Review

Impact of preformulation on
drug development
Sonali S Bharate & Ram A Vishwakarma†
1. Introduction Indian Institute of Integrative Medicine (CSIR), Jammu, India

2. Salt form selection and its

Introduction: Preformulation assists scientists in screening lead candidates
stability
based on their physicochemical and biopharmaceutical properties. This data
3. Polymorphic phase transitions is useful for selection of new chemical entities (NCEs) for preclinical efficacy/
4. Crystal habit and druggability toxicity studies which is a major section under investigational new drug appli-
cation. A strong collaboration between discovery and formulation group is
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5. Chirality
essential for selecting right NCEs in order to reduce attrition rate in the late
6. Bulk properties (particle size
and size distribution, flow,
stage development.
dissolution, etc.) Areas covered: This article describes the significance of preformulation
research in drug discovery and development. Various crucial preformulation
7. Hygroscopicity
parameters with case studies have been discussed.
8. Drug stability Expert opinion: Physicochemical and biopharmaceutical characterization of
9. Drug solubility NCEs is a decisive parameter during product development. Early prediction
10. Physical incompatibilities of these properties helps in selecting suitable physical form (salt, polymorph,
11. Biopharmaceutical etc.) of the candidate. Based on pharmacokinetic and efficacy/toxicity studies,
aspects -- absorption, suitable formulation for Phase I clinical studies can be developed. Overall
distribution, metabolism and these activities contribute in streamlining efficacy/toxicology evaluation,
For personal use only.

excretion allowing pharmacologically effective and developable molecules to reach

the clinic and eventually to the market. In this review, the magnitude of
12. Preformulation and IND
approval
understanding preformulation properties of NCEs and their utility in product
development has been elaborated with case studies.
13. Conclusion
14. Expert opinion Keywords: drug development, investigational new drug, preformulation, product recall

Expert Opin. Drug Deliv. [Early Online]

1. Introduction

Preformulation studies (physicochemical and biopharmaceutical properties) play a

significant role in drug discovery and development programs as they provide valu-
able information about druggability during lead identification and optimization.
The role of preformulation data means different things to different groups in the
discovery and development phases. For chemistry team, preformulation testing
provides clues to optimize the chemical structure with respect to solubility, perme-
ability and stability [1,2]. For a biology group, preformulation information (perme-
ability, solubility and stability) is useful to decide optimal drug exposure for
pharmacokinetic and toxicological studies. The analytical team gets feedback from
the preformulation group on developing stability-indicating assays and setting drug
specifications. After lead selection, as a molecule moves further toward the develop-
ment phase, preformulation data provides guidance to bulk manufacturing, formula-
tion development and clinical evaluation protocols [3]. The bulk manufacturing team
uses data generated from preformulation studies on salt selection, purity of the poly-
morph and particle size specifications. The formulation team utilizes maximum data
from preformulation testing to design an appropriate dosage form. Physicochemical
properties are used for improving drug solubility, permeability and stability, selecting
appropriate excipients and processing conditions to design and evaluate the final dos-
age form. The clinical evaluation team utilizes preformulation data along with data
from preclinical animal studies to understand the pharmacokinetics of the drug in

10.1517/17425247.2013.783563 © 2013 Informa UK, Ltd. ISSN 1742-5247, e-ISSN 1744-7593 1

All rights reserved: reproduction in whole or in part not permitted
 S. S. Bharate & R. A. Vishwakarma
Article highlights.
. Preformulation research helps in selection of NCEs for
further development cycles and is a major component of
the CMC section of IND application.
. Significance of preformulation properties of APIs in drug
discovery and development has been exemplified with
case studies wherein preformulation studies helped in
product development. Case studies of product recall due
to inadequate preformulation data are also discussed.
. Physicochemical characterization of chemical moieties at
an early stage of development will reduce development
costs and timelines.
. Negligence toward assessment of any of the
Expert Opin. Drug Deliv. Downloaded from informahealthcare.com by Universita Studi di Torino on 04/03/13
preformulation parameters may lead to a significant
impact on total development activities.
This box summarizes key points contained in the article.
humans through the maximum absorbed dose and the biophar-
maceutics classification system (BCS) paradigms. Therefore, a
strong preformulation team in the drug discovery setting is crit-
ical for optimizing the pharmaceutical properties of a drug.
This can considerably reduce attrition rate, time and cost of
developing a new drug [4-7]. An alternative approach would be
For personal use only.
to have quick proof of concept for first-time-in-man studies to
make a decision on the drug candidate. This early ‘proof of
concept’ is an alternative view for rapid, slimmed down devel-
opment both in terms of cost and time.
It has been shown that inadequate preformulation data is
one of the reasons for product recalls. The information
required on preformulation will, to a certain extent, be dic-
tated by the final product, the proposed route of administra-
tion and the development plan. A schematic overview of
preformulation characteristics is depicted in Figure 1.
An extensive number of research papers have been pub-
lished on different techniques and protocols for estimation
of preformulation parameters [8], as well as there are several
reviews [9-11] on the theoretical aspects of preformulation.
Szunyogh et al. [10] reviewed (written in Hungarian language)
the importance of decreased particle size in preformulation.
Hageman [9] has discussed the assessment of key physico-
chemical properties and how they are implicated in both dis-
covery enablement and in final product developability of
selected candidate. Ashizawa [11] reviewed (in Japanese lan-
guage) the importance of physicochemical profiling and pre-
formulation studies at the drug discovery stage. However,
there is no review available where the significance of individ-
ual preformulation parameters in product development is
elaborated using case studies of drug candidates. Furthermore,
a critical analysis of preformulation-related product recalls is
missing in earlier reviews. The present review provides a crit-
ical account on how understanding of chemical and pharma-
ceutical properties of medicinal chemistry leads plays a vital
role in product development. This has been elaborated using
case studies of drug candidates wherein preformulation data
2 Expert Opin. Drug Deliv. [Early Online]
proved to be crucial in the drug’s preclinical/clinical develop-
ment. Additionally, case studies of product recalls have been
discussed wherein some additional preformulation data would
have avoided these withdrawals. Preformulation requisites and
regulatory aspects of preformulation for investigational new
drug (IND) applications have also been discussed. The chem-
ical structures of drugs (1 -- 28) discussed in the following
sections are shown in Figure 2.
2. Salt form selection and its stability
The chemical, biological, physical and economic characteris-
tics of drug candidates can be manipulated by converting
them to a salt form. Pharmaceutical chemists and formulation
scientists modify these characteristics by selecting an appropri-
ate salt form of a drug substance. This permits development
of dosage forms with good bioavailability, manufacturability
and stability along with patients’ acceptance. Salts are most
frequently employed for altering aqueous solubility; however,
selected salt forms also influence a range of other properties
such as melting point, chemical stability, hygroscopicity, solu-
tion pH, dissolution rate, crystal form and mechanical prop-
erties [12,13]. Pharmaceutical salts frequently exist as hydrates,
which create difficulties during formulation development,
particularly in wet granulation. It often becomes difficult to
monitor and control the anhydrate/hydrate form during the
wet mixing, fluid bed drying and aqueous film coating pro-
cesses. This factor has led to the deselection of a particular
salt form in further development process many times [14,15].
During the drug development process, discovery of new
crystal forms offers an opportunity to select an optimal form
of a drug candidate [16]. Cocrystals have also been utilized
for modifying the physicochemical parameters of drugs.
Tsutsumi et al. [17] studied miconazole salts and cocrystals
for improving physicochemical properties. They reported
hemisuccinate as a novel cocrystal form of miconazole with
an improved dissolution rate and superior stability, making
it an alternative for nitrate salt. Pharmaceutical cocrystal strat-
egy compliments the salt formation to enhance bioavailability
of drug product and stability [18].
The information generated from preliminary crystallization
studies is often helpful to the process chemistry group, which
investigates possible manufacturing routes for the molecule of
interest. The manufacturing route may be the same as used by
the discovery chemistry group, but it can also significantly dif-
fer. Both the process chemistry and preformulation groups
extensively collaborate for the next 12 -- 18 months in order
to ensure a viable synthetic route for the chosen salt form of
the drug candidate. A significant portion of this batch is des-
tined for the preparation of 3 -- 4 g of each salt that were
thought to be viable from smaller-scale studies. A similar-
sized portion of free base/acid is also taken for comparison
purposes. The combination of individual studies undertaken
on each of these 3 -- 4 g portions varies depending on the
type(s) of dosage form ultimately required for marketing.

