1 s2.0 S0168365911004809 Main PDF

Journal of Controlled Release 156 (2011) 281296
Contents lists available at ScienceDirect
Journal of Controlled Release

j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / j c o n r e l
Review
Recent advances and novel strategies in pre-clinical formulation development: An

overview
Amit K. Shah a, Sunil A. Agnihotri b,
a
b
Product Development, Aurolife Pharma, LLC. 2400 Route 130 N, Dayton, NJ 08810, United States
Product Development, Frontage Laboratories, Inc. 75 E Uwchlan Ave, Exton, PA 19341, United States
a r t i c l e
i n f o
Article history:
Received 1 January 2011
Accepted 15 May 2011
Available online 7 July 2011
Keywords:
Preclinical
Physico-chemical properties
Formulation
Stability
In vivo models
a b s t r a c t
Preclinical proling for a New Chemical Entity (NCE), if carried out carefully, can be a good predictor of human
clinical outcome. Along with the pre-clinical study design a thorough understanding of the physico-chemical
properties of the drug candidate and a careful selection of the formulation development strategy are of high
importance.
The study scientist can experience various challenges in executing a pre-clinical study. This review article
provides an overview of the signicance of pre-formulation study parameters and their relevance to preclinical
studies. Various physico-chemical properties such as solubility, partition co-efcient, and permeability are
attributes critical to the performance of the drug substance. This article presents unique formulation
development strategies for the successful completion of pre-clinical studies. Formulation development approach
for a pre-clinical study involves taking into consideration various important factors such as duration of the study,
Biopharmaceutics Classication System (BCS) of the drug, intended duration of action and the desired route of
administration. These parameters play key role in the selection of solubilizers, surfactants, co-solvents and
optimum pH for the formulation. Two most common routes of administration in the early screening of
pharmaceuticals viz., oral and intravenous are emphasized. The article also describes recent advances in
preclinical formulation development including selected examples of in vivo preclinical models for anti-cancer,
anti-viral, anti-diabetic and anti-hypertensive drugs. Adherence to the regulatory requirement is also the key to
successful completion of the preclinical development. An overview of preclinical formulation development along
with basic concepts and the recent studies conducted in the past decade are presented in this review.
2011 Elsevier B.V. All rights reserved.
Contents
1.
2.
3.
4.
5.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . .
1.1.
Challenges in preclinical formulation development . . .
1.2.
Stages of pre-clinical studies . . . . . . . . . . . . . .
Pre-formulation studies in preclinical development phase . . .
2.1.
Drug solubility . . . . . . . . . . . . . . . . . . . .
2.2.
Partition co-efcient and in vitro permeability . . . . .
2.3.
Dissociation constant . . . . . . . . . . . . . . . . .
2.4.
Physical form of the drug substance . . . . . . . . . .
Formulation development in pre-clinical development phase . .
Formulation development strategies . . . . . . . . . . . . .
4.1.
Duration of the study . . . . . . . . . . . . . . . . .
4.2.
Biopharmaceutics classication system of drug substance
4.3.
Desired route of administration . . . . . . . . . . . .
4.3.1.
Oral route . . . . . . . . . . . . . . . . . .
4.3.2.
Intravenous and intraperitoneal route . . . . .
4.4.
Intended duration of action . . . . . . . . . . . . . .
Stability of pre-clinical formulations . . . . . . . . . . . . .
Corresponding author. Tel.: + 1 484 870 5597.

E-mail address: agnihotrisunil@gmail.com (S.A. Agnihotri).
0168-3659/$ see front matter 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.jconrel.2011.07.003
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6.
A.K. Shah, S.A. Agnihotri / Journal of Controlled Release 156 (2011) 281296
Recent advances in pre-clinical studies . . . . . . . .

6.1.
Formulation development . . . . . . . . . . .
6.2.
In vitro modeling . . . . . . . . . . . . . . .
6.3.
Pharmacodynamics in pre-clinical studies . . . .
7.
In vivo pre-clinical models . . . . . . . . . . . . . .
7.1.
Anti-cancer . . . . . . . . . . . . . . . . . .
7.2.
Anti-viral . . . . . . . . . . . . . . . . . . .
7.3.
Anti-diabetic . . . . . . . . . . . . . . . . .
7.4.
Anti-hypertensive . . . . . . . . . . . . . . .
8.
Interspecies differences in physiology and pharmacology
9.
Regulatory requirements in pre-clinical studies . . . .
10.
Analytical testing in pre-clinical studies . . . . . . . .
11.
Conclusion . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . .
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.
1. Introduction
Preclinical drug development is a phase of research that is
undertaken on appropriate animal models before clinical trials (testing
on humans) can actually begin. Preclinical studies are used to evaluate
the safety of a new compound so that the appropriate compounds are
brought into human trials. Compounds must be effective in the
treatment of disease, relatively free of unwanted side-effects and have
treatment regimens (dose, dosage form and frequency of administration) compatible with the target patient population and disease.
Preclinical drug development is a risk-based approach in which
nonhuman (animal model) safety and efcacy information is extrapolated to a potential human outcome. Actually, the preclinical
development agenda for many novel therapies is even more risky in
predicting clinical results when very limited data is available to support
the use of the animal model under study. In the end, results of preclinical
studies (nonhuman models) are validated by conducting clinical studies
(human studies). A thorough understanding of the pharmacological and
toxicological preclinical drug response with respect to dose, frequency,
and route of administration allows scientists to initiate and continue
human trials under rational and ethical conditions.
Typical pre-clinical conditions contain a starting dose and a dose
frequency that produces an intended level of pharmacologic response,
a safe dose escalation scheme which permits differentiation of
response as a function of drug exposure, an understanding of when
potential toxicity may outweigh potential pharmacologic benet, and
an information of pharmacokinetics and response variability [1]. The
nonhuman model will generate a body of evidence and condence
whether the drug candidate is worthy of further development or
whether it should be terminated from the development pipeline. A
thorough understanding of any nonhuman model is fundamentally
important so that drug-related outcomes can be separated from
normal, endogenous variability or other processes unrelated to the
drug. Rodents, canines, and nonhuman primates have become
common preclinical models because of the established understanding
of these animals and their underlying physiology [24].
Understanding of similarities and differences between nonhuman
and human physiological systems is vital to acquire quality information
from preclinical agenda. Virtually every study and every decision to be
made on the development of a drug candidate will be predicated on the
assumption that preclinical models are a predictor of human exposure.
Ultimately, the understanding of preclinical drug disposition (distribution, metabolism, and excretion) coupled with an understanding of cell
or tissue specic activity/toxicity completes the knowledge base for a
drug candidate to move into and through clinical evaluation.
Testing of new drugs in humans cannot begin until there is profound
evidence that the drug (or the drug product) can be used with
reasonable safety in humans [5]. The basic goal of preclinical
development phase is to assess potential therapeutic effects of the
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substance on living organisms and to gather sufcient data to determine

reasonable safety of the substance in humans through laboratory
experimentation and animal investigation [6]. The FDA requires no prior
approval for investigators or pharmaceutical industry sponsors to begin
a preclinical investigation on a potential drug substance. Investigators
and sponsors are, however, required to follow Good Laboratory
Practices (GLP) regulations [7]. GLPs govern laboratory facilities,
personnel, equipment, and operations. Compliance with GLPs requires
procedures and documentation of training, study schedules, processes,
and status reports, which are submitted to facility management and
included in the nal study report to the FDA. Preclinical investigation
usually takes one to three years to complete. If at that time enough data
are gathered to reach the goal of potential therapeutic effect and
reasonable safety, the product sponsor must formally notify the FDA of
its wishes to test the potential new drug on humans. A general
preclinical development program is outlined in Fig. 1.
Unlike the past, when the majority of research compounds had a
relatively small molecular weight and acceptable solubility, the
number of larger and less soluble molecules displaying permeabilityand/or solubility-limited absorption has increased tremendously
during the last few years. Therefore, it is not surprising that traditional
formulation approaches such as disperse and dose are no longer
adequate in pharmacology laboratories. Furthermore, the use of
different formulation systems for the various in vivo studies in early
development may lead to completely different exposures and hence
inconclusive results. Therefore, a structured approach to efcient
selection and development of preclinical formulations is mandatory.
1.1. Challenges in preclinical formulation development
Incomplete physico-chemical characterization, limited availability of
drug, short timelines and unfavorable drug properties (e.g. poor
solubility and poor permeability) are major challenges in pre-clinical
formulation development. To address these challenges a systematic
development plan that entails simultaneous development of conventional and advanced delivery systems is important. Many times
variability in the quality of drug candidate such as particle size
distribution, crystallinity, and polymorphic form pose additional
hurdles. Additionally, different requirements regarding formulation
properties may require scientists to develop several different formulations at various stages of preclinical studies. Consequently, scientists
planning early in vivo studies need to address several challenges to allow
fast and successful candidate proling [8]. The scientist must;
1. Dene strategies to select and provide well-characterized formulations yielding sufcient exposure and tolerability.
2. Dene strategies to overcome drug delivery challenges due to
unfavorable biopharmaceutical or physico-chemical properties of
early candidates.
Drug
Discovery
Preclinical
Development
Clinical
Trial
API
Manufacturing
Formulation
Analytical/
Bioanalytical
283
affect various properties such as solubility, bioavailability, and

stability of the drug substance [10,11]. A thorough salt screening
process may result in one or more salt form which are less challenging
to synthesize and are favorable for formulation development. Once a
lead drug candidate is identied, an understanding of various physicochemical properties such as aqueous solubility, pH solubility, pKa
(dissociation constant), and log P is essential prior to initiating any
formulation development activities.
IND
PK/PD/ADME
Toxicity/
Safety
GMP/GLP/CTM
Fig. 1. A general preclinical development program. IND, Investigational new drug; GMP,
Good manufacturing practice; GLP, Good laboratory practice; CTM, Clinical trial
material.
3. Dene strategies to achieve optimum data comparability for

different studies (Harmonization/Standardization).
4. Follow a structured approach to efciently select and develop
preclinical formulation.
1.2. Stages of pre-clinical studies
There are three primary stages of pre-clinical study such as
pharmacological study, pharmacokinetic study, and dose ranging
study (toxicological study). A brief description of each study is given
below:
1. Pharmacology study is carried out to evaluate the effect of drug on
the body, i.e. to determine the pharmacological or physiological
effect of the drug on the body. It also assists in determining the
target organ and/or tissue, determine the effect of concentration
and the drug concentration-activity relationship, identify the need
for safety monitoring for future studies.
2. Pharmacokinetic (PK) study is carried out to evaluate the effect of
body on the drug, i.e. the fate of the drug upon administration,
effects such as Absorption, Distribution, Metabolism, and Excretion
which is universally known as ADME. Recently, the interaction of
the drug with the body has also been related to the process of
release of drug from the formulation. This is termed as Liberation.
Therefore, PK is described newly as LADME.
3. Toxicology study is carried out to evaluate the extent and effect of
systemic absorption of drug candidate. Toxicology study can be
further categorized into various stages such as single dose toxicity
study, multiple dose/repeat dose toxicity study, dose escalation
study, carcinogenicity study, and reproductive toxicity study. A
fairly recent terminology toxicokinetics has been introduced to
evaluate preclinical toxicity. Toxicology study assists the scientist
in establishing the No Observable Effect Level (NOEL) or the No
Observable Adverse Effect Level (NOAEL) for a new chemical
entity. The toxicokinetic data for either NOEL or NOAEL can provide
a safe starting and maximum dose for Phase I clinical studies to the
clinical investigator [9].
2. Pre-formulation studies in preclinical development phase
Pre-formulation study is the key to developing a successful
formulation. Physico-chemical characterization of a new drug
substance should be performed regardless of its intended use. Upon
identication of a lead candidate the drug discovery group needs to
invest resources in identifying and synthesizing appropriate salt form
of the drug candidate. Salt form of the drug substance can signicantly
2.1. Drug solubility

