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12
ROLE OF HPLC IN
PREFORMULA
TION
Irina Kazakevich
12.1 INTRODUCTION
Preformulation is a bridge between discovery and development where devel-
opment scientists participate in selection and optimization of lead compounds.
It is very critical at this stage to evaluate the developability of potential drug
candidates in order to select new chemical entities and decrease the number
of failures during future drug development.
On average, only one out of ten new chemical entities (NCE) entering first-
in-human testing reaches registration, approval, and marketing stage. The
reasons for failures of development compounds include problems with bio-
pharmaceutical properties, clinical safety, toxicology, efficacy, cost of goods,
and marketing (see Figure 12-1) [1, 2]. The biopharmaceutical properties such
as gastrointestinal and plasma solubility, lipophilicity (LogD), permeability,
first-pass metabolism, systemic metabolism, protein binding, and in vivo
bioavailability are related to the solubility, chemical stability, and permeabil-
ity of drug candidates and have to be considered at discovery lead selection
before recommendation to the development stage.
A major challenge in any drug discovery program is achieving reasonable
bioavailability upon oral administration; therefore, any information that high-
lights potential problems with cell permeability and absorption is valuable
when reviewing structural families as leads for drug discovery. Lipinski et al.
[3] have reviewed 2245 compounds selected from the United States Adopted
577
HPLC for Pharmaceutical Scientists, Edited by Yuri Kazakevich and Rosario LoBrutto
Copyright © 2007 by John Wiley & Sons, Inc.
Name (USAN), International Nonproprietary Name (INN), and World Drug
Index (WDI),
comparing calculated physical properties and clinical exposure.
Four parameters were chosen that were associated with solubility and per-
meability, namely, molecular weight, octanol/water partition coefficient, the
number of hydrogen bond donors, and the number of hydrogen bond
acceptors. It was concluded that compounds are most likely to have poor
absorption when molecular weight is >500, the calculated LogP is >5, the
number of hydrogen bond donors is >5, and the number of hydrogen bond
acceptors is >10. Lipinski has referred to this analysis as “rule of five” because
the cutoffs for each of the four parameters were all close to five or a multiple
of five. The rule of five can serve as qualitative absorption/permeability
predictor.
The absorption of drug molecules in the gastrointestinal tract is dependent
upon the pK
a
of the compound and the pH of the gastrointestinal region
(Figure 12-2). Almost 63% of all drugs are ionized in aqueous solution and
can exist in a neutral or a charged state, depending on the pH of the local
environment [4].
Based on the major goal of preformulation—identification of possible
failure in future development—numerous studies are performed to fully char-
acterize prospective drug candidates. The major analytical technique in each
preformulation group is liquid chromatography. Ninety percent of all ana-
lytical equipment in preformulation groups are HPLC systems equipped with
UV and MS detection systems. HPLC is a fast and reliable method for con-
centration and identity determination by UV and/or MS detection, respec-
tively.The type of HPLC methods differ based on the specific preformulation
tests that will be described below.
In the early stage of preformulation, characterization of the drug molecule
involves ionization constants and partition coefficient determinations, aqueous
and nonaqueous kinetic and equilibrium solubility determination, pH solubil-
ity profile, chemical stability assessment, and salt and polymorph screening.
Assessment of biopharmaceutics and toxicological screening are also essential
578 ROLE OF HPLC IN PREFORMULATION
Figure 12-1. Reasons for attrition from 1991 to 2000. (Reprinted with permission from
reference 1.)
at this stage. At the later stage of preformulation, after recommendation of
NCE to development,
the development support from preformulation group
involves a more detailed solid-state characterization program, elaborating on
moisture sorption, compressibility, melting point, particle size, shape, and
surface area assessments, as well as excipient compatibility and prototype for-
mulation stability evaluation.
Further information on the role of preformulation in drug development
process can be found in several excellent monographs [6–8] with the focus on
pharmaceutical aspects of process development.
12.2 INITIAL PHYSICOCHEMICAL CHARACTERIZATION
(DISCOVERY SUPPORT)
During the early discovery stage the medicinal chemists use in vitro activities
and fast in vivo small animal studies to discover the best compound to develop.
The support from development scientist consists of providing information
about LogP, pK
a
, and LogD for ionizable drugs and aqueous solubility. These
physical characteristics can affect the absorption of drug candidate and, there-
fore, drug bioaivalability.The requirements for HPLC analysis at this stage are
speed and efficiency of the separation. It is critical to mention that at the early
stage of discovery, very little information is available about the properties of
INITIAL PHYSICOCHEMICAL CHARACTERIZATION (DISCOVERY SUPPORT) 579
Figure 12-2. Physical properties of the gastrointestinal tract. (Reprinted with permis-
sion from reference 5.)
molecule and only a few milligrams of compound is available for characteri-
zation.