Analytical
A) Molecular structure B) Chemical analysis
determination I) Quantification II) Purity
a) Elemental a) TLC a) Synthetic impurities
b) NMR b) HPLC b) Residual solvent
Reverse phase
c) Mass spectrometry c) Residual water
Chiral
d) IR d) Heavy metals
e) UV/Vis e) Degradants 1
microbiology
Candidate drug
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Pharmaceutical
A) Biopharmaceutical properties B) Chemical compatibility
a) Monolayer adsorption I) Processing
b) ADME II) Excipients
C) Regulatory stability
Figure 1. Map of preformulation studies revolving around the candidate drug. Studies would commence in the analytical
segment and swing around to the pharmaceutical segment as the compounds’ development progressed. As early studies
For personal use only.
affect later studies, the dotted pointer indicates one linkage, the determination of the UV/VIS spectrum is useful for HPLC
method development, which is influenced by synthetic impurities and degradants, which then allows degradation studies to
be conducted [8].
Occasionally, it may be necessary to undertake a pharmacoki-
netic evaluation of each salt in comparison with the free acid/
base. At this stage in the development process, the aim is to
quickly manufacture 50 -- 200 g of promising candidate(s)
for initial clinical evaluation. The alternative approach is to
proceed with the free base or acid form, if the drug candidate
displays efficacy in animal studies. In such cases, the extensive
process chemistry efforts can be skipped and the medicinal
chemistry synthetic route could be employed to quickly pre-
pare free base/acid form. Formulation strategies could be
employed in order to achieve adequate exposure of the drug.
Dosage forms most commonly used for drug substances
encountered during preliminary clinical investigations are tab-
lets/capsules, inhalation dosage forms and injections [19]. Of
the many salts synthesized, the preferred form is selected by
a pharmaceutical chemist primarily on a practical basis (raw
materials employed, ease of crystallization process and per-
centage yield). Other basic considerations include stability,
hygroscopicity and flowability of the resulting bulk drug.
The following representative examples illustrate how and
why selection of proper salt forms is necessary during prefor-
mulation and before going forward to product development.
The process for producing a stable chlortetracycline (1) sus-
pension was developed at Lederle Laboratories between
1951 and 1954 [20]. Berge et al. [21] observed that hydrochlo-
ride salt was highly unstable in aqueous solution, but calcium
salt was very stable. They also reported that potassium salt of
Expert Opin. Drug Deliv. [Early Online]
Impact of preformulation on drug development
Physical
Physical analysis
a) Melting point f) Microscopy
b) Hygroscopicity i) Particle shape
c) Differential scanning calorimetry ii) Particle size
g) Powder
d) Thermogravimetry
i) Compaction
e) X-ray crystal determination
2 ii) Flow
Physicochemical
A) Chemical stability B) Physicochemical properties
3
a) Oxidation I) Log P
b) Light II) pKa
c) pH III) Solubility
Degradants
d) Temperature a) Solvent
e) Solvent b) pH
c) Dissolution
penicillin G (2) was much less hygroscopic than sodium salt;
however, penicillin G potassium tends to have an unpleasant
metallic taste [20]. Serajuddin et al. [22] observed that hydro-
chloride salt of the poorly water-soluble drug REV-5901
(3, an orally effective peptidoleukotriene antagonist) is unsta-
ble due to conversion of anhydrous and monohydrate forms
to a hydrate form at > 40% relative humidity. On loss of
water at 40 -- 60 C, the hydrate form became unstable, which
was also confirmed by accelerated stability studies. However,
the basic form of REV-5901 was stable and pharmaceutically
acceptable crystalline solid. Engel et al. [23] performed a salt
selection study on LY333531 (4), a competitive reversible
inhibitor of protein kinase Cb, which was being evaluated
for treatment of diabetic complications (e.g., retinopathy
and erectile dysfunction). LY333531 as a free base has poor
aqueous solubility and thus its salt form was desired to
enhance solubility and bioavailability. Seven salts (hydrochlo-
ride, mesylate, tartrate, sulfate, succinate, acetate and phos-
phate) were initially crystallized and evaluated for various
physical properties such as particle size and morphology,
hygroscopicity, crystallinity and aqueous solubility in order
to find out feasibility for further development. Of the seven
salts selected, five salts (sulfate, succinate, tartrate, acetate
and phosphate) were eliminated due to poor crystallinity,
low solubility and difficulty in improving chemical purity
during preparation. The two remaining mesylate and hydro-
chloride salts were comprehensively analyzed for additional
3
 S. S. Bharate & R. A. Vishwakarma

O
H O
OH O O OH OH S H N
OH N N
OH
H2N N
N O N
O O O
O
H H OH
N HO Cl NH
2 Penicillin G 3 REV-5901
O
1 Chlortetracycline 4 LY333531
Cl N HO O S
OH H O
NH O
O O-
7 L-649923
Cl OH S
O
O O
5 Diclofenac 6 O OH
O N
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OH H H
O NH2 O O
H N
N H
N N N OH N
O OH
OH HO OH H
O N N S O
N HO O N 8 Nelfinavir
10 Thiamine
9 Theophylline HO
O
O O O O OH OH
N Cl OH 11 Erythromycin OH
N H2N
S H HO
Cl
O HO O HO
O H H OH
O O N OH
HN N
NH HN +
N N 16 Oxytetracycline
N O O-
N
13 Chloramphenicol O
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N S S O
15 Phenylbutazone
12 Ritonavir OH
HO
H
14 Rotigotine O O
Cl N O
O
O N
S NH HN N
HN S S N
O H2N N O
O O O
N O N O
H
H O
17 Tolbutamide 18 Hydrochlorothiazide
N
N O 19 Irbesartan
O O
N N O
OH O
O S N O
NH OH
NH2 O H
I I 20 Rapamune
F OAc
21 RPR 200765 HO O
O I
N HO O
22 NBI 75043 I 23 Levothyroxine O
H3CO OH
F
N O HH
H3CO OCH3 OCH3 O
Cl OH F 24 Fluocinonide
25 Combretastatin A4 OH
HO O NH NH
O O HN N NH2
27 Warfarin
OH O
26 Flavopiridol 28 Metformin

Figure 2. Chemical structures of drugs 1 -- 28 discussed in preformulation-related case studies.