Two important factors in the entire development process are drug
solubility and bioavailability. Poor water solubility has been attributed
to almost half of the 150,000 new molecular entities (NMEs)
synthesized annually by pharmaceutical companies, and is also
claimed to reduce the performance of more than 10% of successfully
marketed drugs [12].
Drug solubility is an important parameter for both oral and
intravenous administration. For oral drug delivery, aqueous solubility
in various pH media is one of the most important property of the drug
substance which can signicantly affect the drug absorption and
subsequently the bioavailability [13]. A careful selection of the buffer
system and its concentration is essential for pH solubility screening.
The type of the buffer salt and its concentration can have a signicant
impact on the solubility of the drug substance. For instance, a pH 4.5
buffer can be prepared using either acetate or citrate salt. These two
buffers at identical concentrations my show a difference in the
solubility of the drug substance attributed to the structural properties
of the drug substance and/or the salt form. The concentration of the
buffer system also must be selected carefully. A very high buffer
concentration may lead to erroneously high solubility number but
may not be feasible for i.v. administration.
Various techniques are available for solubility determination of
drug substance. The oldest and the most commonly used solubility
measurement technique was developed by Higuchi and Connors [14].
Saturation solubility of the drug substance can be carried out by
adding aliquots of drug substance to a small volume of the solvent
(usually 24 mL). The mixture is then allowed to equilibrate by
shaking or rotating at ambient temperature for a period of at least
24 h or until un-dissolved particles are observed at the bottom of the
vial/tube. The mixtures are centrifuged and/or ltered, the supernatant withdrawn and analyzed for assay. Most researchers like to
carry out the saturation solubility study at ambient temperature,
whereas some like to perform the study at 37 C. There are
advantages of each technique. If the solubility study is performed
at room temperature it will provide true solubility which can assist
the formulator in designing a robust formulation. In this particular
case saturation solubility at elevated temperature may be erroneous
because more amount of drug might go in solution at higher
temperatures which may eventually (and slowly) precipitate out at
ambient temperature and give incorrect solubility values, therefore
causing problems during formulation development. However, pH
solubility study at 37 C will provide accurate prediction of in vivo
solubility when the drug is traveling through the GI tract. Hence,
the study design lies on the primary objective of the study. In
addition to the pH solubility of the drug, equilibrium solubility
in various solvents that can be used for animal studies should be
determined to identify the correct solvent for future formulation
development.
In early drug development, a quick and robust tool is required to
analyze the solubility of the drug substance. For this reason several
high-throughput techniques are available for solubility screening.
These techniques can assist the pre-formulation group to successfully
screen thousands of compounds in a short period of time. A list of
these techniques are presented [15] in Table 1.
284
Table 1
High-throughput solubility determination techniques (reproduced from Ref. [14]).
Organization/ Technique
inventor
Description
Symyx
Ninety-six-well library format, wide range

of solvents, determination of pH solubility,
pKa, log P and temperature solubility. Can
measure up to 192 compounds a week
Solubility, temperature solubility, and
chemical stability screening
Saturation/Equilibrium solubility
High throughput low volume liquid
chromatography
Intrinsic solubility, pH solubility prole,
pKa determinations. FDA recognized
Thermodynamic solubility, multiple
wavelength scanning [16]
Aqueous and non-aqueous
saturation/equilibrium solubility [17]
Automated solubility
determination
platform
Bruker Optics SpecScreen xHTS

Anachem
Nanostream
ReactarrayTM
Nanostream CL
pION
pH metric technique
Chen et al.
UV plate reader
Chen and
Venkatesh
Miniature device
2.2. Partition co-efcient and in vitro permeability

The ratio of the equilibrium molar concentration of a drug in the
organic phase to that of the aqueous phase is termed as the partition
coefcient (Log P) of the drug. Log P is a key property in determining
hydrophilic or lipophilic characteristics of the drug substance. With
the advent of the high-throughput screening several molecules that
are identied in early stage are lipophilic and hence cause subsequent
challenges in solubility [18,19]. To some extent lipophilicity can
describe the extent of solubilization of a solute in a given solvent [20].
Most of the new drugs passing the initial screening by high
throughput technology such as molecular modeling are highly
lipophilic and are of relatively larger molecular weight as opposed
to drug candidates in the past, therefore, these molecules pose a
bigger challenge due to their solubility and permeability limitations
[18,21,22]. As a result the actual solubility/permeability of the drug
substance should be known. The molar aqueous solubility of an
uncharged solute can be calculated according to Eq. (1)[20].
LogSu = 0:8 logP0:01MP25
Where, Su, log P, and MP are the aqueous solubility, Octanol-water

partition coefcient, and melting point of the drug substance
respectively. As presented in the above equation the aqueous
solubility of a solute decreases with an increase in the log P and MP.
Octanol-water partition coefcient is also termed as Kow. Primarily
Kow plays an important role in the Quantitative Structure-Activity
Relationship (QSAR). The correlation between the chemical structure of
a drug and its biological activity has been studied primarily by Hansch
and Fujita [23]. QSAR in general can be described by the following
equation [24,25].
BR a bB cC
Where, BR is the biological response; B, C, are molecular

properties and a, b, are tting parameters. A large number of
molecular properties, such as molecular weight, log Kow, molar
refraction, etc. contributes to the above equation. Also, large fractions
of the QSAR's were established with only log Kow as the independent
variable, as represented by the following equation:
BR a blogKow
Secondly, Kow plays an important role in determining the hydrophobicity of the molecule, and subsequently it's partitioning into the
biological membrane. This, in turn, affects the bioavailability and
biological response of the drug candidate. Partition coefcient is also a
good indicator of diffusion or permeation across the cell membrane,

which is the rate-limiting step in the drug transport process.
There are two basic method of measuring lipophilicity. One of the
method traditionally used is octanol-water partition co-efcient [26]. In
this method the drug substance can be dissolved in the most soluble
phase (either octanol or water) and mixed with the other. The mixture is
placed in a shaker or a rotator and allowed to equilibrate for 24 to 48 h.
Aliquot of each media is withdrawn at a xed time interval and the drug
content is analyzed by HPLC. The second method relies on a library of
known log D value. The sample log D value is determined based on a
library of know log D standards [27]. Sample analysis is carried out by
liquid chromatography.
Due to poor physico-chemical properties of drug substance, the
ultimate aim of the drug development process is to increase the cellmembrane permeability, and to achieve signicant intestinal absorption. Also, regardless of the route of administration, cell membrane
permeation is required for a drug molecule to reach the general
circulation. Even after the intravenous, intramuscular, or any other
parenteral routes of administration, the drug can permeate in the
extracellular space via paracellular route, but the drug has to cross the
cell membranes in order to reach its biological target [28]. Therefore, in
vitro permeability study also plays a key role in determining a
formulation strategy. As a result, a reliable screening method is required
to assess the permeability of the drug. Various in vitro models are
available for the prediction of membrane permeability. These include,
excised tissue (perfused intestinal segments, everted sac, intestinal
mucosa), membrane vesicles (caco-2 cells, Madin-Darby canine kidney,
HT29), and articial membrane methods (immobilized articial
membrane, PAMPA). The major advantages of articial membrane
permeability over biological preparations are reproducibility and the
possibility of high throughput screening. Whereas, the major limitations
are, lack of enzymes, transporters, and paracellular pathways [29].
Generally, for highly active compound with high permeability
(P 10 6 cm/s) a successful pre-clinical trial depends on the solubility
of the drug substance [30].
2.3. Dissociation constant
Dissociation is the process in which the compound separate into
smaller ions, particles, etc. The portion of the drug dissociated can be
represented by dissociation constant (pKa), also known as ionization
constant. Ionization plays an important role in determining the
permeability of the drug molecule across various physiological
membranes. The drug can be transported across the physiological
membrane by either passive transport or carrier mediated transport.
The drug molecule containing ionizable group which can ionize between
pH 2 and 8 are very important because these can inuence the solubility,
membrane transport, protein binding, and volume of distribution [31].
For the drug containing ionizable group salts can be formed relatively
easily. Salts can enhance the rate of solubilization of the drug substance
at a given pH, however, the intrinsic solubility is equivalent to that of free
base or free acid. Solubilization of the drug in the form of a salt may
change the pH of the media if the buffering capacity of the solvent is
insufcient. Dissociation constant can be measured by traditional acidbase titration. However, for this to be effective the drug must be at least
slightly soluble in aqueous media. When performing acidbase titration,
care should be taken that the titration is not merely of the acid and the
base, and pKa of the drug substance is determined. High-throughput
techniques are also available for measuring ionization constant. One of
the recent high throughput techniques for measuring ionization
constant include capillary electrophoresis [32,33].
2.4. Physical form of the drug substance
In solid state, the drug substance can exist in amorphous or
crystalline state. Crystalline drugs can also exist in various polymorphic
forms. Characterization of polymorphism is critical for formulation