Therefore, it is important to choose the most efficient column and the
simplest mobile phase. Also, recommended is the use of more contemporary
HPLC systems as UPLC from Waters employing columns with dimensions of
50 × 2.1mm, 1.8-µm particle size and the Fast 1200 system from Agilent with
column dimensions of 50 × 4.6mm, 1.8-µm particle size, respectively, to
enhance the turnaround time for sample analysis. Other platforms would
include using Chromolith Speedrod
®
monolithic columns at high flow rates.
Also, taking into consideration the short column length, gradient elution
should be recommended for all HPLC methods at this stage of drug candidate
characterization. The post-run equilibration time is not significant in the case
where short columns are used, and dwell volume is improved significantly for
a new generation of HPLC systems.
Many types of modeling techniques are available in the discovery phase of
drug development, from structure activity relationships (SAR) to physiology
based pharmacokinetics (PBPK) and pharmacokinetics-/pharmacodynamics
(PK/PD) to help choosing some of the lead compounds. Some tests that are
carried out by discovery include techniques related to structure determina-
tion, metabolism, and permeability: NMR, MS/MS, elemental analysis,
PAMPA, CACO-2, and in vitro metabolic stability. Although they are impor-
tant as a part of physicochemical molecular characterization under the bio-
pharmaceutics umbrella, they will not be discussed here. The reader can find
relevant information in numerous monographs [9, 10].
12.2.1 Ionization Constant, pK
a
Most potential drug candidates are weak bases or acids. Solubility and many
other properties of the drug molecule is dependent on its ionization state.
Acids are usually considered to be proton donors and bases are proton accep-
tors. Any drug molecule with basic functionality in aqueous media holds the
following equilibrium:
(12-1)
where the ionization equilibrium constant could be expressed as
(12-2)
It is obvious from the above equilibrium that the ratio of ionic to nonionic
form of the drug in the solution is controlled by the proton concentration,
which is commonly represented by pH values (negative logarithm of proton
concentration). Taking the negative logarithm of expression (12-2), the well-
known Henderson–Hasselbalch equation could be obtained:
K
a
=
[]
⋅
[]
[]
+
BH
BH
+
BH B H
+
↔+
+
580 ROLE OF HPLC IN PREFORMULATION
(12-3)
T
his allows for the estimation of the prevailing drug form at a particular pH.
Ionic form of any organic molecule is usually more soluble in aqueous media,
while the neutral form is usually more hydrophobic and thus shows an
increased affinity for lipids.
Variation of the ionization state of the molecule at different pH has typical
sigmoidal shape (as shown in Figure 12-3). Corresponding expression for this
dependence could be derived from equation (12-2) and the mass balance of
the ionic and nonionic form of the drug:
(12-4)
If one assumes quantity q equal to 100, then concentration of B or BH
+
forms
will numerically be equal to the percentage of corresponding form in the solu-
tion and solving equation (12-3) with expression (12-4) one will get the expres-
sion for BH
+
concentration expressed as a percent of ionized form
(12-5)
The inflection point of this curve corresponds to the point where pH = pK
a
,
and it is a common way for the determination of the drug pK
a
values.
Several different techniques are usually employed for pK
a
determination.
They were described in detail by Comer [11].
BH
ppH
ppH
+
−
()
−
()
[]
=
⋅
+
100 10
110
K
K
a
a
q =
[]
+
[]
+
BBH
ppH
BH
B
K
a
=+
[]
[]
+
log
INITIAL PHYSICOCHEMICAL CHARACTERIZATION (DISCOVERY SUPPORT) 581
Figure 12-3. Dependence of the relative amount (in the form of a percent) of proto-
nated form on the pH of aqueous media.
In practice the most common technique to determine pK
a
value is by
employing potentiometric titration based on the detection of the variations of
either the conductivity or current at fixed applied potential at various pH
values
.The automated potentiometric titration system well known as a GLpK
a
or PCA200 from Sirius Analytical [12] is considered to be a good approach
for pK
a
determination with water-soluble drugs at pH 2–8 for the new drug
candidates when the amount of drug substance is limited. For poorly water-
soluble compounds it is advised to use GlpK
a
with D-Pass or Sirius Profiler
SGA as a pH/UV method for determination of compounds that have inher-
ently lower concentration in the solution media.