4 Expert Opin. Drug Deliv. [Early Online]


parameters such as polymorphism, stability, purification, fil-
terability and relative bioavailability in dogs. Results obtained
from these efforts indicated that mesylate salt can be devel-
oped as the optimal salt form of LY333531.
Apart from salt form selection, the stability of the selected
salt is imperative. Chemical and physical aspects associated
with preparation of pharmaceutical salt are formation of
hydrates, polymorphism and amorphization. These aspects
are also important from a technological point of view, as
they affect solubility, dissolution rate and bioavailability of
drugs. Therefore, each pharmaceutical salt must undergo a
careful preformulation study in order to define experimental
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conditions of its stability. Fini et al. [24] prepared salts of diclo-
fenac (5) with various salt-forming agents in order to find the
optimally stable and soluble salt form. Diclofenac salts with
eight different cyclic aliphatic amines, such as pyrrolidine,
piperidine, morpholine, piperazine and their N-hydroxyethyl
analogs, were prepared and characterized. However, these
salts were found to possess poor aqueous solubility as com-
pared with diclofenac sodium. Hence, each pharmaceutical
salt must undergo a careful preformulation study in order to
define its solubility and stability. Ionizable and non-ionizable
prodrugs can also be prepared to improve bioavailability of
drug candidates. However, use of free base form of the
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molecule with alternative formulation strategy (nanodelivery
systems, such as nanoparticles, liposomes, nanoemulsions/
microemulsions, micelles and longer circulating half-life of
the drugs by PEGylation) is equally straight forward. Typi-
cally, the salt formation is considered as the most practical
approach to enhance bioavailability.
The maleate salt of a basic drug dibenzo-(b,f)-oxepen-
10-yl-methyl-methyl-prop-2-ynyl-amine (6) was found to be
more stable than free base. This selected salt was developed
for oral administration in the treatment of neurodegenerative
disorders, such as Parkinson’s disease and amyotrophic lateral
sclerosis. Hard gelatin capsules of various potencies (0.25,
2.5 and 10 mg) were selected for Phase I clinical studies. After
initial clinical testing using capsules in Phases I and IIA, scien-
tists decided to switch the dosage form from capsule to tablet
with the same potencies for the Phases IIB and III clinical
studies, for commercialization purpose. Tablets were devel-
oped with a minimal change in composition from the capsule
formulation to maintain continuity between developmental
activities of two dosage forms and to ensure that their stability
would be similar. Tablet formulations of the maleate salt of
this drug showed major loss in potency and lack of mass bal-
ance upon storage under accelerated stability testing. No such
stability problem was observed in capsules that were similar in
composition. Interestingly, the tablet was found to be stable
when it was encapsulated in a capsule shell. It was identified
that the reason behind the instability of the drug in tablets was
conversion of salt to its free base form in the microenvironment
of tablet formulation [25-27].
The free base of an orally active leukotriene D4 antagonist
L-649923 (7) was found to be highly unstable because of its
Expert Opin. Drug Deliv. [Early Online]
Impact of preformulation on drug development
propensity to undergo intramolecular esterification to form
g-lactone. In order to avoid formation of g-lactone, formula-
tion efforts were undertaken wherein calcium salt of drug and
tablet dosage form were chosen for formulation development.
A direct compression method was employed in tablet
manufacturing. A wet granulation process was avoided by
using excipients with low water content and adding sodium
carbonate as an alkalizing agent. This case demonstrated the
importance of considering microenvironment pH of the solid
to stabilize the drug product. Understanding degradation
mechanisms should be the basis for selecting the right salt
and developing a chemically stable solid dosage form [28,29].
The possible impurities in new chemical entities (NCEs)
are also a major concern during the drug development pro-
cess. For profiling of genotoxic impurities, a rapid and conve-
nient method for chemical analysis should be developed at an
earlier stage [30]. The recent example of the anti-HIV drug nel-
finavir mesylate (Viracept), which was withdrawn from the
market has shown the importance of impurity profiling [31].
The oral formulation of nelfinavir mesylate (8) was with-
drawn from the European market in mid-2007 due to elevated
levels of ethyl methanesulfonate which is a potential cancer-
causing contaminant. The ethyl methanesulfonate was gener-
ated as a by-product of the starting material, methane sulfonic
acid [32]. This issue resulted in a recall from European regula-
tors to assess risk mitigation strategies for all marketed prod-
ucts employing sulfonic acid counter-ions to ensure that
sulfonate esters that could be potentially formed are controlled
to threshold of toxicological concern-based limits [31,33].
3. Polymorphic phase transitions
3.1 Phase transitions during product development
Transformations between solid phases in dosage forms often
affects the drug release process and, therefore, it is important
to understand the mechanisms and kinetics of phase transfor-
mations and factors that may influence them [34]. The risk of
change to the physical form of the drug substance can be min-
imized by selecting a stable physical form of active pharma-
ceutical ingredient (API); however, it may also adversely
affect the bioavailability of the API. Thus, based on the collec-
tive information on the bioavailability of stable, metastable
and least-stable forms of API, an appropriate form should
be selected. After selection of a suitable form of the drug,
the process of formulation and conditions also decides the
solid state of the drug in the formulation. This phenomenon
is referred to as processing-induced phase transitions. In the
finished tablet dosage forms, in situ transformation also
affects tablet properties such as porosity, interparticulate
interactions and hardness. In situ phase transitions of
API in formulation has been exemplified with theophylline
(9) [35], thiamine hydrochloride (10) [36] and erythromycin
(11) [37], etc. Wet massing is one of the important steps in tab-
leting, wherein Tantry et al. [38] observed complete phase