development studies. Various drug polymorphs can affect the solubility,
bioavailability and stability of the drug substance. For drug substances
existing in various polymorphs there may be only one active form and
also one or more polymorph may be meta-stable and can be converted
to a more stable polymorph. Generally, optical microscopy is utilized to
distinguish between the crystalline and amorphous forms of material.
Crystalline solids generally display birefringence, whereas amorphous
solids usually do not display birefringence. Polymorphs can be
characterized by Differential Scanning Calorimetry (DSC) studies. DSC
studies coupled with X-Ray Powder diffraction (XRPD) can show a
difference in the structural morphology of the drug substance.
Additionally, melting point, glass transition temperature (Tg), and the
decomposition temperature can be determined to obtain valuable
information about the drug substance.
Table 2
Commonly used pre-clinical excipients for oral and i.v. use along with their LD50 values.
Excipient
Route of
Functional
administration category
LD50
Gelucire
44/14
Oral
Rat oral: 20 g/kg
PEG 400
Oral/i.v.
0.9% NaCl
i.v.
Labrasol
Oral, i.v.
3. Formulation development in pre-clinical development phase
Hydroxy
Oral, i.v.
Propyl -Cyclodextrin
It is estimated that the development of a new drug from discovery to

marketing approval in the United States typically takes 1015 years. On
average the cost to research and develop each successful drug is
estimated to be $800 million to $1 billion [34,35]. For every 500010,000
molecules that enter the research and development pipeline, approximately 250 molecules enter preclinical program, only ve molecules
are actually tested in clinical trials and ultimately only one receives
approval [34,35]. The curtailment of this enormous wastage of resources
and consequent reduction in the overall costs of drug development
should clearly be the priority for the pharmaceutical industry.
As a result following the successful completion of the preformulation study, a well-dened formulation development path
must be established based on the requirement of the pre-clinical
study. A detailed description of the formulation development
strategies that can be adopted in pre-clinical studies are presented
in the following sections.
Labral 1944CS
Oral, i.v.
Polysorbate 80
Oral, i.v.
Cremophor RH 40
Oral
Cremophor EL
i.v.
Soluphor P
Oral, i.v.
Solutol HS15
i.v.
Vitamin E TPGS
Oral
Transcutol HP
Ethanol
Propylene glycol
Labrafac
Oral
Oral, iv
Oral, i.v.
Oral
4. Formulation development strategies

The nature of studies conducted in preclinical phase depends on
the target disease, route of administration and the anticipated
treatment duration in humans. Therefore, administration by different
routes range from a single dose studies to a very long time exposure.
Regardless of species and route of administration the basic goal is to
administer high but justiable doses in order to establish safety
margins for clinical studies (in human) and to determine the target
organs of toxicity [36].
Based on the study requirement, various strategies should be
adopted to develop formulation for pre-clinical studies. The formulation scientist must consider several possibilities prior to initiating
formulation development studies for non-human studies. The
formulation development plan for pre-clinical study should be carried
out based on the intended use of the drug in humans, the end-user.
For e.g. for a new drug if the intended nal dosage form is i.v., the preclinical formulation development should also be geared towards
development of an i.v. formulation.
During pre-clinical formulation development several excipients
can be explored for suitability for the desired study design. Examples
of excipients include llers, diluents, solvents, wetting agents,
emulsiers, preservatives, absorption enhancers, release modiers,
avors and coloring agents, among others. For the selection of
excipients, apart from their intended use, regulatory considerations
such as approval and safety of the excipient need to be examined
carefully. Generally, it is preferable to select excipients that have been
previously used in the marketed products with a relevant route of
administration. The list of excipients used in marketed products can
be found at the FDA's Inactive Ingredient Database website at http://
www.accessdata.fda.gov/scripts/cder/iig/index.cfm. Pre-clinical study
285
Solubilizer, coemulsier,
bioavailability
enhancer
Solubilizer
Solubilizer,
tonicity
adjustor
Solubilizer,
bioavailability
enhancer
Solubilizer,
stabilizer
Solubilizer, coemulsier,
bioavailability
enhancer
Surfactant
Solubilizer,
emulsifying
agent
Solubilizer,
emulsifying
agent
Solubilizer,
permeation
enhancer
Solubilizer,
surfactant
Solubilizer,
emulsier,
bioavailability
enhancer
Solubilizer
Co-solvent
Solubilizer
Oily co-solvent
Rat i.v.: 7.3 g/kg

Mouse i.v.: 8.6 g/kg
Mouse oral:
28.9 g/kg
Rat oral: 22 g/kg
Rat i.v. 1 g/kg Mouse

i.p.: 0.33 g/kg Rat
oral: 18.8 g/kg
Rat oral: N 20 mL/kg
Rat oral: 34.5 mL/kg

Mouse oral: 25 g/kg
Rat oral: N 16 g/kg
Mouse i.v.: N 12 g/kg
Mouse i.p.: N 6.4 g/kg
Rat oral: 6.4 mL/kg
Mouse i.v.: 2.54 mL/kg Rabbit oral:
N 10 mL/kg
Rat oral: 5 mg/kg
Rat oral: N 20 mg/kg

Rat oral: N 7 g/kg
Rat
Rat
Rat
Rat
oral: 7.5 mg/kg

oral: 3.6 g/kg
oral: 20 g/kg
oral: N 10 mL/kg
design may require the use of excipients which have minimal safety
records. Use of these excipients can add signicant risk to the product
development process which may require additional preclinical and
clinical studies. All the excipients can have toxicity effect on the
studied species, some more than other. As a result, the rst approach
in developing a formulation should be a simple solution. However, if
the drug is not soluble in water the formulation strategies described in
the paper can be followed. A list of excipients that can be used for oral
and i.v. administration is provided in Table 2 along with their LD50
values [3742].
The following sections provide a detailed description on various
formulation development strategies that can help the pharmaceutical
scientist to develop successful formulation for pre-clinical studies.
4.1. Duration of the study
Most commonly used animal models for pre-clinical studies
include mouse, rat, rabbit, and dog. Each species reacts differently to
commonly used excipients. The response generated upon administration of these excipients is variable depending on the route of
administration. Tolerance to these excipients administered by
different routes also depends on the functional class of excipient
such as solubilizer, surfactant, co-solvent, etc. A good example of this
case is a comparison of poly (ethylene glycol) 400 (PEG400) with
286
Soluphor P. PEG400 is well tolerated via intravenous (i.v.) or oral

route of administration in comparison to Soluphor P. Also, some of the
excipients may cause toxic effects upon single dose administration or
multiple dose administration. Therefore, the formulation development strategy should consider whether the study is a single dose
(acute) or multiple dose (chronic) study. A detailed calculation must
be carried out to evaluate the NOEL (No Observable Effect Level) for
each excipient to be used in pre-clinical study. This in turn will ensure
that the excipients being used in the formulation does not exceed the
daily LD50 value for that particular animal species. A list of LD50 values
of most commonly used excipients is given in Table 2. The maximum
percentage of excipient that can be used in a formulation can be
calculated according to Eq. (4).
Max: % of excipient in the formulation
NOEL
100
Number of doses per day Dose per day
4
Where, NOEL is no observable effect level (g/kg/day) and dose/day
is maximum amount of dose to be administered in one day.
In addition to the LD50 values, possible synergistic effect of the
excipient should also be evaluated to ensure that these excipients do
not produce any toxic effects for the duration of the study.
4.2. Biopharmaceutics classication system of drug substance
Knowledge of the Biopharmaceutics Classication System (BCS) of
the drug substance is very important to determine the formulation
development strategy. For highly soluble and highly permeable drug
substance (BCS Class I) with immediate release dosage form, and a
narrow therapeutic index, it is very likely to exceed the maximum
toxic dose (MTD) upon administration. Therefore, in this particular
case it is necessary to retard the drug release from the formulation. As
a result a controlled release or modied release dosage form is highly
desirable. For this particular scenario, formulation for non-human
study can be developed by using matrix type of tablet formulation,
hot-melt wax granulation, or barrier system. Each type of formulation
approach has its own advantage and disadvantage. A matrix tablet
formulation is relatively easy to process without use of any specialized
equipment; however, it is less exible in terms of modifying or
altering the release prole. For hot-melt wax granulation technique
with identical amounts of controlled release agent the release prole
can be curtailed to the desired rate by preparing different size
granules. In the case of bead coating, beads can be coated with various
release retarding polymers and subsequently can be mixed in various
proportions to obtain the desired release prole. The only limitation of
hot-melt granulation and bead coating technique is that these
processes require specialized equipment which may not be costeffective in early stage of drug development.
Considering an alternate scenario, for highly soluble but poorly
permeable drug substance (BCS Class III) incorporation of surfactant
or bioavailability enhancers is essential to enhance the systemic effect.
These excipients play a major role in modifying the bioavailability of
the drug. Certain excipients have been reported to enhance the
bioavailability of the drug substance. Vitamin E TPGS has been
hypothesized to increase the bioavailability of certain drugs by
enhancing the solubility of the API and by acting as a weak P-gp
inhibitor [4345]. Studies have shown that Polyethylene Glycol (PEG)
can either increase or decrease the oral absorption of drugs. Similar to
Vitamin E TPGS, PEG-300 at a concentration of 20% inhibited P-gp in in
vitro cell studies of Caco-2 monolayer and MDR1-MDCK cells [46].
However, contrary to this study, a dose of 10 g of PEG 400 have shown
to reduce the oral bioavailability of ranitidine in humans due to
reduction in small intestinal transit time and probably due to inux of
uid into the gut lumen due to the osmotic effect of PEG [47]. As a
result a thorough understanding of the properties of the drug
molecule and its possible interaction with the excipients is essential