HPLC is another convenient method for measurement of the NCE pK
a
values. As was shown by Melander and Horvath [13], the retention of any
ionizable analyte closely resembles the curve shown in Figure 12-3. Chro-
matographic determination of the pK
a
could be accurately performed with
very limited amount of sample. Fast HPLC method with optimum analyte
retention is suitable for this purpose, but the influence of the organic mobile-
phase modifier on the mobile phase pH and analyte pK
a
should be accounted
for in order to provide the accurate calculation of the respective pK
a
value.
Detailed discussion of the HPLC-based methods for the pK
a
determination is
given in Chapter 4.
In the case of sufficient drug supply the old-fashioned solubility method can
be used for pK
a
determination based on the different equilibrium solubility
at different pH values. This method is very precise, but time- and drug-
consuming, and is described in detail in reference 6.
Drug substance often contains several ionizable groups that may signifi-
cantly complicate experimental measurement of the pK
a
. All different types
of pK
a
determination methods are essentially based on the measurement of
the titration curve. If the pK
a
values of several ionizable groups in the mole-
cule are within 2 pH units from each other, experimental measurement
become very tedious. Recent advancements in the molecular computational
methods and developments of physicochemical databases for a large number
of known compounds allow computer-based prediction of the pK
a
values on
the basis of known physicochemical correlations and fast computer screening
of known values for related or structurally similar compounds from the data-
base. Detailed discussion of these programs is given in Chapter 10.
12.2.2 Partition and Distribution Coefficients
One of the most important physicochemical parameters associated with oral
absorption, central nervous system (CNS) penetration, and other pharmaco-
kinetic parameters is lipophilicity of organic compounds, which determines
distribution of a molecule between the aqueous and the lipid environments.
The lipophilicity in the form of LogP was included in Lipinski’s rule of five as
one of the major characteristics of drug-like organic molecules. It was stated
that LogP should be not more than five for drug candidates to have a good
582 ROLE OF HPLC IN PREFORMULATION
oral absorption property. In Table 12-1, some LogP values for various types of
dosage forms are given.
T
he partition coefficient itself is a constant and is defined as the ratio of
concentration of compound in aqueous phase to the concentration in an
immiscible solvent, as the neutral molecule. In practical terms the neutral mol-
ecule exists for bases >2 pH units above pK
a
and for acids >2 pH units below
pK
a
. In practice, log P will vary according to the conditions under which it is
measured and the choice of partitioning solvent. LogP is the logarithm of dis-
tribution coefficient at a pH where analyte is in its neutral state. This is not a
constant and will vary according to the protogenic nature of the molecule.
The choice of partition solvent has been a subject of debate. Different type
of solvents have been used for the determination of partitioning coefficient
[14], but the majority of the data are generated using water–n-octanol parti-
tioning. Octanol was chosen as a simple model of a phospholipid membrane.
However, it has shown serious shortcomings in predicting blood–brain barrier
or skin penetration. Other solvents such as chloroform, cyclohexane, and
propylene glycol dipelargonate (PGDP) have been used for modeling biolog-
ical membranes.
Octanol is a hydrogen-bonding solvent, and thus it shows certain specificity
in its ability to dissolve some components. For example, K
0
w
for phenol in
hexane is only 0.11 while in octanol it is equal to 29.5. There were several
attempts to rationalize solvent effects using solubility parameters [15], dielec-
tric constant [16], and others,but none appear to be consistent.n-Octanol gives
the most consistent results with other physicochemical properties and drug
absorption in gastrointestinal tract.
The classical measurement of LogP is the shake flask method [17].A known
amount of drug is dissolved in a flask containing both octanol phase and
aqueous buffer at controlled pH to ensure the existence of only nonionic form
(at least two units from the drug pK
a
). The flask is shaken to equilibrate the
sample between two phases. There must be no undissolved substance present
in both phases. After the system reaches its equilibrium, which is time- and
temperature-dependent, the concentration of drug is analyzed by HPLC in
both phases. Partitioning coefficient is calculated as
(12-6)
K
c
c
w
w
0
0
=
INITIAL PHYSICOCHEMICAL CHARACTERIZATION (DISCOVERY SUPPORT) 583
TABLE 12-1. Preferable Dosing Form for Different
LogP Regions
LogP Dosing Form
Low <0 Injectable
Medium 0–3 Oral
High 3–4 Transdermal
Very high 4–7 Toxic buildup in fatty acids
This method allows for the accurate determination of K
0
w
only within the −
1000 to +1000 region or approximately within six orders of magnitude span.