transition of stable anhydrous theophylline to theophylline
5
 S. S. Bharate & R. A. Vishwakarma
monohydrate at elevated temperatures, which resulted in a
decrease in its dissolution rate. There was complete conver-
sion of stable anhydrous theophylline (A) to theophylline
monohydrate (M) upon addition of water. During the drying
process, dehydration of the M form resulted in formation of
metastable anhydrous theophylline (A*), which was trans-
formed to A. An increase in drying temperature accelerated
the A* to A transition; however, addition of PVP (a crystalli-
zation inhibitor) inhibited this transition. Interestingly, a
phase transition was noticed with the variation in method of
preparation of granules. The fluid-bed process produced gran-
ules that consisted almost completely of form A (stable form
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of theophylline), whereas the high shear method yielded mix-
ture of A and A* forms. This in situ transition led to decreased
dissolution rates of these tablets. Therefore, this phase transi-
tion between stable and metastable forms of API can have a
pronounced impact on product performance.
Chakravarty et al. [36] monitored phase transformation in
thiamine hydrochloride tablets on storage, which were formu-
lated by direct compression and wet granulation methods. Thi-
amine hydrochloride was converted from non-stoichiometric
hydrate (NSH) to hemihydrate (HH) in stored tablets. There
were significant differences in the physicochemical properties
of tablets such as tablet microstructure, compact physical prop-
For personal use only.
erties and product behavior. A marked increase in disintegra-
tion time, tablet volume and hardness upon wet granulation
of NSH with microcrystalline cellulose (MCC) was also
observed. However, when tablets of NSH-MCC were formu-
lated by dry processing via direct compression, no change was
observed in properties except the in situ solid form converted
to needle-like HH crystals in all stored tablets. Longer storage
also led to crystal growth with reduced changes in properties.
Abbott Laboratories [37] reported dissolution failure of
erythromycin tablet formulation due to formation of in situ
isomorphic dehydrate. Specific lots of 12-month stability
samples of erythromycin dihydrate tablets (labeled claim
250 mg) failed in dissolution specifications. Lot 15, which
had a very good initial dissolution (> 99% at 60 min) failed
uniformly at the 12-month time point (71% released at
60 min). The tableting process for erythromycin involved
milling and drying, which was responsible for altering crystal-
linity and water content. During formulation development,
erythromycin dihydrate gets dehydrated, which gradually
binds with hydroxyl-rich excipients such as Mg(OH)2, and
thus results in delayed dissolution process.
3.2 Effect of polymorphism on bioavailability
To determine stability/instability of a drug, knowledge of
solid-state reactions and polymorphism [39] is important.
The selected solid form of a drug for development must
remain stable in formulation throughout the shelf life of the
API [40]. For formulation development, generally crystalline
form of drug with well defined and reproducible physical
properties is preferred [41]. The polymorphic form of a drug
shows variation in solubility, melting point, density, hardness,
6 Expert Opin. Drug Deliv. [Early Online]
crystal shape, optical and elemental properties (e.g., vapor
pressure etc.) [42]. Moreover relative bioavailability of solid
compounds may vary markedly from one form to another.
The crystal structure affects stability and developability of
the crystallized product. The ritonavir (12, Novir) [43] story
illustrates the importance of transitions among crystal forms
(conformational polymorphism). In 1998, Abbott experi-
enced a catastrophic example of physical instability of the
drug ritonavir [44,45]. Until 1998, Abbott had successfully man-
ufactured hundreds of batches of the life-saving anti-HIV drug
ritonavir. These batches were formulated by dissolving ritona-
vir in polyethylene glycol/ethanol/polyoxyethylene/other com-
ponents in a soft-gelatin capsule. In the spring of that year,
batches of the soft gelatin capsule started failing dissolution
specifications. Abbott quickly established that these failures
were caused by crystallization of a new, previously unknown,
less soluble polymorph of ritonavir. This new polymorph
(form 2) had about fourfold less solubility than the old form
and was supersaturated in formulation. Ultimately, Abbott
had to withdraw the soft-gel product from the market to
resolve the formulation issue. Abbott scientists then investi-
gated the reason for the sudden appearance of form 2. The
prima facie reason for appearance of form 2 was formation of
a poorly purging cis-impurity that acted as the template for
the formation of cis-ritonavir that was less soluble. Investiga-
tion at a ritonavir-manufacturing site in Italy identified a
possible reason for the appearance of form 2 as a result of acci-
dental seeding with this form [46]. Such transformations,
because they affect the solubility of the drug, can complicate
first-in-human trials and toxicological studies [45,47,48].
Another example of solid-state instability is chloramphenicol
palmitate (13), which when administered in suspension form
showed unsatisfactory clinical effect due to presence of biolog-
ically less active polymorphic form A [42]. It was discovered
that this compound exists in two different crystalline forms [49]:
the form A (b polymorphs -- biologically less active) [50] and
metastable form B (a polymorph -- active modification).
Upon oral ingestion of the suspension formulation of this
drug, blood levels of form A were found to be seven times
higher than form B [51]. The British and United States Pharma-
copoeias specify a limit test which places an upper permitted
limit of 10% of less active b polymorph in a chloramphenicol
palmitate mixture (chloramphenicol palmitate oral suspension
USP) [52].
In April 2008, Schwarz Pharma recalled transdermal
patches of the dopamine agonist rotigotine (14) (Neupro
patch) in the United States and some in Europe because of
problems with the delivery mechanism and a quality defect.
Formation of crystals of rotigotine in Neupro (a matrix design
rotigotine transdermal system) led to the product recall and
subsequent discontinuation. The crystallization occurred
because of the appearance of a new unknown polymorph of
rotigotine within the patch. This new polymorph was found
to be thermodynamically more stable and less soluble than
that of the original form; therefore, it produced crystals in