in selecting suitable excipients for pre-clinical formulation
development.
4.3. Desired route of administration
4.3.1. Oral route
Oral route is the most convenient, cost-effective, and a safe route of
administration. Pre-clinical evaluation via oral route of administration
should be performed if the desired route for future clinical studies is
oral. It is essential to know the bioavailability of the drug when
administered orally. The oral PK data must be compared against a
single dose IV administration to determine the absolute bioavailability
of the active from the oral formulation. Oral PK study can be carried
out using various formulations such as solutions, suspensions, tablets,
and capsules.
4.3.1.1. Oral gavage. This method of dosing is carried out in order to
deliver a xed volume of the dose directly in the stomach. Oral gavage
is mostly given in rodents and this procedure ensures complete
dosing of the drug product. Formulations such as solutions and
suspensions can be administered by this method. The usual dosing
volume is not more than 10 mL/kg.
4.3.1.2. Oral solution and suspension. Traditional dosing is carried out
either in the form of solution, suspension, or powder in the animal
feed. Solutions can be prepared if the solubility of the active is not a
limiting factor. Oral solutions can be prepared by use of co-solvents,
solubilizers, surfactants or by pH adjustment of the aqueous media.
Oral suspension can be prepared if the drug substance has limited
solubility in the solvent of choice. For dosing the oral solution and/or
suspension, palatability of the formulation is very critical. Various
avors are available for enhanced palatability and to mask the taste of
surfactants, solubilizers, and oils used in the oral liquid formulation.
4.3.1.3. Oral solid dosage form. Mini-tabs and/or capsules are the most
convenient oral solid dosage form for pre-clinical study. Mini
capsules size 9 are available for dosing into rodents such as rats.
Pure API lled in size 9 capsules can be dosed in to rats by using a
dosing syringe. Administering the drug in this manner will accurately
predict the bioavailability of the drug substance without any
interference of additional excipients. This is an excellent tool for
early drug screening. Most of the time neat API dosed in capsules
provides an accurate prediction of PK data. One of the recent studies in
beagle dogs [48] showed that the AUC0 inf for pure Active
Pharmaceutical Ingredient (API) lled in capsules was not signicantly different than a liquid formulation containing multiple
surfactants. This study shows that having a drug substance in a
readily available form may increase the rate of absorption but does
not affect the extent of absorption. The above study suggests that in
certain cases the drug response is dependent on physicochemical
properties such as log P and permeability of the drug substance..
For oral dosage form, the physical state (form) of the API dictates
the formulation strategy. APIs in solid state are relatively easy to
handle during compounding trials and during manufacturing. APIs
with lower bulk density and static in nature are difcult to handle. The
most difcult to handle APIs are those that are available in semisolid/gel form. Formulation development process can be fairly
challenging for semi-solid APIs. For such API for low dose, e.g.
b10 mg dose or b10% of the total unit weight, one approach is to
adsorbed the API on to inert excipient with uniform particle size and
to further process the granules by compressing them in to tablet or ll
in a capsule. However, with higher drug loading a semi-solid/gel API
must be formulated in an oral solution, suspension or syrup dosage
form.
4.3.1.4. Self-emulsifying drug delivery system. Self-emulsifying drug

delivery system (SEDDS) or self-microemulsifying drug delivery
system (SMEDDS) is a relatively newer drug delivery system with
specic application of oral delivery of very hydrophobic drugs.
SMEDDS formulation system consists of the drug dissolved or
suspended in an oil phase along with a surfactant and a co-surfactant
or solubilizer. The basic principle of this system is that when such
system is diluted with an aqueous phase under gentle agitation, a ne
oil-in-water (o/w) emulsion is formed instantaneously. As a result
when these formulations are administered orally, the composition
and the digestive motility of the stomach/intestine provide the
required aqueous phase and the agitation for self-emulsication in
vivo in the lumen of the gut [49]. Upon introduction into the aqueous
media the surfactant and the co-surfactant preferentially get adsorbed
at the interface, which reduces the interfacial energy as well as
provides a mechanical barrier to coalescence. This lowering of free
energy required to form emulsion subsequently improves the
thermodynamic stability of the formed microemulsion [5052]. In
order to develop and select a SMEDDS various parameters to be
evaluated [53] are (i) Solubility of the drug in various component such
as oil, surfactant, co-surfactant, (ii) Areas of self-emulsifying region as
obtained by the phase diagrams, and (iii) The droplet size distribution
of the resultant emulsion following self-emulsication.
Solubility of the drug substance in individual components will
provide background information on identifying the lead solubilizer
and surfactants. Following this study phase diagrams should be
plotted to determine the precipitation potential of the microemulsion. Phase diagrams should be plotted with the different ratios
of surfactant/co-surfactant mixture mixed with drug dissolved in oil
phase. Traditionally, turbidity method is used to develop selfemulsifying drug delivery system. In this method water is slowly
added to the mixture under gentle stirring to evaluate the precipitation potential. Observations are made to determine the different
phases of solution such as going from clear to turbid to clear. Based on
the observations the phase diagram is plotted and the ratio of
oil/surfactant/co-surfactant system can be selected. However, this
method often results in erroneous result because the time required for
complete emulsication is too short and therefore it is not possible to
accurately measure the rate of change of turbidity [54,55]. On the
other hand, refractive index (RI) and/or percent transmittance (%T)
can accurately predict the transparency of the formulation. The RI and
%T of the test solution is compared with that of water which has a
value of 1.333 and 100% respectively. If the RI of the sample is similar
to 1.333 or if the %T is 99% then the system is considered transparent
[56]. Droplet size distribution of the formulations can be characterized
by using light scattering technique. An emulsion with a smaller
droplet size has larger surface area and hence may result in higher
solubility and faster absorption of the drug. However, care should be
taken that the amount of the surfactant/co-surfactant in the
formulation should not exceed the daily toxic dose in that animal
species.
4.3.1.5. Surfactant systems. Solubility and permeability are two
important properties of a compound which are necessary for
successful transport of compound into the systemic circulation by
oral route. Since cell membrane is composed of bi-layer of
phospholipids, a certain degree of lipophilicity is a requirement for
drug absorption through intestinal wall. While high lipophilicity is
advantageous in terms of permeability, but intrinsically it translates
into poor aqueous solubility. Since the rst step in the oral absorption
process is dissolution of the compound in the gastrointestinal
contents (which is an aqueous component), poor aqueous solubility
is a major concern for oral formulation of compounds. As a result, the
formulation may require the use of surfactants in order to solubilize or
suspend these hydrophobic molecules. Various anionic and nonionic
surfactants are available for pre-clinical studies. In addition to the
287
solubility enhancement properties, surfactants may produce substantial effect on the absorption and metabolism of the drug substance.
This may subsequently affect the pharmacodynamic and pharmacokinetic outcome. Bittner et al. investigated the effect of oral predosing of inactive ingredient Solutol HS15 on the pharmacokinetic
prole of intravenously administered Colchicine in rats [57]. It was
found that after oral pre-treatment with Solutol HS15, colchicine
plasma clearance decreased by a factor of two and its maximum
plasma concentration increased almost two-fold in comparison to the
control group. It was concluded that absorption of Solutol HS15
and/or its degradation products into the systemic circulation seems to
be a major contributor to the observed effects. A similar study [58]
evaluated the impact of the surface-active formulation ingredients
Cremophor EL, Tween 80 and Solutol HS15 on the intrinsic
clearance of Midazolam in rat hepatocytes and microsomes. It was
concluded that cytochrome P450 3A mediated metabolism of
Midazolam seemed to be prevented by all the above surfactants at
concentrations above 0.03%.
A review [59] of pharmacological effects of Cremophor EL and
Tween 80 have also been demonstrated to inuence the disposition
of solubilized drugs that are administered intravenously. The overall
resulting effect is a highly increased systemic drug exposure and a
simultaneously decreased clearance, leading to alteration in the
pharmacodynamic characteristics of the solubilized drug. Pharmacokinetic experiments revealed that this effect is primarily caused due to
reduced cellular uptake of the drug from large spherical micellar-like
structures with a highly hydrophobic interior, which act as the
principal carrier of circulating drug. Within the central blood
compartment, this results in a profound alteration of drug accumulation in erythrocytes, thereby reducing the free drug fraction
available for cellular partitioning and inuencing drug distribution
as well as elimination routes.
Generally, high amounts of surfactants are required to disperse or
dissolve hydrophobic drug molecules. The tolerability of such
solubilizing aids or vehicles is particularly an issue for pre-clinical
studies, in which formulations with much higher concentrations and
volumes (mg/kg) are administered compared to clinical formulations.
Vehicles are generally tested in a control group to verify their
tolerability, but sometimes a vehicle interacts with the drug, e.g. the
toxic effects of a drug can depend on the vehicle used. Such
confounded effects may also be a problem for studies of metabolism,
pharmacodynamics or pharmacokinetics [60]. These examples highlight the need for thorough characterization of excipients in view of
their potential interference with pre-clinical studies. These examples
also emphasize the need for new excipients which are inactive or
posses lower biological activity in comparison to the commonly used
surfactants.
4.3.2. Intravenous and intraperitoneal route
4.3.2.1. I.V. solution. For early animal studies intravenous administration is popular route second to oral administration. Solutions (pH
adjusted) or co-solvent systems are highly desirable and are easy to
compound. It is relatively easy to compound an IV formulation of a
drug substance available in HCl or acetate or any other salt form. For
these drug substances the general strategy should be to compound
them in phosphate buffered saline (PBS) solution. After solubilizing
the compound at a desired concentration, it is necessary to adjust the
pH preferably between 47 and osmolality to about 290 mOsm/kg
respectively. The pH adjustment should be carried out using weak
base or a salt of weak acid such as NaHCO3 of low molar strength so
that the ionic strength of the nal product is not high, which may
cause irritation upon administration. Compounding the formulation
in PBS ensures the correct osmolality of the formulation for IV
administration. Solution dosage form is highly desirable, however,
due to limited solubility and higher log P of recently developed
288
molecules an injectable suspension or an emulsion formulation is also

preferred [61].
4.3.2.2. Suspension and nanosuspension. Until a few years ago
suspension formulation were developed for intra-muscular (IM) or
intra-peritoneal (IP) drug delivery. However, in the recent years IV
suspensions are also being developed. Each preceding route has
some advantages and disadvantages. Advantages of IM injection
include (i) Consistent absorption in comparison to oral administration
(ii) Possibility of developing sustained release depot injection and
reduce the frequency of administration, and (iii) Most viable for
unconscious or vomiting patients. On the other hand the disadvantage
of IM injection include (i) Pain at the site of injection and possibility of
muscle damage.
The advantages of IP route are based on its similarity to IV route i.e.
larger drug absorption area. On the other hand IP administration may
cause damage of organs upon injection and a chance of peritonitis that
can be caused by the drug or the vehicle.
IV injectable suspensions have gained noticeable attention in the
recent years due to the following reasons [61,62] (i) High drug loading
in comparison to IV solution which facilitates lower injection volume,
(ii) Reduction in toxicity by replacing toxic solubilizers with low
concentration of surfactants, (iii) Possibility of altering the PK of the
drug leading to higher dosing and reduced frequency of administration, and (iv) Possibility of drug targeting.
Nanotechnology is one of the fastest-growing technologies which
is contributing signicantly to the progress of medical science. One of
the strategies in preclinical formulation is nanosizing, which refers to
the reduction of the active pharmaceutical ingredient (API) particle
size down to the sub-micron range. Recent advances in milling
technology and our understanding of such colloidal systems have
enabled the production of API particles of 100200 nm size in a
reproducible manner. The sub-micron particles are stabilized with
surfactants or polymers in nanosuspensions which can be further
processed into standard dosage forms, such as capsules or tablets,
suitable for oral administration. These nanoformulations also offer
increased dissolution rates for poorly soluble compounds which in
turn improve bioavailability, reduce variability and food effects for
orally administered drug molecules [63].
Two most critical aspect of an IV suspension is the particle size of
the drug in the suspension and stability of the formed particles Two
approaches are mainly employed to produce drug nanoparticles: top
down and bottom up technologies [61,64]. The top down approach
is by far the more popular approach which is commonly referred as
nanosizing. This approach relies on mechanical attrition to render
large crystalline particles into nanoparticles. Examples of the top
down approach include Elan's NanoCrystal wet-milling technology
[65] and SkyePharma's Dissocubes high-pressure homogenization
technology [64,66]. The bottom up approach relies on controlled
precipitation/crystallization [61]. The process involves dissolving the
drug in a solvent and precipitating it in a controlled manner to
nanoparticles through addition of an anti-solvent (usually, water).
This technology is available from DowPharma (Midland, MI, USA) and
BASF Pharma Solutions (Florham Park, NJ, USA). A hybrid approach is
also feasible. Baxter's NANOEDGE technology employs both bottom
up and top down approaches through microprecipitation and
homogenization [64]. For further reading the readers referred to a
recent review article [63].
Instability of suspensions may be caused either due to high
surface energy of the formed particles or due to crystal growth.
Synthesis of nanosuspension by homogenization imparts high
energy to the formed particles. As a result the change in the Gibbs
free energy associated with the formation of larger surface area and
additional interface, the formed nanosuspension are deemed to be
thermodynamically unstable and will tend to minimize the total
energy by agglomeration [67]. This aggregation leads to instability of
the suspension. Aggregation of the sub-micron particles can be