T
hese experiments could be complicated by solubility and equilibration kinet-
ics and the properties of a substance. For example, if a studied compound
has a property of nonionic surfactant, it will be mainly accumulated at the
water–organic interface, and shaking of this two-phase system will create a
stable emulsion difficult for analytical sampling. The ultracentrifugation at
speed of 14,000rpm for 15–20min can be enough in most cases to separate
two phases. Actual equilibration of the system is tested by several measure-
ments of the equilibrium concentration at different time intervals.
Because of the wide range of partitioning coefficient values, in most cases
the decimal logarithm of K
0
w
is used, and it is denoted as LogP:
(12-7)
The biggest challenge for the use of HPLC in the LogP measurement is the
determination of the drug concentration in the octanol phase. If the octanol
solution is being injected onto the reversed-phase column, it can modify the
stationary phase, shift the analyte retention, and lead to an incorrect mea-
surement due to the retention shift. To avoid this problem the dilution in the
corresponding mobile phase is recommended. Also, when LogP is more than
four, the concentration of drug in water phase is very small, causing a detec-
tion problem with UV detection. This becomes even more troublesome if the
compound of interest has a weak UV chromophore. The use of MS detection
and proper ionization mode is recommended to increase the sensitivity.
Direct HPLC experiment can be used for estimation of LogP, but this tech-
nique is valid only for neutral molecules or for ionized molecules analyzed in
their neutral state [18]. The following is a brief description of this method.
Compounds with known LogP is injected onto C18 hydrophobic column,
and the respective retention factors are used to create a calibration curve. The
estimation of LogP for unknown compounds can be made on the basis of this
calibration curve. This method is straightforward, but requires the previous
knowledge of pK
a
values for ionizable compounds to avoid the possible ion-
ization that will lead to incorrect determination of values of LogP. Recently,
an automated isocratic liquid chromatography system, dedicated to the
measurement of LogP,Profiler LDA, was introduced into the market by Sirius-
Analytical, Ltd. There were numerous attempts to use the retention time of
compound in correlation with its distribution properties in RP HPLC [19, 20].
The retention factor was used to calculate a distribution coefficient between
stationary phase and mobile phase. In case of Sirius Profiler LDA automated
system, a set of molecules with known LogP values was used to calibrate the
system and convert the chromatographic retention time into octanol/water
partition coefficients. The system could cover the LogP range from −1 to 5.5
by choosing between three different methods and different column lengths
LogP =
()
log K
w
0
584 ROLE OF HPLC IN PREFORMULATION
ranging from 1 to 25cm, but was recently removed from the market.The well-
known automated pH titrator from Sirius
, GlpK
a
, can be used as well to deter-
mine the octanol/water partition coefficient. The measurement is based on a
two-phase acid/base titration in a mixture of water/octanol [21].
Partition coefficient discussed above represents oil/water equilibrium dis-
tribution of only neutral forms of a substance. The distribution at different pH
is described by LogD, which is the logarithm of the ratio of the concentrations
of all forms of analyte in oil and water phases at particular pH. Logarithm of
distribution coefficient at pH 7.4 is often used to estimate the lipophilicity of
a drug at the pH of blood plasma.
As follows from the definition, the distribution coefficient is dependent on
the pH. It is usually assumed that in the oil-phase drug molecule could exist
in only nonionic form; thus the distribution coefficient, D
0
w
, for basic drug B
could be written as
(12-8)
If LogP and pK
a
for a studied drug is known, then it is possible to express D
0
w
as a function of pH of aqueous phase through these values using equations
(12-3) and (12-6)–(12-8). Resulting expression is
(12-9)
Figure 12-4 represents the comparison of the pH dependencies of ionic form
of a basic drug with LogD.
Log D pH LogP Log
ppH
w
K
a
0
110
()
()
=−+
[]
−
D
B
w
0
=
[]
[]
+
[]
oil
water
+
water
BBH
INITIAL PHYSICOCHEMICAL CHARACTERIZATION (DISCOVERY SUPPORT) 585
LogD
Figure 12-4. Normalized dependence of the protonated form of the base (solid) and
its LogD dependence on the aqueous pH (dashed).
At high pH, the neutral form of a drug (basic compound) has a distribu-
tion coefficient equal to its partitioning coefficient.