patches which were visible to the naked eye as ‘snowflakes’.
These ‘snowflake’ crystals consisted of pure rotigotine, the dif-
ference being that rotigotine molecules in this new polymorph
had a slightly different packing structure with each other com-
pared with the original rotigotine form. As only soluble mol-
ecules of rotigotine can cross the skin, crystals must dissolve in
order to release drug molecules that can be absorbed. If this
does not occur, the amount of drug available to the patient
could be reduced. Therefore, the net effect of the ‘snowflake’
phenomenon is the possibility of reduced drug delivery and
therapeutic efficacy [53-55].
Although polymorphic behavior alters the solubility of
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drugs, it does not necessarily alter the bioavailability as evi-
denced by phenylbutazone (15). In 1949, phenylbutazone
was used as a potent antirheumatic drug. As it is highly hydro-
phobic in nature (log P = ~ 4.15), it has very poor solubility in
acidic pH. It was observed that phenylbutazone has three
polymorphic forms namely form I, x and y. The maximum
drug concentration of form y was significantly higher over
the form x, because form y exhibited faster dissolution rate
(50% more) over the other forms. However, it was also
reported that there was no significant difference in bioavail-
ability parameters of form I and x as the rate of absorption
of these crystals was identical [56-58].
For personal use only.
The two forms of oxytetracycline, a dihydrate form and
HCl salt form have varying stability and water solubility.
The former was more stable compared to HCl salt form and
the aqueous solubility was opposite. Therefore, the relatively
insoluble hydrated base in the official preparation (British
Pharmacopoeia, 1973) may not be entirely satisfactory due
to its poor solubility. Thus, HCl salt was used for the prepa-
ration of formulations. However, Brice and Hammer in
1969 [59] reported that 16 lots of oxytetracycline hydrochlo-
ride (16) capsules (250 mg) from 13 suppliers showed drug
serum levels lower than that of the innovator product which
was thought to be associated with its instability. Further, in
1974 Barber et al. [60] observed the presence of different poly-
morph (form A) in the formulation, which had a slow dissolu-
tion rate compared with other polymorphs in 0.1 M HCl. For
example, tablets prepared using form A exhibited ~ 55% disso-
lution at 30 min, whereas the tablets prepared with form B
exhibited complete (~ 95%) dissolution at 30 min [61].
4. Crystal habit and druggability
Crystal engineering studies are important in the pharmaceuti-
cal industry to develop stable and robust solid dosage forms.
Solid-state properties have a great impact on physicochemical
and biopharmaceutical properties of drugs. The solid state is
important as it affects filtration, flow, tableting and dissolu-
tion. Although there may not be significant differences in bio-
availability of drugs with different crystal habits, it is of
importance from a technological point of view. The injection
of a suspension-containing drug in crystal form is influenced
by its habit. Crystal habit also influences the ease of
Expert Opin. Drug Deliv. [Early Online]
Impact of preformulation on drug development
compression of the tablet and the flow properties of the solid
form of the drug. For example, plate-like crystals of tolbuta-
mide (17) created powder-bridging in the hopper of the tablet
machine and also led to capping problems during tableting [62].
Bristol-Myers Squibb recalled antihypertensive combination
of hydrochlorothiazide (18) and irbesartan (19) (Avapro tab-
lets) in January 2011, due to problems associated with dissolu-
tion of irbesartan. This reduced solubility of irbesartan in
certain batches was the result of formation of a secondary crys-
tal form of irbesartan. The formation of the less soluble crystal
form was due to tablet manufacturing changes. Detailed inves-
tigations revealed that an increase in granulation time resulted
in formation of these secondary crystals [63].
5. Chirality
Determination of chirality is a part of typical development
track activity for preformulation monitoring. Generally, one
of the enantiomers does not have desired pharmacological
properties and thus unwanted enantiomer should be elimi-
nated from the drug formulation. Once the separation tech-
nique is available, the inactive form should be excluded
considering economical point of view. Therefore, for develop-
ment of NCE, the resolution method of an optically active
compound should be available at earlier stages of the discovery
process. As a regulatory requirement for IND submission, the
enantiopure form of the drug is essential. Thus, the selection
of right enantiomer for the market product must be decided
before patent application and IND submission. Racemic
mixture can only be considered if the unwanted enantiomer
racemizes rapidly (metabolically) to the desired enantiomer.
Heavy investment on wrong enantiomer can be avoided by
selecting a therapeutically active enantiomer [64]. For example,
sirolimus (20) (Rapamune) marketed by Pfizer (formerly by
Wyeth) is an immunosuppressive agent that was approved by
the Food and Drug Administration (FDA) in 1999. It is avail-
able as a clear, viscous oral solution containing 1 mg/ml siro-
limus and coated tablets of 1 mg sirolimus as an active
ingredient. Inactive ingredients in the Rapamune oral solu-
tion are Phosal 50 PG and polysorbate 80 (nonaqueous
vehicles). Sirolimus is chiral (15 stereogenic centers) and is
produced as a single defined stereoisomer in solid state using
asymmetric synthesis. However, in aqueous solutions this was
interconverted into three isomers (A, B and C). Therefore,
selection and use of nonaqueous vehicles during product devel-
opment eliminated major concerns with regard to solid-
state properties of the drug substance, such as the potential
for polymorphic forms, particle size, surface area, intrinsic dis-
solution rate and oral bioavailability issues of sirolimus [65-67].
6.Bulk properties (particle size and size
distribution, flow, dissolution, etc.)
Particle shape and size affect both primary and bulk powder
properties and play a decisive role in controlling dissolution [68].
7
 S. S. Bharate & R. A. Vishwakarma
Knowledge of particle size and size distribution, flow proper-
ties and dissolution are key aspects that should be studied in
the field of dissolution science and during the formulation
development process [69]. The melting point of pharmaceutical
salts is often intrinsically related to their physicochemical prop-
erties. The APIs with low melting points often exhibit plastic
deformation during the formulation development process,
which can cause both caking and aggregation, and has an
impact on both flow and compressibility performance. These
in turn affect many critical quality attributes of the drug
product (with low dose) such as disintegration and in vitro
dissolution rates, friability and content uniformity [70].
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Bastin et al. [19] demonstrated the importance of flow prop-
erties by studying physicochemical properties of various salts
of the anti-arthritic drug RPR 200765 (21). Among four
salt forms investigated (mesylate, camphorsulfonate, hydro-
chloride and hydrobromide), mesylate salt demonstrated
good handling properties such as good flow (required during
formulation stage) and a non-hygroscopic nature, allowing
straightforward capsule and tablet development. These flow
properties were superior to the alternative salts studied
(hydrochloride, hydrobromide and camphorsulfonate) [71].
7. Hygroscopicity
For personal use only.
Hygroscopicity of drug substances is an important parameter
to be considered in product development. The moisture
uptake rate varies for different drugs and excipients and is
dependent on environmental conditions and it often affects
stability of the drug in the dosage form [72]. NBI-75043 (22)
is a highly selective and potent H1 receptor antagonist that
was under evaluation for safety and efficacy in the treatment
of insomnia. Initially a tartrate salt of NBI-75043 was pro-
duced and dried to a constant weight, but it was found to
be ethanol solvate as determined by C, H and N in gas chro-
matography residual solvent analyses. In addition, the tartrate
salt was a hygroscopic solid with a low melting point
(~ 94 C). Moreover, ethanol solvate was found to readily
exchange ethanol for water within 1 week under accelerated
stability conditions (40 C/75% relative humidity). Due to
these inherent negative properties for API, this salt form of
NBI-75043 was recognized as being unacceptable for long-
term studies. Neurocrine Biosciences found that the besylate
salt of NBI-75043 is crystalline in nature and exists in a single
polymorphic form with a higher melting point (165 -- 168 C)
and, thus, was found to be suitable for product development [73].
8. Drug stability
Stability screening of drug candidates plays an important role
in drug discovery. It provides an overview of the stability of
the compound in a variety of pharmaceutical situations and,
thus, helps to identify potential liabilities that may affect the
drug development paradigm. The information obtained
from these studies can be used to: i) provide feedback to the
8 Expert Opin. Drug Deliv. [Early Online]
chemistry team for modification of labile groups to improve
stability; ii) help development scientists to determine develop-
ability of the compound; iii) provide guidelines on compound
handling and storage; and iv) provide information to guide
stabilization strategies [3,48,74].
An orally administered levothyroxine sodium (23) product
did not remain potent and stable through their expiration
dates. Levothyroxine sodium was first introduced into the
market before 1962, without an approved new drug applica-
tion (NDA), apparently in belief that it was not a new drug.
After that almost every manufacturer had regular recalls due
to reason of potency or stability issues. It has been reported
that levothyroxine sodium was unstable in the presence of
light, temperature, air and humidity [75,76]. As these products
were marketed without NDAs, manufacturers need not have
FDA approval each time after reformulating levothyroxine
sodium products. Manufacturers performed formulation
changes such as changed excipients, physical appearance of
coloring agents and other product aspects, which significantly
changed the potency of the drug (either decreasing or increas-
ing by as much as 30%). In a few cases, these formulation
changes resulted in toxicity to people who were on the same
dosage for years. With the occurrence of potency issues, the
US FDA classified all orally administered drug products of
levothyroxine sodium as new drugs and made it mandatory
for all existing products to undergo the NDA process to
ensure consistent potency. As the drug is necessary to millions
of Americans, the FDA allowed marketing of these products
without an approved NDA until August 2000 and gave suffi-
cient time to submit NDAs [77]. Teva Pharmaceuticals recalled
60 mL bottles of 0.05% fluocinonide (24) topical solution
USP in the United States because of degradation/impurities
which led to subpotency [78].
9. Drug solubility
Solubility of a drug substance is a fundamental property that
should be evaluated early in the discovery phase. In reality, it
is difficult to predict the aqueous solubility due to the compli-
cated solubilization procedure and solid-phase chemistry of
the drug candidate. A variety of approaches are employed at
different phases of drug discovery and development such as
in silico, kinetic and equilibrium solubility. Thermodynamic
or equilibrium solubility has been considered as the ‘gold
standard’ for solubility determination [79], but kinetic solubil-
ity is more appropriate for early phase drug discovery [80].
A direct relationship exists between solubility of a molecule
and its dissolution rate. Drug penetration through lipid mem-
branes depends on the lipophilicity of the molecule and, thus,
may be correlated with the partition coefficient value. Absorp-
tion of a drug depends on the correct balance between these
two opposite properties [81]. A lack of solubility affects the
ability of drug to achieve efficacious and toxicologically rele-
vant exposures in animals. The solubility of compounds to
be tested is critical for all high-throughput screening (HTS)