prevented by use of anionic and nonionic surfactants. These
surfactants include phospholipids, polysorbate 80, and poloxamers
[68]. The process in which the nanoparticles in the suspension
dissolve and re-grow as larger particles is termed as Ostwald
ripening. Ostwald ripening can be cause by a large variability in the
particle size distribution in the nanosuspension and can be
minimized by reducing the drug particle size distribution [69]. A
careful selection of the type and concentration of viscosity builders
or surfactant stabilizers such as carboxy methyl cellulose is the key
to successful production of nanosuspensions [70].
4.3.2.3. Complexation with cyclodextrins. The ability of cyclodextrins to
form inclusion complexes has been studied extensively and applied to
modify the physicochemical properties of various drug molecules
[7173]. Cyclodextrins have been used for their ability to improve the
chemical (photo-degradation, hydrolysis, decomposition and oxidation) and the physical stability of drugs [7477]. Complexation with
cyclodextrin has also demonstrated an increase in the aqueous
solubility, dissolution, and bioavailability of the poorly soluble
drugs [7880]. The three major cyclodextrins are -cyclodextrin,
-cyclodextrin, and -cyclodextrin. The -cyclodextrin can be
administered parenterally as it is, however they have lower
solubilizing capacity in comparison to the other two cyclodextrins.
Underivatized -cyclodextrins cannot be used for parenteral administration for two reasons; rst, because of their low solubilizing
capability (0.10.2 g/100 ml dissolved complex), and second, because they are not metabolized, and accumulate in the kidneys as an
insoluble crystalline cholesterol complex, which causes hemolysis of
the human erythrocytes and severe nephrotoxic symptoms. Hydroxyalkylated derivatives of -cyclodextrins, such as, hydroxyethyl-, 2hydroxypropyl-, and 3-hydroxypropyl-CD's greatly increase the
solubility of many compounds, as well as, have strongly reduced
hemolytic properties. Out of these three derivatives, 2-hydroxypropyl- cyclodextrin (HPCD) is well tolerated parenterally, even in
extremely high doses [81] and therefore, is the most favorable of all
the CD derivatives. Also, at 25 C the aqueous solubility of HPCD is
1 g/ml, whereas that of CD is only 18 mg/ml. HPCD has a high
inclusion afnity, low toxicity, and ability to alter the phase
solubility behavior in favor of isotherms of the A-type [82,83]. A
number of previous studies have demonstrated that (2,6-di-Omethyl)-CD (DIMEB), randomly methylated CD (RAMEB) and CD's have better or almost similar solubilizing capacity compared to
that of HPCD [82,84]. However, these cyclodextrin derivatives
cannot be used as injectables due to their potential toxicity, when
administered parenterally [81].
Usually a 1:1 stoichiometry of drug: cyclodextrin prevails in
dilute solutions however a higher order complex may be formed
with an increase in the concentration. In cases when the stoichiometry is not certain, apparent constants are calculated and used as
an estimate of the extent of complexation. For ionizable substrates,
the extent of association varies with the pH. The stoichiometry of
complexes has been studied extensively, and numerous methods are
available in the literature for studying the complexation. These
methods include, calorimetry [85,86], articial neural network [87],
membrane permeation technique [88], spectrophotometry [89],
potentiometry [90] and mass spectrometry, and NMR techniques
[91].
4.3.2.4. Sterility and endotoxin levels of parenteral products. IV/IP/IM
formulations cannot be addressed without the mention of two
important parameters; sterility and endotoxin limit of the formulation. Sterility assurance of the nished product cannot be stressed
enough when performing pre-clinical study, especially, while performing dose ranging or acute toxicity study. The following steps must
be taken at a minimum to ensure sterility of IV preparation for preclinical study:

1. Formulations for IV preparation should be prepared in a bio-safety
cabinet or a laminar ow hood (LFH), collectively referred as hood
in the following sections.
2. The hood must be operated according to the manufacturer's
recommended procedure, e.g. Do not obstruct the ow pattern in
the hood by placing large objects in front of the air outlet, no
sudden movements in the hood, the door of the hood should be
always below the sash level etc.
3. Prior to preparation of IV formulation the hood must be carefully
wiped down by Hydrogen Peroxide solution such as SteriPerox
ensuring wiping of the walls and the oors of the hood and allow it
to air dry.
4. The glass wares used in the preparation of formulation and lling of
nal product should be de-pyrogenated. Aseptic handling technique should be employed during compounding of the injectable
products.
5. All the excipients used in the preparation should be low endotoxin.
According to USP 33 b85N the endotoxin limit for parenteral drugs
is dened on the basis of administered dose according to the following
Eq. (5).
EU Limit K=M
Where, EU is the Endotoxin Unit, K is the threshold human

pyrogenic dose of endotoxin per kg of body weight, and M is the
Maximum recommended human dose of product per kg of body
weight in a single hour period.
The USP 33 further states that K is 5 USP-EU/kg for any route of
administration other than intra-thecal (for which K is 0.2 USP-EU/kg
body weight). Although the equation for USP EU calculation is intended
for human administration it is recommended to use the equation to
calculate the EU limit for pre-clinical animal study as well. The
endotoxin limit for animal models commonly used in a pre-clinical
study is given in Table 3.
4.4. Intended duration of action
Based on the acute toxicity and/or the desired duration of action
pre-clinical studies can be carried out for extended release dosage
form. Oral solid dosage form is a conventional dosage form for human
administration. For oral drug delivery the extended release dosage
form can vary in its duration of action from 6 h to 24 h. An extended
release dosage form has several advantages such as: it prevents dose
dumping, prolongs the effect of the drug, and decreases the frequency
of administration, hence improving patient compliance. A delayed
release dosage form is required if the active ingredient in the
formulation is highly unstable in acidic pH of the stomach. Preclinical study for such type of dosage form should be carried out
according to the intended human study. In one of the studies once
daily dose of Aceclofenac was prepared and evaluated for pre-clinical
and clinical studies [93]. In this study matrix tablets were successfully
prepared using hydroxypropyl methyl cellulose (HPMC) and were
tested at a dose of 10 mg/kg p.o. for anti-inammatory and analgesic
activity of Aceclofenac in rats. The study also included controls groups
and rats that received pure Aceclofenac. Pharmacokinetic and subacute toxicity studies were also carried out in rats.
Another study was carried out on anti-diabetic drug Metformin.
Metformin has unique pharmacokinetic and pharmacodynamic
properties. One of the studies showed that despite similar Area
under curve (AUC) prole, orally administered metformin produced
augmented effect in comparison to i.v. bolus dose [94]. In this study
Metformin was administered via i.v. bolus and p.o. bolus to diabetic
rats. The calculated AUC values for these modes of administration
289
Table 3
Endotoxin limits for drugs for Pre-clinical Research for commonly used animal models
(Adopted from reference [92]).
Model
Body weight (kg)
Dose (mg/h)
EU/mg
Mouse
0.03
Gerbil
0.09
Rat
0.45
Rabbit
Monkey
Baboon
12
0.001
0.010
0.025
0.001
0.010
0.025
0.001
0.010
0.025
0.010
0.025
0.050
0.250
0.500
0.100
0.250
0.500
0.100
150
15
6
450
45
18
2250
225
90
2000
800
400
160
80
40
240
120
60
were 6.19 1.19 mg min/mL and 5.65 0.65 mg min/mL respectively. The results suggest that the magnitudes of systemic exposure were
similar for these two modes of administration. However, the overall
magnitudes of glucose lowering effect described by AUEC were 22.7
9.0 mg min/dL and 53.2 18.4 mg min/dL for i.v. and p.o bolus doses
respectively. These results suggest that the glucose lowering effect for
the p.o. dose is almost twice that of the i.v. bolus dose. A PK/PD
modeling study indicated that the magnitude of the glucose lowering
effect of metformin in liver is related to the drug concentration in the
portal vein [95]. Metformin exhibits a ip-op pharmacokinetics, i.e.
the rate of absorption is slower than the renal elimination rate which
may have lead to higher portal drug concentration for a longer period
for oral administration. The study suggested that for intraduodenal
bolus and infusion a higher portal vein concentration was achieved for
metformin which subsequently enhanced the exposure of liver/portal
biophase to Metformin in comparison to i.v. infusion. Based on the
comparison of the glucose lowering effect of intraportal and the
intraduodenal administration the authors also demonstrated that in
addition to elevated portal exposure, GI administration of metformin
also leads to a higher exposure of the GI biophase of the drug.
Metformin has poor colonic absorption [96,97] as a result a
gastroretentive dosage form was developed to demonstrate the
importance of pharmacokinetic and pharmacodynamic properties in
comparison to other modes of administration [98]. As a result it is
extremely important to understand the physicochemical properties of
the drug substance and the target product prole.
5. Stability of pre-clinical formulations
In order to ensure proper dose administration, it is important to
establish the stability data for the formulation used in the study. The
formulations prepared in the laboratory for animal dosing must have
adequate stability until completion of the study. The drug must be
stable in the formulation during the following situations: (i) During
compounding/manufacturing which may require heating the excipients up to 60C to solubilize the drug, (ii) If the formulation is
prepared at a site other than the testing facility the formulation must
be stable during transportation/shipping of the product, and (iii) For
the duration of the study. Short-term stability of 2448 h for a single
dose study, however, long-term stability is essential for a 14-day or a
28-day toxicity study at the recommended storage condition.
Physical and chemical instability of formulation can occur due to
oxidation, hydrolysis, pH sensitivity, or excipient incompatibility.
These processes may occur individually or in combination with one or
more pathways. All these degradation processes can be accelerated at
290
elevated temperatures which can lead to chemical and/or physical

