evident the active ingredient was commercially available via the internet,
eventually leading to the failure of the company [18]. The rights to Pyridorin have
since been acquired by Nephrogenex (http://www.nephrogenex.com), and the drug
candidate is still in clinical development.
10.6 PRECLINICAL STUDIES
Although IND application has three general areas as discussed above, different
disease indications differ in terms of the details that are required for each section.
While guidance can be found on the Center for Drug Evaluation and Research
(CDER) website [19], the requirements are under constant evaluation, and changes
are often implemented. It is important to arrange to meet with the FDA to review the
plans for generating an IND package in a pre-IND meeting to ensure that the planned
studies will provide the data needed. If a company does not have a designated
development organization, there are contract research organizations (CROs) that can
design and conduct the required studies. Preclinical studies require chemical, animal
pharmacology, and medical knowledge for the CMC , nonclinical pharmacology and
toxicology, and clinical sections, respectively. It is advisable to designate an in-house
project manager with some experience, or to work closely with a consultant with
experience in preclinical development.
It is important to realize that preclinical drug development is a process in which
data are collected aimed at terminating a compounds development. The drug
candidate needs to pass the battery of safety and toxicology analysis. As mentioned
above, some of the parameters analyzed are precautionary and should not end a
compounds development. The whole process is designed to filter out compounds that
are not suitable to be tested in humans since no improvements are made to the drug
candidate once in this process.
It is not possible to design one generic preclinical study package. There are
indications such as antiinfectives in which case the drug needs to be safe, and most
likely will be administered for one or a few weeks. In this case, 1 month toxicological
studies will suffice. Conversely, the safety and toxicological hurdles for an oncology
drug candidate, while typically administered for a longer period of time than an
antiinfective, will be lower than for most indications. Tarceva, marketed by OSI
Pharmaceuticals and Roche for advanced nonsmall cell lung cancer and pancreatic
cancer causes a rather significant rash which would not be acceptable for most other
indications [20].
Alternatively, drugs for diabetes presumably will be dosed daily for most or the
remainder of the patient’s life and the toxicological studies need to include dosing for
1 year. For some classes of diabetes drug candidates, such as peroxisome proliferator-activated receptor (PPAR) agonists, the FDA requires a 2 year carcinogenicity
study in two species to be completed before dosing in man for longer than
6 months [21].
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10.6.1 Example 1: IND Enabling Data Package to Support 1 Month
Dosing in Man
A generic IND enabling data package for the animal pharmacology and toxicology
studies to dose humans for 1 month is displayed in Figure 10.3. This preclinical data
package can be generated within 1 year with the toxicity studies requiring the longest
duration (9 months).
10.6.2 Example 2: Peroxisome Proliferator-Activated Receptor Agonist
for Type-2 Diabetes
A generic IND enabling data package for the animal pharmacology and toxicology
studies to dose a PPAR agonist for Type-2 diabetes in humans for more than 6 months
is displayed in Figure 10.4. Due to chronic toxicity studies that require >2 years to
generate data, this preclinical data package is assembled in 3.5 years.
There have been several failures for PPAR agonists in late stage clinical trials due
to an increase in the incidence of cancers. This has led FDA to request the 2 year
carcinogenicity study to be completed in two species before the drug is given to
humans for more than 6 months. Since Phase 3 trials will include dosing for more than
6 months, the carcinogenicity studies have to be completed before the Phase 3 trials
can be initiated. A 2 year carcinogenicity stud y actually requires 3 years to complete.
Hence, assuming Phase 1 and 2 studies require 2.5–3 years to complete, the
carcinogenicity studies should be initiated approximately at the time of IND filing
to avoid a delay in development. Sine carcinogenicity studies may cost as much as
fivefold the cost of an initial Phase 1 study, candidate selection for development is
critical at a very early stage.
A complete set of experiments for an IND enabling package is displayed in
Table 10.2. Rat and dog are assumed as the two species for toxicological assessments.
Although many of the items outlined in Table10.1 are conducted to select a preclinical
candidate, the tests need to be repeated under GLP conditions to be included in an IND
application.
Below are brief discussions of the parameters that were not discussed previously.
10.6.3 Mass Balance
A radiolabeled sample of the compound is synthesized for a mass balance study.
Typically, rats are dosed with the radiolabeled compound and urine and feces are
collected over a period of time for analysis of radioactivity. Recovery of the radiolabel
as the intact compound or metabolite in urine, feces as well as tissue and organs should
be 100% to account for absorption, distribution, metabolism, and excretion of the
compound.
10.6.4 Animal Pharmacology and Toxicology Studies
For a detailed discussion on animal pharmacology and toxicology studies, see
Chapter 7.
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