test systems. As most of the in vitro test conditions also
require a pH of 7.4, there is a real chance that the concentra-
tions apparently being tested are in fact not being attained. It
is difficult in HTS set up to ensure that the actual test concen-
trations have been achieved. Therefore, many times such tests
are typically run at relatively high concentrations to ensure
adequate safety margins (e.g., hERG channel liability) [82].
This characteristic also affects future developability and for-
mulation efforts for compounds [83].
Beijnen et al. [84] reported the formulation of investiga-
tional cytotoxic drugs. Combretastatin A4 (NSC-81373, 25)
is a natural product, isolated from dry stem wood of the South
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African tree Combretum caffrum, with excellent in vitro anti-
cancer activity, but no significant in vivo activity. Formulation
of combretastatin A4 was problematical due to limited aque-
ous solubility and chemical instability of the drug in solution,
which was aggravated by light. The most promising formula-
tion was one using the PET solvent system. PET is a mixture
of polyethylene glycol 400 (60%), polysorbate 80 (10%) and
ethanol (to 100%). In a preliminary murine toxicological
study, severe irritation at the injection site was noticed.
Attempts were also made to use other formulation approaches
such as using emulsions; however, the physicochemical prop-
erties of combretastatin A4 did not allow these attempts to
For personal use only.
succeed. Subsequently prodrug approach showed promise
wherein the phosphate ester proved to have adequate solubil-
ity and stability in aqueous solution; however, this candidate
was found to be inactive in vivo and current research is
directed toward identifying other suitable prodrugs.
Flavopiridol [5,7-dihydroxy-8-(4-N-methyl-2-hydroxypyr-
idyl)-6¢-chloroflavone hydrochloride, 26] is a flavonoid with
weak electrolyte properties and poor aqueous solubility
(0.02 mg/mL). Flavopiridol is currently under consideration
as a parenteral treatment for breast tumors [85]. A number of
methods have been tried to improve solubility of flavopiridol
such as cosolvency, complexation and pH control. Because of
its low intrinsic solubility, precipitation of the solubilized
drug, when diluted with blood upon injection, was a problem.
Li et al. [86] studied precipitation of the drug in parenteral for-
mulation containing up to 10 mg/mL of flavopiridol using a
static serial dilution technique. There was no precipitation
in formulations containing 2 and 5 mg/mL of flavopiridol
in 30% 2-hydroxypropyl-b-cyclodextrin (HP-b-CD) -0.1 M
citric acid at pH 5.8 and 10 mg/mL of flavopiridol in 30%
HP-b-CD--0.1 M citric acid at pH 3 [86,87]. This improve-
ment in solubility of flavopiridol was thought to be due to
better solubility of the HP-b-CD complex of flavopiridol at
the abovementioned pH.
10. Physical incompatibilities
Assessment of possible incompatibilities between drug and
excipients is a key element of preformulation. Formulation
of a drug substance involves being blended with different exci-
pients to improve its manufacturability and to maximize the
Expert Opin. Drug Deliv. [Early Online]
Impact of preformulation on drug development
product’s ability to effectively administer the drug dose.
Most excipients have no direct pharmacological action but
they impart useful properties to the formulation. Apart from
this, they can also give rise to inadvertent and/or unintended
effects such as increased drug degradation. Physical and chem-
ical interactions between drugs and excipients may affect
chemical nature, stability and bioavailability of drug products,
and subsequently their therapeutic efficacy and safety [88].
Incorporation of additive(s) to an intravenous fluid may
alter inherent characteristics of the drug substance present,
resulting in parenteral incompatibility. Physical compatibility
of various components in parenteral nutrition (PN) solutions
has been a concern, particularly solubility of minerals such as
calcium and phosphate [89]. A safety alert was issued by the
FDA in 1994 regarding hazards of precipitation associated
with PN. Admixtures were thought to contain precipitates
of calcium phosphate and patient autopsies revealed diffuse
microvascular pulmonary emboli containing calcium phos-
phate [90,91]. During January 1994, two patients at a federal
hospital died suddenly while receiving a ‘three-in-one’ (i.e.,
amino acid, carbohydrate and lipid) peripheral PN admixture.
Clinical diagnosis in both patients was pulmonary embolism.
The autopsies revealed amorphous deposits of calcium phos-
phate in pulmonary microvasculature of both patients [92].
The size of the particles was not evaluated, but the reason
behind the deaths was considered to be due to a faulty mixing
sequence, deficient inline filtration and a short time from
compounding to administration [93,94].
An example of therapeutic incompatibility is Coumadin
tablets. Bristol-Myers Squibb recalled Coumadin (warfarin
sodium, 27) crystalline 5 mg tablets (lot number 9H49374A)
because of efficacy issues. In these formulations, isopropanol
was used to keep the active ingredient in a crystalline state.
However, poor quality isopropanol had a risk of Coumadin
being less potent or overly potent and certain lots were recalled
because of isopropanol not meeting quality specifications.
Recalls in these batches were decided because if a drug becomes
less potent, there is an increased risk of blood clots, heart attack
or stroke, and if it becomes too potent there is more risk of
bleeding [95].
Metformin (28) (Starlix), a product of Novartis, which is
intended for treating type II diabetes, is an example of charge
interaction reported by Hollenbeck et al. [96]. Novartis
observed low percentage recovery during the analytical
method development and validation of the combination
bilayer product Starlix. The purpose of bilayer was to separate
the drug (metformin) from croscarmellose sodium. When
these two are present in solid state, the charge interaction is
not expected due to low moisture content in the tablet
(< 2%). However, in solution form, the positively charged
metformin and negatively charged croscarmellose sodium
underwent a charge interaction, which led to a 4 -- 8% loss
in metformin recovery in the tablets. In solution form, this
recovery was very low (between 55 -- 73%) confirming the
interaction. This issue was resolved by incorporating arginine
9
 S. S. Bharate & R. A. Vishwakarma
into the formulations, which is a competitor for binding sites
on croscarmellose sodium. Arginine has stronger interaction
with croscarmellose sodium than metformin, thus, enabling
a complete recovery of metformin in tablet formulation [97].
For a detailed account on interactions and incompatibilities
of APIs with pharmaceutical excipients, a specific review [88]
on this topic should be referred to.
11. Biopharmaceutical aspects -- absorption,
distribution, metabolism and excretion
A total of 90% of compounds reaching an IND filing fail to
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be a NDA [98]. The key factor pursued in many lead discovery
and optimization programs is high in vitro potency/activity
(as low as in the nanomolar range). At preclinical stage, an
optimization of physicochemical and biopharmaceutical
properties such as solubility, selectivity, cellular activity and
ADMET properties may reduce the failure rate in clinical
trials [99-106]. Compounds that are identified as poorly soluble
and/or which show poor bioavailability as well as cross-
species metabolism necessitates additional formulation efforts
at the developmental stage [106-111]. The most cited example is
lopinavir (29), which is a potent antiretroviral drug. However,
it has high plasma protein binding and poor bioavailability.
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Formulation in combination with ritonavir (a fixed dose com-
bination, Kaletra), a CYP3A inhibitor, was then developed
to overcome bioavailability issues [112,113].
Another antiretroviral drug, saquinavir (30) belongs to the
class of protease inhibitors. Two formulations have been
marketed; a hard-gel capsule formulation of mesylate salt
(Invirase) was approved in 1995, wherein ritonavir in com-
bination with saquinavir was administered in order to increase
saquinavir bioavailability. This hard gelatin capsule formula-
tion was poorly absorbed and led to viral resistance in many
patients. For this reason, in order to improve bioavailability,
another formulation (Fortovase) was approved in 1997 -- an
orally administered microemulsion in the form of a soft-
gel capsule formulation of saquinavir. In May 2005, Roche
announced market withdrawal of Fortovase owing to reduction
in demand, possibly because Invirase boosted with ritonavir
was more effective compared with Fortovase [114]. Bioavailabil-
ity of other protease inhibitors such as atazanavir (31) [115]
(Reyataz marketed by Bristol-Myers Squibb was approved
in 2003); darunavir (32) [116] (Prezista formerly known as
TMC114 was approved in 2006) and etravirine (33) [117]
(Intelence was approved in 2008, marketed by Tibotec, a sub-
sidiary of Johnson & Johnson) were found to be increased
when given in combination with lopinavir/ritonavir. Chemical
structures of drugs 29 -- 33 are shown in Figure 3.
12. Preformulation and IND approval
Drug research and development is a unique multidisciplinary
process to identify novel therapeutic agents. The process of
taking NCE from concept to clinic and eventually to
10 Expert Opin. Drug Deliv. [Early Online]
commercialization involves several steps that traditionally
occur in a series [118]. The average cost of taking a candidate
from the discovery stage to commercialization is estimated
to exceed US$800 million, with an average development
time span of ~ 15 years, and a success rate of only 1 in
5,000 discovery candidates reaching the market [119]. HTS
and combinatorial chemistry technologies have been devel-
oped in the past two decades to speed up initial drug discovery
stages such as synthesis of a large number of diversely oriented
compounds and the ability to screen them quickly in a shorter
time. In contrast, these technologies have produced large
number of ‘hits’ with poor physicochemical and ADMET
properties which are responsible for high attrition rate at fur-
ther stages of drug discovery. Several complementary in silico
and in vitro strategies have also emerged to screen these com-
pounds and assess their potential to become lead candidates.
Some of the advantages of HTS may be combined with
analyses of structure--activity relationships to direct rapid,
sometimes automated, iterative chemistry. Prediction of
potency at target, potency at likely toxicity sites, potency in
cellular assays and prediction of ADMET at early stages of
drug discovery research is now gaining importance [120,121].
Compounds emerging as ‘hits’ from these screening processes
are characterized further and tested in vivo for safety and
efficacy [107]. A number of drugs have been removed from
the market due to safety/efficacy concerns and economic
factors [122]. Elements that are essential to take a new lead
candidate through the preclinical development process are
numerous. These include synthesizing sufficient compound
for safety assessment, formulating and characterizing drug
product, determining the drug’s bioavailability and metabolic
profile, and conducting safety studies to determine the drug’s
toxicity. Better understanding of the chemical and pharma-
ceutical properties of newly synthesized molecules, leading
to better pharmacokinetic parameters, certainly reduces the
failure rate of preclinical and early clinical molecules [123,124].
After pharmacological and toxicological screening, the best
compound(s) from the biopharmaceutical point of view could
be selected based on physicochemical parameters such as sol-
ubility, dissolution rate, lipophilicity, hygroscopicity, pKa,
polymorphism, stability and particle characteristics at earlier
stages with the highest possible accuracy. These properties
continue to be referenced throughout API scale-up process
chemistry and good manufacturing practices [125,126]. There-
fore, at an early stage, physicochemical studies have a signifi-
cant part to play in anticipating formulation problems and
in identifying a logical path in dosage form technology.
Such studies may also result in reduction of development
costs [127,128]. Successful implementation of this process results
in generation of sufficient data to file an IND application [129].
12.1 Preformulation requisites
Drug research is a very complicated endeavor which includes
several diverse disciplines united with a common goal of
developing novel therapeutic agents. Due to growing