instability. Chemical instability causes a decrease in the potency of the
active and a simultaneous increase in the degradation product.
Physical stability is critical in suspension and emulsion formulations.
Physical instability in suspension can cause particle agglomeration
and in emulsion it may cause phase separation.
Early formulation stability can be enhanced by taking following
precautions [99].
1. The formulations must be prepared fresh whenever possible. This
technique should be avoided for repeat dosing because freshly
prepared formulation may differ in potency and hence the dosing
at each time point may not be accurate.
2. Store the formulations away from light, in amber vial or covered in
aluminum foil.
3. Store the formulations at refrigerated storage condition (28 C)
whenever possible. While storing in a refrigerator, prior to
administration, the formulation must be observed visually to
evaluate any precipitation of drug substance in the vehicle. If
precipitation occurs the drug can be re-dissolved by gently swirling
or vortexing the entire mixture.
4. Store the formulation away from oxidation. Use appropriate size
storage container to minimize the head space or purge the head
space with nitrogen especially for formulation such as liposomes or
emulsions.
6. Recent advances in pre-clinical studies
6.1. Formulation development
Surfactant systems are essential for any poorly soluble drug
candidate to be formulated in a liquid dosage form. Various categories
of surfactants available are anionic, cationic, or non-ionic in nature.
Cationic surfactancts can be readily absorbed by the cell membrane
which can lead to hemolysis [100]. Various anionic and non-ionic
surfactants are widely used in pharmaceutical preparations including
pre-clinical formulations. Although however, anionic and non-ionic
surfactants are considered relatively safe, these surfactants can also
cause cytotoxicity if used in excess of their recommended concentration [101]. As a result, a surfactant-free delivery system would be ideal
for pre-clinical or human use. In one of the recent studies the authors
have developed surfactant-free formulation by using octenyl succinate modied starch for oral drug delivery [48]. The formulation and
the manufacturing strategy thereof signicantly reduced the surface
tension of a highly hydrophobic drug. This study demonstrates the
utility of a surfactant free system, which can be a promising tool to
deliver surfactant-free drug product. However, with this delivery
system it is critical to evaluate whether a surfactant-free system can
work universally for drug substance with varied physical properties.
The in vivo effect of a model molecule compounded with a typical
surfactant base system v/s surfactant-free system must also be
evaluated.
In preclinical studies, an extensive formulation screening would
not only bind substantial resources, but is also uncertain in terms of its
relevance for humans. Thus an additional biopharmaceutical assessment program is required in the preclinical development phase. The
biopharmaceutical assessment program consists of two steps. The rst
step is a computer simulation that should help to elucidate the
hurdles for oral bioavailability, as well as identify critical parameters
of a drug formulation. The simulation may also reveal some
parameters that only have a limited impact on the biopharmaceutical
performance of a formulation, which is of equal importance. This rst
step is only an in-silico model, but the physiology of the human
situation is simulated. The second part of the biopharmaceutical
assessment is to test proof of concept formulations in statistically
designed pharmacokinetic experiments. A seven compartment phys-
iological based model was developed by Yu et al. followed by

incorporation of absorption step which led to the compartmental
absorption and transit model [102]. The limitation of this model was
that this model did not incorporate particle solubilization. As a result,
this model can be applicable to only freely soluble drug. Modication
was made to this model in which various parameters such as pH
dependent solubility, and drug dissolution and precipitation steps
were added. This model was termed as the Advanced Compartmental
Absorption and Transit model [103]. Based on this model, Kuentz et
al., have used GastroPlus TM to simulate the absorption process based
on pre-formulation data. The input data for this model involved a
physico-chemical drug characterization including drug solubility
measurements in simulated physiological media, as well as permeability determination. Further computer simulations were conducted
to determine the sensitivity of the prediction model to changes in
selected input values. Thus, oral bioavailability prediction was studied
as a function of the particle size and drug solubility. Two preclinical
formulations were developed, one formulation was a capsule lled
with the micronized drug, whereas the other formulation was a
surfactant solution of the drug. These two formulations in a 2 3
screening factorial plan were compared over the clinically relevant
dose range in fasted and fed dogs. The results of the computer
simulation indicated that a fraction of the dose is dissolved in the
stomach and precipitates partially in the small intestine. The
simulation predicted almost full drug absorption during the GI transit
time. Interestingly, the simulation implies that the stomach drug
solubility had little impact on overall fraction absorbed. The results
also showed that changes of particle size and reference solubility
within two orders of magnitude hardly affected the oral bioavailability. This in-silico deduction was subsequently compared with the
results of the dog studies. Indeed a surfactant drug solution showed
no clear biopharmaceutical superiority over a solid capsule formulation on the average of both dose strengths in fasted and fed dogs.
GastroPlus TM TM simulation together with the statistically designed
dog study provided a thorough biopharmaceutical assessment of the
new CNS drug.
The computer simulation GastroPlus TM requires input of numerous data such as MW, log P, pKa, particle density, and Peff. It also
required a series of assumptions such as stomach and intestinal transit
times, intestinal surface area etc. As a result this simulation may not
be able to predict accurate in vivo absorption and permeability due to
large number of variables. Another commercial software program
known as PK-SIM is also available for physiological based absorption
modeling.
A very promising study was carried out recently for targeting
Insulin to liver in which Hepatic-directed vesicle insulin (HDV-I) was
developed by scientist for subcutaneous and oral delivery of insulin
[104]. The formulation contains a proprietary hepatocyte-targeting
molecule (HTM). Following its oral administration, HDV prevents the
degradation of Insulin in the upper gastrointestinal tract, whereas
HTM targets the delivery of insulin to hepatocytes similar to
physiological insulin delivery [104]. The results of the study showed
that peripheral administration of HDV-I to hyperglycemic dogs
efciently delivers insulin to the liver, which in turn promotes a
100-fold increase in the glucose uptake in comparison to regular
porcine or human recombinant insulin. A 28-Day subcutaneous
repeated-dose toxicity study in rats also suggested that HDV was
safe to administer with no adverse effect. This study suggested that
HDV-I could be a novel break-through in insulin therapy.
6.2. In vitro modeling
Recently, special consideration has been given to the use of human
tissue or primary human cells in in-vitro modeling. In-vitro modeling
using human tissue could offer valuable preclinical research data and
possibly increased safety [105]. Improvement in cell culture
techniques along with increasing availability of human tissues have

led to increased use of human primary and immortalized cells in
toxicology and pharmacodynamic studies. Human in-vitro models are
being used with physiological models to predict quantitative
metabolism, transport, clearance, and pharmacodynamic outcome.
Biopta, a Scottish contract research organization (CRO), which
provides human tissue-based preclinical development services has
secured 265,000 in funding [106]. Biopta claims that modeling in
human tissues rather than animals provides drug makers with more
accurate data on which to base go or no-go development decisions.
The rm recently tested a promising new drug for asthma which had
only been tested in animals the drug was totally ineffective in human
lung tissues. This would not have been known until a phase II clinical
trial when 20 to 100 patients had been tested, wasting time, effort,
and upwards of 500,000 in clinical trial costs. These savings, coupled
with the drug industry's need to quickly replenish pipelines as
efciently as possible, are driving demand for innovative preclinical
technologies.
6.3. Pharmacodynamics in pre-clinical studies
Over the years, prediction of pharmacokinetic properties of drugs
in humans has been relatively within reach to predict based on the in
vitro and pre clinical data largely due to the advancement of
physiologically-based pharmacokinetic modeling [107,108]. In some
instances pharmacodynamic approach is better suited than the
traditional pharmacokinetic approach due to various reasons such
as drug resistance or the site of drug distribution.
For example, for an anti-microbial agent, repeated dosing exposure
could have an impact on outcome and a possibility of development of
resistance to a particular drug. Therefore, in such instance a pharmacodynamic modeling could be highly benecial to support the dose
selection of a new drug candidate [109]. In one of the studies PK/PD
modeling technique was applied to compare sustained-release
microsphere formulation against the subcutaneous injection for a
recombinant growth hormone. The sustained-release microspheres
was successful in maintaining the growth hormone level above the EC50
[110]. This study provided the dosing guidance for subsequent clinical
trials. Gabrielsson, et.al. [111] with the aid of simulation modeling have
demonstrated that compounds with a strong potential for giving a clear
response may fail to produce adequate effect due to the lack of optimum
study design or due to an inadequate number of time points. As a result
an integration of pharmacodynamic parameters into the traditional
pharmacokinetic studies will be benecial. Such modeling allows for the
characterization and prediction of the time course of the drug action
instead of the cumulative concentration, which in turn provides a
scientic basis for the development of dosage regimen. A schematic
representation is presented in Fig. 2.
An extensive program to include PK/PD must be developed and
followed during the early pre-clinical studies. This program should be
helpful during further drug development activities. At a minimum the
specic aims of this program should include [113]; development of
mechanism-based models for efcacy/toxicity, valuation and prediction of in vivo potency and intrinsic activity, drug interaction
evaluation, and optimization of dosage form and dosage regimen.
The evaluation of anti-tumor agents in immune-decient mice

transplanted with human tumors is the major model system for drug
development. In its most simple iteration, tumors are grown
subcutaneously, and the model allows rapid and quantiable
assessment of anti-tumor activity relative to mouse toxicity. Logically,
precedence should be given to those agents that show the greatest
anti-tumor activity in the preclinical setting, assuming the preclinical
data are predictive of drug activity in human studies. The challenge
lies in being able to extrapolate these results to the clinic [114].
There are many reasons why preclinical results do not predict
human efcacy mainly (i) Differences in interspecies pharmacology and
(ii) Criteria used to advance an agent in preclinical trials may not be as
stringent as those used to evaluate response rates in the clinical setting.
Peterson and Houghton [114] suggest that if certain aspects of the
study design are given careful consideration, it may be valid to predict
clinical results derived from preclinical work with the xenograft model.
These aspects include (a) the development of early-passage models of
the appropriate human cancer, rather than the use of ancient cell lines
that have been in culture for decades; (b) the use of clinically relevant
response criteria to evaluate a new entity; (c) the assessment of tumor
responsiveness relative to drug systemic exposure; and (d) a rational
consideration of the major/minor strengths and weaknesses of the
model.
It has been well recognized that when human cancers are
transplanted into mice they retain many characteristics of the original
tumor (histology, chromosomal abnormalities, and surface antigen
expression). Although subcutaneous tumors metastasize infrequently
this rate is increased when transplanted to orthotopic (natural) sites.
Peterson and Houghton [114] have discussed several examples
[115117] that showed activity in preclinical childhood tumors but
failed to extrapolate the similar effects in clinical trials. Evaluation of
systemic exposure (AUC) revealed that the systemic exposure was 5fold higher in rodents than can be achieved in patients. These
interspecies differences in the systemic exposure (AUC) caused
differences in the results of preclinical and clinical studies.
As described above, perhaps the greatest challenge in achieving
relative uniformity between the research conditions present in the
preclinical and clinical settings is accounting for the pharmacokinetic
differences between mouse and man. In order to develop a predictive
model for the preclinical evaluation of anthracycline cardiotoxicity and
the means of preventing it, Pouna et al. [118] have studied the functional
parameters of perfused hearts isolated from rats receiving repeated
doses of several anthracyclines. The anthracyclines studied were
doxorubicin, epirubicin, pirarubicin and daunorubicin, and we also
studied a liposomal formulation of daunorubicin (DaunoXome) and the
co-administration of dexrazoxane (ICRF-187) and doxorubicin. Anthracyclines were administered intraperitoneally and general toxicity
towards the animals as well as alterations of left ventricular contractility
and relaxation ex vivo was evaluated. The results demonstrated that
feedback
pharmacokinetics
drug
delivery
input
7.1. Anti-cancer
Historically, cancer drug development has been a roller coaster.
Numerous agents have shown exciting activity in preclinical models
and yet have had minimal activity clinically. These disappointments
have led to reasonable skepticism about the true value of both
syngeneic and xenograft rodent tumor models in accurately identifying agents that will have important clinical utility.
drug
elimination
output
-----------------
7. In vivo pre-clinical models
291
drug
concentration
in
plasma
b
i
o
p
h
a
s
e
pharmacodynamics
drug
effects
pathophysiology
disease
parameters
Fig. 2. Schematic representation of the relationship between the drug dose, plasma
concentration (pharmacokinetic), drug effect (pharmacodynamic) and clinical effect
(Adopted from reference [112]).
292
Epirubicin and daunorubicin were signicantly less cardiotoxic than

doxorubicin, and neither pirarubicin nor DaunoXome caused signicant
alterations in cardiac function. There was a direct relationship between
the decrease in cardiac contractility or relaxation and anthracycline
accumulation in the heart, evaluated after the treatment schedule.
Authors conclude that the isolated perfused rat heart appears to be a
valuable model for screening of new anthracyclines and of strategies for
circumventing anthracycline cardiotoxicity.
7.2. Anti-viral
Black et al. [119] have tested the antiviral activity of HIV-1
protease inhibitors in vivo in the Rauscher murine leukemia virus
(RMuLV) model. RMuLV-infected mice were treated twice a day with
either an active (SKF 108922) or inactive (SKF 109273) compound for
fourteen days by the intraperitoneal route. Compared with excipient
control, SKF 108922, formulated with hydroxypropyl--cyclodextrin
(HPBCD), reduced virus-induced splenomegaly, viremia, and serum
reverse transcriptase (RT) levels, while SKF 109273 was inactive. The
HPBCD vehicle by itself enhanced replication of RMuLV. The effects of
changing the formulation and the route of administration were also
examined. SKF 108922, formulated in HPBCD, had similar antiviral
activity when administered either by the intraperitoneal route or
subcutaneous route. However, SKF 108922 administered as a colloidal
suspension in Cholesterol Sulfate (CS) had no detectable antiviral
effect. Measurements of the plasma concentrations of SKF 108922
solubilized in HPBCD exceeded 1000 nM within 10 min after SC
administration where as administration of the same dose formulated
with CS was not detected in plasma. Information on optimal
conditions for administering these agents should prove useful in
guiding their clinical application. Therefore, RMuLV should provide a
good model for the preclinical evaluation and development of this
class of agents for the treatment of HIV.
7.3. Anti-diabetic
Stepensky et al. [98] have examined the pharmacokinetic (PK) and
pharmacodynamic (PD) rationales to develop controlled release (CR)
formulations of metformin. An experimentally induced model of type
2 diabetes (NIDDM) was produced by intraperitoneal injection of
streptozotocin. Rats with blood glucose below 140 mg/dl following
overnight fast and above 300 mg/dl at fed conditions were selected for
the experiment. Unrestrained diabetic rats received the drug as
intravenous bolus (i.v.), oral solution (p.o.), intra-duodenal bolus, 4-h
infusion, or intra-colonic bolus. In addition, two CR-gastroretentive
dosage forms (CR-GRDF) that released the drug over 3 or 6 h (in vitro),
and retained in the rats' stomach for 810 h were also developed. The
rate of elimination of metformin following intravenous and p.o. bolus
modes of administration were distinctly different, metformin exhibited ip-op PK after oral administration. The colonic absorption was
low but sustained and was associated with highly variable glucoselowering effects, thus providing a PK rationale to develop CR-GRDF. In
addition, the glucose-lowering effect was greater following p.o. versus
i.v. administration, despite equivalent AUC, indicating a rst pass PD
effect, thus, adding a PD rationale to develop metformin CR-GRDF.
When administered to the diabetic rats, CR-GRDFs produced bioavailability and extent of glucose lowering effects that were similar to those
of the duodenal infusion and p.o. metformin administration. These
ndings are attributed to the adsorption of metformin to the intestine
that yields slow and prolonged absorption even following p.o.
administration of drug solution. The data indicates that unless the CR
formulation could signicantly extend the absorption period, it is not
likely to improve glucose-lowering efcacy.
Stepensky et al. [98] conclude that the streptozotocin induced
diabetic rat model was found to be an effective strategy for the
evaluation of the PK and PD of CR-gastroretentive dosage forms,
thereby providing a means to assess the PK and PD rationale to

develop these formulations in order to optimize drug treatment.
Although initially there seemed to be both a PK and a PD rationale to
develop such formulations, the actual ndings indicate that the
differences in input rate of the drug to the upper GI region did not
signicantly affect the extent of metformin action. This interesting
nding evolves most probably from the afnity of the positively
charged drug to the GI wall, thus yielding a slow rate of drug
absorption, even following p.o. administration of drug solution. The
work stresses the importance of establishing a PK and PD rationale
prior to the development of CR formulations.
In another study [120], insulin gel was evaluated by intranasal
administration in streptozotocin induced diabetic rat model as well as
in healthy human volunteers. The insulin gel was formulated using
the combination of carbopol and hydroxypropyl methylcellulose as
gelling agent. The in vivo efcacy of insulin gel administered
intranasally was assessed by measuring the blood glucose levels and
serum insulin levels at specied time intervals in rats and humans.
The use of bioadhesive nasal gel containing insulin not only promoted
the prolonged contact between the drug and the absorptive sites in
the nasal cavity but also facilitated direct absorption of medicament
through the nasal mucosa. Absorption of the drug through the nasal
mucosa was high in the rst 0.5 to 1.5 h of the study with a sharp
decline in blood sugar and rise in insulin values corresponding to that
decline in blood sugar.
7.4. Anti-hypertensive
Generally, rabbit and dog are regarded as suitable animal models
for intraoral administration since the oral cavity of both are
histologically similar to man [121]. Dali et al. [122] have reported a
sublingual rabbit model and its potential utility in preclinical
development of anti-hypertensive drugs in intraoral dosage forms.
First, the delivery parameters: the effect of dosing solution volume,
dosing solution pH, and method of delivery, that is, solution spray
versus instillation were characterized. Second, systemic absorption
was compared to published results of sublingually administered drug
in human. Using propranolol as a model compound the effect of
formulation and dosing variables was explored as a means to
characterize the limiting parameters of this model. In addition,
verapamil and captopril were selected as reference compounds to
compare this model to sublingual absorption in humans. Rabbits were
dosed sublingually and systemic absorption was measured over time.
Sublingual absorption of propranolol was dependent on dosing
solution pH and volume. Intra-oral spray device did not affect the
overall exposure compared to instillation using a syringe. Despite
species and dosing regimen differences the relative bioavailabilities of
propranolol and verapamil were very similar in rabbits and humans.
In contrast, captopril absorption from the sublingual cavity of rabbits
was low and did not agree with that observed in man. It is likely, that
the physiochemical properties of captopril and the barrier properties
of the sublingual mucosa do not favor absorption in rabbits. Although
differences might be expected between dosing conditions and data
obtained in humans versus rabbits, the relative bioavailabilities of
propranolol and verapamil were very similar suggesting that the
rabbit model may be suitable for assessing intra-oral drug delivery in
early development.
8. Interspecies differences in physiology and pharmacology
Preclinical animal data are an integral component of the product
development process, being used for predicting the potential for drug
toxicity and for estimating rst-time doses in humans. These
extrapolations are based on an assumption of a correlation between
the exposureresponse relationship in animals and man. Unfortunately, there is no single animal species that can serve as the perfect
surrogate for human subjects, and the appropriate surrogate species