HN
N O
N HN NH
O O
O OHO O O
29 Lopinavir
O
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S N
H O HN N
O
O OH
O O
H N
32 Darunavir
Figure 3. Chemical structures of drugs 29 -- 33 discussed in ADME-related case studies.
complexity of drug substances, development of a quality drug
product and a reproducible manufacturing process becomes
more challenging and costly. The method should yield consis-
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tent high-quality drug products that should not be affected by
variation within the drug substance, excipients and raw mate-
rials. In the early stage of product development, a fundamen-
tal understanding of the physicochemical properties of the
drug substance potentially influences performance and manu-
facturability of the drug product. The study of physicochem-
ical properties of a drug substance is usually conducted during
preformulation research. In general, preformulation work can
start as early as during biological screening, in which diverse
compounds are screened for desired activity. However, an
extensive physicochemical characterization of a drug sub-
stance will not usually start until one or more potential candi-
dates are identified and taken into clinical testing during the
investigational stages. The information and knowledge gained
from preformulation studies also help in accelerating develop-
ment of new therapeutic entities. For example, selection of
compounds that have physical properties favorable for oral
absorption and certain types of process operations can facili-
tate rapid progress of these compounds at later stages of
drug product development. Generally, preformulation studies
start with an exploration of solubility, permeability, stability,
hygroscopicity, particle size, polymorphism and so on. Some
of these characteristics are interrelated and their analysis may
need to be considered in combination or simultaneously [130].
Drugs penetrate across the epithelium via transcellular or
paracellular pathways. Lipophilic drugs prefer the transcellular
route and hydrophilic drugs penetrate primarily through the
paracellular pathway, which involves passive or altered diffu-
sion through intercellular spaces [131]. In active transport,
membrane proteins act as carrier molecules to transport drugs
across cell membranes [132]. Factors that affect poor solubility
Expert Opin. Drug Deliv. [Early Online]
Impact of preformulation on drug development
O
NH2 N
O HO HN
O OH
NH HN N
N H3CO NH NH
NH
O HN OCH3
O
30 Saquinavir 31 Atazanavir
H2N Br
NH2
N O
N
NH
33 Etravirine
and thereby poor bioavailability are high crystallinity/
high melting point and hydrophobicity or high log P value.
The n-octanol/water partition coefficient is the ratio of con-
centration of compound in n-octanol to that in water under
equilibrium. Log P is a parameter that determines lipophilic-
ity of a molecule and has wide application in prediction of
biological activities and ADMET [133]. In general, if log
P = 0, there is equal distribution of the drug in both phases.
Log P > 0 is indicative that the drug is lipid soluble and
log P < 0 means the drug is water soluble. Therefore, the
higher the log P value, the higher the affinity for lipid mem-
branes. Values of log P that are too high (> 5) or too low
(< 3) may be associated with poor transport characteris-
tics [134,135]. Physicochemical and pharmacokinetic properties
of a drug will decide rationality of product development or
suitable drug delivery systems.
For topical and transdermal delivery, physiochemical
factors such as solubility, crystallinity, molecular weight
(< 600 Da), melting point (< 200 C) and Log P (between
-1 and 4) must be considered. The higher value of Log P
points to a potentially higher ease of penetration of the com-
pound into cells of living organisms [136-138]. There is not an
absolute standard for solubility or dissolution for drugs given
orally. The acceptable level of solubility will differ from com-
pound to compound based on dose and permeability. If the
dose is low and permeability is good, then even poorly soluble
compounds will be well absorbed. However, high-dose drugs
that are poorly soluble are much more likely to have limited
solubility. These compounds are better candidates for solubil-
ity enhancement and prodrug strategies. Typically, partition
coefficients in the range of 100 -- 1,000 (log P = 2 to 3) are
required for efficient passive transcellular transport. Extremely
lipophilic compounds (log P > 3.5) are associated with
decreased absorption [139]. Compounds that have a low
11
 S. S. Bharate & R. A. Vishwakarma
aqueous solubility (< 1 mg/mL) may already possess inherent
sustained-release potential as a result of their low solubil-
ity [140]. For ophthalmic drug delivery, favorable parameters
of drug are: log P value of 2 to 3 and molecular weight
is < 500 Da [141]. In the case of a nasal delivery system, nasal
absorption drops-off sharply for lipophilic drugs with a
molecular weight > 1,000 Da [142]. Drugs in a nonionized
state are absorbed due to a high apparent partition coefficient
(more lipophilic). Commonly intranasal drugs are adminis-
tered as molecularly dispersed forms such as solutions. The
allowable volume of solution for intranasal administration
is relatively low and, therefore, drugs with low aqueous
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solubility and/or requiring high doses may present a problem.
The profile of an ideal drug candidate for nasal delivery
is molecular weight < 500 Da, log P < 5 and aqueous
solubility < 50 mg/mL [143]. Drug solubility and dissolution
rate are important, especially if it is administered as a solid
dosage form (e.g., a powder). Powder morphology and parti-
cle size influences amount and pattern of drug deposition
inside the nasal cavity [144-146]. Based on physicochemical
drug properties, a suitable drug delivery approach can be
selected and/or drug properties can be modified in terms of
solubility, log P value, crystallinity, hygroscopicity and so on
to achieve required criteria for a targeted delivery system.
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12.2 Regulatory requirements
A preformulation report of a new molecule constitutes deter-
mination of chemical, manufacturing and control (CMC)
properties, analytical profiling, thermodynamic and physico-
chemical properties, pharmaceutical and mechanical proper-
ties, solid-state characteristics, biopharmaceutical properties,
excipient compatibility studies and stability. These drug
substance properties should be emphasized in the CMC/
pharmaceutical development section of IND. This provides
early insights to both industry and regulators regarding the
potential significance of physicochemical properties on drug
product quality and manufacturing processes [147]. It is now
accepted that properties of a given drug substance are to be
thoroughly investigated early during the phases of develop-
ment and results of these findings must be included in the
CMC section of IND. The CMC section consists of informa-
tion pertaining to chemical composition, manufacturing
methods, stability and controls used for manufacturing the
drug substance and drug product. One should know chemical
stability as well as activity of the product. This information is
assessed to ensure that the company can adequately produce
and supply consistent and active batches of the drug. Efforts
are currently under way within the International Conference
on Harmonisation to develop a common technical document
that will harmonize CMC regulatory requirements for global
development and marketing. Preparation of CMC sections
according to European Union and United States guidelines
results in two formally different NDAs. Components of CMC
sections of the EU Marketing Authorisation Applications and
US NDA are very much the same [64,148,149].
12 Expert Opin. Drug Deliv. [Early Online]
13. Conclusion
In summary, the article has provided critical account on the
significance of preformulation studies in the drug develop-
ment process. To facilitate clinical development and to reduce
attrition rate, a thorough study of physicochemical properties
of drug candidates is desired. This also serves as the foundation
for developing robust formulations.
14. Expert opinion
Collaboration of medicinal chemists and formulation scien-
tists in drug discovery research is essential to identify a drug
candidate that has the best chance of clinical and eventually
commercial success. The partnership between medicinal
chemists and formulation scientists in drug discovery produ-
ces NCE with structural modifications in order to improve
solubility, stability, permeability and oral bioavailability.
The role of formulation scientists has been recognized for pre-
paring formulations for preclinical in vivo studies and for
identification of final solid-state forms of drugs. Apart from
this, the team work between pharmaceutics and medicinal
chemistry departments at earlier stages of drug discovery
increases the speed of preclinical evaluation and reduces attri-
tion during the development process. The performance of
solid dosage forms is reliant on the physicochemical proper-
ties of the active ingredient and excipients. Before going for
Phase I clinical studies, excipients used for the formulation
development process should be identified. The purpose is to
ultimately prevent unnecessary and very costly changes
including time and overall development cost in subsequent
steps. The goal is to identify critical parameters important in
the development of a stable, effective and safe drug delivery sys-
tem or dosage form. Table 1 depicts examples of drug products
wherein preformulation data showed impact on the drug’s
development or its recall from the market. Examples of prod-
uct recall are primarily because of inadequate preformulation
data where different/more preformulation data might have
avoided market loss.
Most companies perform salt screening in order to have
a molecule with appropriate physicochemical properties
such as crystallinity, low hygroscopicity, dissolution rate, sol-
ubility, bioavailability and stability. Improper salt form selec-
tion may lead to instability in the final product as seen for
REV-5901 (3) where hydrochloride/sulfate salt was found to
be unstable. The most commonly used pH modifiers for
acidic drugs are sodium, calcium, potassium and so on. For
basic drugs, they are hydrochloride, mesylate, sulfate, citrate
and hydrobromide. REV-5901 is a base with a pKa value of
3.7 and the pH solubility profile of REV-5901 showed that
the pHmax of the compound was 1 indicating that the salt
form would exist only at pH < 1. Therefore, only two salt
forms (hydrochloride and sulfate salts) could be prepared.
A salt with weaker acids such as phosphoric, acetic, lactic
and tartaric acid was not feasible. Another interesting point