needs to be evaluated for each situation [123].
Selection of the animal species to be used for toxicity testing
should factor the potential interspecies differences that can inuence
systemic drug exposure and target cell sensitivity. These include
potential differences in drug absorption, clearance, distribution, and
metabolism. These factors can determine whether or not a species will
exhibit toxicity or drug carcinogenicity [6].
9. Regulatory requirements in pre-clinical studies
The U.S. Food and Drug Administration (FDA) operate an active
program designed to understand and utilize preclinical models as
predictors of human xenobiotic exposure. Nonprot organizations
outside of FDA, such as the Health and Environmental Sciences
Institute, also contribute to the development of more predictive
and alternate models of safety assessment. One of the most notable
and active preclinical assessment initiatives is the National Center
for the Replacement, Renement and Reduction of Animals in
Research.
The established technologies and study designs carry the value of
being validated, generally well-controlled, and having reference to
historical databases. New technologies are typically not validated and,
by their denition, do not have a relevant historical database for
reference. Also, new technologies carry an inherent risk in their value.
Interpretation of data obtained from these technologies must be
limited and not overweighted when making human dose regimen
decisions.
The International Congress on Harmonization (ICH) has established a basic repertoire of guidelines that outline the technical
requirements of acceptable preclinical drug development (www.ich.
org). Also, the Center for Drug Evaluation and Research (CDER) has
compiled a series of guidelines to assist the innovator with
development issues and these guidelines may be found at the FDA
website (www.fda.gov). With the ongoing implementation and
renement of guidelines from ICH, the geographic regions of the
United States, Europe, and Japan have standardized approaches to the
drug development process. However, while these guidelines provide a
exible and innovative basis for preclinical drug evaluation, they
serve as a minimum requirement for achieving drug approval.
As described in the previous sections there are three major types of
pre-clinical studies: Pharmacological, Pharmacokinetic, and Toxicological studies. Each stage of pre-clinical study can be carried out
either regulated or un-regulated. Regulated pre-clinical study is
termed as Good Laboratory Practice (GLP). GLP is not required for
pharmacological study or in vitro pharmacokinetic studies. However,
GLP must be followed for safety studies such as animal toxicity,
genotoxicity and toxicokinetics [124].
During the submission of an IND application the applicant must
submit information such as a description of pharmacological effect,
mechanism of action(s) and information on absorption, distribution,
metabolism, and excretion (pharmacokinetic) if available. This
information can be omitted in the ling if it is not available [125].
The toxicity study should generally be carried out in accordance
with Good Laboratory Practices (GLP) [125,126]. Upon completion of a
toxicology study a nal fully audited report must be submitted to the
FDA prior to initiating any human study. At a minimum such report
should be submitted within 120 days of beginning the human clinical
trials [125]. Various toxicity studies that are usually carried out but are
not limited to: single dose toxicity study, multiple dose/repeat dose
toxicity study, dose escalation study, carcinogenicity study, and
reproductive toxicity study. A fairly recent terminology toxicokinetics has been introduced to evaluate preclinical toxicity. Toxicokinetic studies can provide information on repeated-dose toxicity
studies and subsequently help design studies for human safety
assessment [127]. Toxicokinetic evaluation is mainly associated with
293
toxicology screening, however overlaps with other areas of pharmacokinetics [9]. The primary objective of the toxicity study is to
determine the systemic absorption of the administered compound.
However, in addition to quantifying the active, quantitation of
metabolite becomes critical in the following circumstances [124].
1. If the drug metabolizes in to a pharmacologically or toxicologically
active metabolite.
2. If the administered drug is a pro-drug which metabolizes to form
an active moiety.
3. If the administered drug metabolizes extensively and measurement of the metabolite is the only means of quantifying the drug
following its administration.
Generally, various parameters that are measured during a nonclinical study [9] are Cmax, tmax, area under the curve (AUC) and half
life (t1/2).
Generally, acute toxicity information is obtained from single dose
toxicity studies in two mammalian species using both the clinical and
parenteral route of administration [128]. However, such information
can also be obtained from dose escalation or short duration dose
ranging studies if carried out appropriately [129,130] . Other equally
appropriate studies include the studies that can achieve large
exposure, achieve saturation of exposure, or use the maximum
feasible dose. Availability of this acute toxicity information does not
require the scientist to perform separate single dose studies. The
repeated dose toxicity study should also be conducted in two
mammalian species (one non-rodent) and the duration of this study
should be derived from the duration, therapeutic indication and the
scope of the proposed clinical trial [128].
10. Analytical testing in pre-clinical studies
Various analytical techniques are used in analyzing the test
samples for pre-clinical studies. Formulation samples can be analyzed
using techniques such as HPLC coupled with various detectors such as
UV, PDA, uorescence, or refractive index. Some samples can also be
analyzed by wet chemistry methodology such as titration. Testing of
plasma and blood samples from animal models can be performed
using techniques such as LC, LC-MS, LC-MS-MS, Q-Tof. The analytical
methodologies used to test samples for toxicology studies must be
validated. The method should be specic to quantitate the analyte in
the matrix which is usually blood, plasma, or tissue. This matrix
should not produce interference during quantication of the drug
substance. Various parameters such as method linearity, accuracy,
precision and sensitivity must be established prior to analyzing the
samples. The limit of quantitation (LOQ) and limit of detection (LOD)
must be established.
Surfactant and bioavailability enhancers used in the formulation
can pose a challenge in the development of analytical method of the
active. The reason being that the most commonly used extraction
solvents such as methanol and acetonitrile are unable to extract the
hydrophobic active from the surfactant/lipid matrix. One of such
excipient is Cremophor EL, which is a commonly used solvents in
pre-clinical drug product development. It is also present in several
marketed products such as Taxol and Vumon . Cremophor EL
consists of a mixture of hydrophobic and hydrophilic groups. Because
of the presence of both hydrophobic and hydrophilic groups it acts as
excellent solubilizer for hydrophobic drugs. However, Cremophor EL
poses a challenge in the quantitation of the active and the impurities
in the drug products via HPLC-UV analysis. Huizing et al.[131] have
shown that Cremophor EL reduces the sensitivity and also interferes
with the resolution of derivatized paclitaxel as well as some
impurities. The authors carried out a Solid Phase Extraction to remove
Cremophor EL prior to analyzing paclitaxel and its metabolites. In
another study Cremophor EL was precipitated and removed from
294
paclitaxel solution via complexation with mercuric chloride to

prevent interference [132].
For oral solid dosage form, dissolution study is very important. In
vitro drug release should be evaluated prior to performing in vivo
studies. USP dissolution test gives a general idea of the tentative
performance of the formulation in vivo. In one of the studies RitonavirPEG8000 solid dispersion were prepared and evaluated for in vitro or
and in vivo studies [133]. In this study Ritonavir, a BCS class IV drug
solid dispersion (SD) were prepared with various drug loading of 10%,
20%, and 30% drug. The in vitro results demonstrated a faster and
complete release of 10% solid dispersion in comparison to the 20% SD,
30% SD, and physical mixtures of Ritonavir and PEG 8000. The in vivo
study in Beagle Dogs showed a higher average Cmax and AUC values of
5.48 g/mL and 14.04 g.h/mL respectively for the 10% solid dispersion in comparison to 20% SD ( 4.42 g/mL and 11.46 g.h/mL) 30% SD
(3.12 g/mL and 7.00 g.h/mL), and crystalline drug (0.40 g/mL and
0.64 g.h/mL). This study shows that with an increase in the drug
loading beyond 10%, in solid dispersion the plasma concentration
decreased. These results were evident from the in vitro dissolution
study.
11. Conclusion
The importance of early formulation development should not be
underestimated. It enables the right selection and optimization of new
drug candidates at different preclinical stages. Pre-formulation study
and formulation development is the key to successful completion of
pre-clinical study which can provide meaningful data and expedites
the evaluation process. A thorough understanding of the physicochemical properties of the drug substance can provide valuable
information to establish the formulation development strategy as well
as crucial to select ideal excipients. The formulation scientist must be
aware of the purpose and evaluate the study specic challenges prior
to initiating formulation development efforts. The formulation
scientist must consider the stage of the preclinical study i.e.
pharmacokinetic, toxicology or dose ranging study, to determine the
maximum amount of inactive ingredients that can be used without
causing adverse effects due to the excipient.
Novel formulations should be explored when conventional
systems are inappropriate in addressing the need for in vivo exposure.
Developing novel formulations requires signicant resources and
time but often prove to be critical in advancing the study. Formulators
need to overcome challenges due to limited availability of drug
substance and tight timelines in order to achieve the aspired goal.
Consideration should be also given to additional parameters such as
the BCS class of the drug and route of administration as well as the
regulatory requirement for the study. Computer simulation is an
upcoming eld for predicting in vivo behavior of the drug substance,
however, for a NCE at least one pre-clinical study must be carried out
to evaluate the accuracy of the computer predictions. A systematic
approach to preclinical study can provide substantial data which can
predict the outcome of the human clinical study, avoid number of
animal studies, shorten development times and lead to lower costs.
Teamwork and excellent coordination between different functional
groups such as pharmacology, DMPK, toxicology, and pharmaceutical
departments is mandatory for the success of the study.
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Journal of Controlled Release 156 (2011) 281296

Contents lists available at ScienceDirect

Journal of Controlled Release

Recent advances and novel strategies in pre-clinical formulation development: An

Corresponding author. Tel.: + 1 484 870 5597.

Recent advances in pre-clinical studies . . . . . . . .

substance on living organisms and to gather sufcient data to determine

affect various properties such as solubility, bioavailability, and

3. Dene strategies to achieve optimum data comparability for

2.1. Drug solubility

Ninety-six-well library format, wide range

Bruker Optics SpecScreen xHTS

2.2. Partition co-efcient and in vitro permeability

Where, Su, log P, and MP are the aqueous solubility, Octanol-water

Where, BR is the biological response; B, C, are molecular

good indicator of diffusion or permeation across the cell membrane,

forms. Characterization of polymorphism is critical for formulation

Rat oral: 20 g/kg

3. Formulation development in pre-clinical development phase

It is estimated that the development of a new drug from discovery to

4. Formulation development strategies

Rat i.v.: 7.3 g/kg

Rat oral: 22 g/kg

Rat i.v. 1 g/kg Mouse

Rat oral: 34.5 mL/kg

Rat oral: N 20 mg/kg

oral: 7.5 mg/kg

Soluphor P. PEG400 is well tolerated via intravenous (i.v.) or oral

molecule and its possible interaction with the excipients is essential

4.3.1.4. Self-emulsifying drug delivery system. Self-emulsifying drug

molecules an injectable suspension or an emulsion formulation is also

the suspension. Aggregation of the sub-micron particles can be

be taken at a minimum to ensure sterility of IV preparation for preclinical study:

Where, EU is the Endotoxin Unit, K is the threshold human

Body weight (kg)

elevated temperatures which can lead to chemical and/or physical

iological based model was developed by Yu et al. followed by

techniques along with increasing availability of human tissues have

The evaluation of anti-tumor agents in immune-decient mice

7. In vivo pre-clinical models

Epirubicin and daunorubicin were signicantly less cardiotoxic than

thereby providing a means to assess the PK and PD rationale to

surrogate for human subjects, and the appropriate surrogate species

paclitaxel solution via complexation with mercuric chloride to

[70] B. Eerdenbrugh, G. Mooter, P. Augustijns, Top-down production of drug

[118] P. Pouna, S. Bonoron-Adele, G. Gouverneur, L. Tariosse, P. Besse, J. Robert,

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