Table 1. Preformulation case studies.
Preformulation parameter
Salt selection and stability
Polymorphic phase transitions
Crystal habit and druggability
Expert Opin. Drug Deliv. [Early Online]
Chirality
Bulk properties (particle
size and size distribution,
flow, dissolution, etc.)
Hygroscopicity
Drug stability
Drug solubility
Physical incompatibilities
Biopharmaceutical aspects-ADME
13
Expert Opin. Drug Deliv. Downloaded from informahealthcare.com by Universita Studi di Torino on 04/03/13
For personal use only.
Drug product Preformulation observations Refs.
Chlortetracycline (1) Calcium salt was very stable compared to hydrochloride salt [21]
Penicillin G (2) Potassium salt was less hygroscopic, but had unpleasant metallic taste [20]
REV-5901 (3) Hydrochloride salt unstable [22]
LY333531 (4) Low aqueous solubility of free base of API; mesylate salt form was optimal [23]
Diclofenac (5) Solubility of sodium salt better than other prepared salt forms [24]
Dibenzo (b,f)oxepen-10-ylmethyl-methyl-prop-2-ynyl-amine Maleate salt was more stable than the free base [26]
hydrogen maleate (6)
L-649923 (7) Free base was very unstable, but the calcium salt was more [28]
pharmaceutically promising than the sodium, ethylenediamine
and benzathine salts
Nelfinavir mesylate (8) Withdrawn from market due to elevated levels of ethyl methanesulfonate [31]
(cancer-causing contaminant)
Theophylline (9) Phase transition of stable anhydrous theophylline to [38]
theophylline monohydrate due to wet granulation, which resulted
in decrease in dissolution rate
Thiamine hydrochloride (10) Conversion of stable thiamine hydrochloride to NSH [36]
due to wet granulation
Erythromycin (11) Conversion to erythromycin dihydrate due to milling and drying [37]
Ritonavir (12) Reduced solubility in the marketed formulation due [151]
to existence of new polymorph
Chloramphenicol palmitate (13) Discovery of presence of biologically less active crystal form [42]
Rotigotine (14) Presence of less stable new polymorphic crystals [54]
Oxytetracycline hydrochloride (16) Discovery of less soluble new polymorph [60]
Tolbutamide (17) Plate-like crystals cause tableting problems [62]
Hydrochlorothiazide (18) and irbesartan (19) Existence of less soluble crystal of irbesartan due to longer granulation time [63]
Rapamune (20) Converts to three isomers in aqueous solution with reduced bioavailability [67]
RPR 200765 (21) Mesylate salt demonstrated good handling properties among the [71]
four salts (camphorsulfonate, hydrochloride and hydrobromide)
NBI 75043 (22) Besylate salt was more pharmaceutically acceptable than tartrate salt [73]
Levothyroxine sodium (23) Unstable in the presence of light, temperature, air and humidity [77]
Fluocinonide (24) Degradation/impurities leading to sub-potency [78]
Combretastatin A4 (25) Limited aqueous solubility and chemical instability -- phosphate [84]
ester was inactive
Flavopiridol (26) Precipitation in the parenteral formulation due to poor solubility [86]
PN Admixture contained precipitate of calcium phosphate, which caused [90]
diffuse microvascular pulmonary emboli.
Warfarin sodium (27) Reduced potency of warfarin sodium due to use of [95]
poor quality isopropanol
Metformin (28) Very low recovery due to interaction between positively charged drug and [96]
negatively charged excipient (croscarmellose sodium)
Lopinavir (29) To overcome bioavailability issue given with ritonavir (a CYP3A inhibitor) [113]
Saquinavir (30) Increase in bioavailability when combined with ritonavir [114]
Impact of preformulation on drug development
 S. S. Bharate & R. A. Vishwakarma
was if the microenvironmental pH rises above 1, the hydro-
chloride/sulfate salt gets converted to free base. Therefore,
REV-5091 was preferred as a free base over the salt forms.
Trace impurities found during salt formation plays a signifi-
cant role in the stability of formulations and may affect
potency of API. The starting material used for salt form prep-
aration may also give rise to formation of impurities as a
by-product as seen in the case of nelfinavir mesylate (8).
Another parameter to be studied during the preformulation
stage is polymorphism and phase transitions. These should be
investigated during initial stages of development. In situ phase
transitions during product development (manufacturing
Expert Opin. Drug Deliv. Downloaded from informahealthcare.com by Universita Studi di Torino on 04/03/13
processes and processing conditions) may affect/alter solubil-
ity of drug and thus the bioavailability. The manufacturing
processes such as wet granulation [as in case with theophylline
(9), thiamine HCl (10)], milling and drying [as observed with
erythromycin (11)] may have pronounced effect on dissolu-
tion rate and stability of drugs. Results of these studies must
be incorporated in the CMC section of NDA. This informa-
tion is required to demonstrate control over manufacturing
processes. Existence of different crystal forms should be stud-
ied because it may impart either harmful or helpful character-
istics to particular formulations in terms of bioavailability.
The most cited example is ritonavir (12) and oxytetracycline
For personal use only.
HCl (16) wherein new polymorphic forms have less solubility
and thus reduced bioavailability. Discovery of a new crystal
form of chloramphenicol palmitate (13) was biologically less
active thus making formulation less effective. In the case of
rotigotine (14), the new polymorph discovered was less stable.
For this reason, polymorphic study is crucial during the pre-
formulation stage and will decide commercial success of the
final product. The crystal structure of a compound will decide
its physicochemical properties such as crystal habit, melting
point, solubility, rate of desolvation, color, mechanical prop-
erties and chemical stability. Plate-like crystals of tolbutamide
(17) were not suitable for tableting and, therefore, not manu-
facturable. Thus, understanding of these properties during
product development will be beneficial.
In the pharmaceutical scenario, particle size and size distri-
bution affects the intrinsic dissolution rate, which is one of the
decisive parameters of drugs after administration. Shape char-
acterization is vital here as different crystal faces dissolve at dif-
ferent rates. At the preformulation stage, one should identify
particle shape and size distribution in order to produce formula-
tions with desired efficacy and with economical production tech-
niques. Good flow properties are preconditions for successful
manufacturing of dosage forms (tablets and hard gelatin capsu-
les). Appropriate fluidity of materials is required for transporta-
tion of powder fill/granules through the hopper of the tableting
machine. Elongated particle shape and small particle size may
cause high tablet weight variations, strength, unacceptable blend
uniformity and difficulty in filling containers and dies. Powder
flow is affected by numerous parameters including purity, crystal-
linity, electrostatic forces, mechanical properties (brittleness,
elasticity), density, size, shape, surface area, moisture content,
14 Expert Opin. Drug Deliv. [Early Online]
direction and rate of shear, storage container dimension and par-
ticle--wall interaction. In the case of RPR 200765 (21), mesylate
salt was found to have better handling properties over sulfonate,
hydrochloride and hydrobromide salts.
Drug stability is an essential component of preformulation
testing and preformulation scientists collaboratively work
with analytical departments to develop stability-indicating
assays. During early stages, a perfect stability-indicating assay
may not be available because initial drug lots may not be pure
and purity is improved as the molecule progresses to subse-
quent development stages. Therefore, the intention of prefor-
mulation scientists is to broadly define conditions under
which the drug would be stable. In the case of levothyroxine
sodium (23) it was not stable throughout its shelf life and after
marketing it was found that the reason behind less potency
was a stability issue with levothyroxine sodium. It was sensi-
tive to light, temperature, air and humidity, thus, loss of
potency was the reason behind product recall.
Determination of solubility during preformulation testing
is a decisive parameter during formulation development. Drug
solubility is one of the physicochemical parameters that receive
a lot of attention during preformulation testing. The BCS para-
digm can be used to develop strategies for enhancing drug solubil-
ity and/or permeability. Generally, the absorption rate constant of
drugs varies by over only ~ 50-fold range (0.001 -- 0.5 mg/min)
whereas drug solubility can vary over six orders of magnitude
(0.1 µg/mL -- 100 mg/mL) [150]. Hence, the formulator has supe-
rior flexibility in altering the drug’s solubility over its permeabil-
ity. Optimization of drug solubility may be more fruitful if
permeability is overcome by increasing drug solubility, to provide
high drug concentration at the absorption site. However, this may
be difficult if the dose is very high. Alternative formulation
approaches could be considered if the drug is poorly soluble. In
the case of flavopiridol (26), researchers formulated flavopiridol
with HP-b-CD and citric acid to keep the drug in a solubilized
form so that it would not precipitate upon injection.
The role of the preformulation team at every stage of the
drug discovery and development process is to select appropriate
methods to guide or alert other development teams about drug
solubility and permeability issues. There have been consequences
observed of formation of new impurities, incomplete mass bal-
ance, destruction of dosage form, unnecessary multiplicity of pro-
totype formulations and changes in physicochemical properties
(stability, solubility, dissolution profile, degree of crystallinity
and hygroscopicity) due to inadequate data on the drug--excipient
interaction. The reason behind this might be incomplete/
insufficient drug--excipient interaction studies at the preformula-
tion stage, which can be one reason for inability to obtain success-
ful drug registration. The bilayer tablet of metformin (28) was
recalled due to a very low recovery of API. This has been attrib-
uted to the use of a negatively charged excipient (i.e., croscarmel-
lose sodium), which had an interaction with metformin.
Throughout the process of drug discovery, a carefully
planned preformulation study provides crucial data and
information regarding drug substance and knowledge of

manufacturing technology before going for formulation
development activities. Negligence toward assessment of any
of the preformulation parameters may lead to a significant
impact on the total development activities.
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Affiliation
Sonali S Bharate1 & Ram A Vishwakarma†2
†
Author for correspondence
1
Indian Institute of Integrative Medicine (CSIR),
Canal Road, Jammu-180001, India
2
Director,
Indian Institute of Integrative Medicine (CSIR),
Canal Road, Jammu-180001, India
E-mail: ram@iiim.ac.in
19