With the decrease of the
pH of the aqueous phase, the degree of drug ionization increases, thus increas-
ing its total concentration in the aqueous phase. As the pH decreases, the
ionic equilibrium is shifted toward the protonated form of a drug, which con-
tinually increases its concentration in the aqueous phase and decreases its
content in oil phase. There is no plateau region in the LogD curve at low pH
for basic compounds (Figure 12-4). On the other hand, for acidic compounds,
there is a plateau region in the LogD curve at low pH (pHs below the pK
a
);
and then as the pH increases, the more ionic equilibrium is shifted toward the
ionized form of the acid, which continually increases its concentration in the
aqueous phase and decreases its content in the oil phase. This results in
the absence of plateau in the LogD curve at high pH (pH > pK
a
) for acidic
compounds.
These are only the theoretical dependencies; real behavior of actual mole-
cule usually is significantly altered due to different types of intermolecular
interactions. Molecular solvation, association, hydrogen bonding, and counte-
rions all have a significant effect on drug ionization constant and partitioning
and distribution coefficients. Detailed and comprehensive discussion of these
effects could be found in the book by Avdeef [22].
12.2.3 Solubility and Solubilization
Aqueous solubility is one of the most important physicochemical properties
of a new drug candidate because it affects both drug absorption and dosage
form development. Only a drug in solution can be absorbed by the gastroin-
testinal track. The rate of dissolution and the intestinal permeability of the
drug molecules are dependent on the aqueous solubility—that is, the higher
the solubility, the faster the rate of dissolution. An excellent monograph
describing the theory of solubility and solubility behavior of organic com-
pounds was written by Grant and Higuchi [23]. For additional information on
solubility, the reader can be referred to references 24–27.
Solubility is expressed as the concentration of a substance in a saturated
solution at a defined temperature. The US Pharmacopeia (USP) gives the
solubility definitions shown in Table 12-2.
Solubility measurements are generally carried out in the early stages of
drug development because it affects drug bioavailability evaluation; in many
cases, solubility-limited absorption has been reported. Only a compound that
is in solution is available to cross the gastrointestinal membrane. The solubil-
ity measurements in aqueous buffered systems at different pHs are used to
mimic gastrointestinal human or animal fluids. Solubility determination in
DMSO is very important at the early stages of lead candidate selection
because of the increasing use of 10mM DMSO solution as a stock solution for
biological testing for very slightly soluble lead candidates [29]. In general,
586 ROLE OF HPLC IN PREFORMULATION
[...]... identification of specific class for the drug candidate is critical for future development of dosage forms Different platforms are used for solubility measurements: UV; HPLC with UV detection; or HPLC with MS detection UV spectrophotometry is the simplest and fastest method, unfortunately with limited applicability In most cases the drug substance available for the study in the preformulation stage is not... SELECTION For ionic drugs the salt form can be considered as an alternative to increase the solubility Drug substance usually is more soluble in aqueous media in its ionic form Low solubility of the neutral form of the drug substance suggests the necessity to formulate it in the form of salt The reader is referred to reference 46 for more information about the properties, selection, and use of salt forms for. .. low solubility compared to original crystal form [51].The product was recalled from market and was reformulated It was a rare example of a dramatic effect of the existence of multiple crystal forms of a commercial pharmaceutical and showed the importance of polymorphic screening for all type of pharmaceutical dosage forms When the existence of polymorphism for new chemical entity is identified, the property... and fast HPLC to determine the assay concentration for each polymorph at the different temperatures are the same as for solubility determination However, for stability evaluation of the different polymorphs a stability-indicating HPLC method should be used 12.6 PREFORMULATION LATE STAGE (DEVELOPMENT SUPPORT) After a new chemical entity has been selected to move forward to development, the preformulation... source of information to formulation and analytical scientists regarding the properties of the recommended drug molecules The development preformulation support provides the additional testing of prototype formulation and excipient compatibility samples as well as guidance for salt form selection and polymorphs screening The major role of this unit is to bridge discovery and development stages HPLC coupled... mechanism To achieve this goal, the best approach is to perform forced degradation studies at the preformulation stage of drug development with most viable candidates, which may include the free base or acid and several corresponding salt forms The FDA and ICH guidance provides very little information about strategies and principles for conducting forced degradation studies, including problems with poorly... salt counterions are shown in Table 12-6 Salt form selection is mainly covered by solid-state charactezation methods, and HPLC is only used to determine the solubility and solid/solution stability of different salt forms The requirements for HPLC method development is the same as for solubility/stability determination described previously, and the same HPLC method may be applied 12.5 POLYMORPHISM Polymorphism... described model to perform drug–excipient compatibility testing prior to Phase I to eliminate potential future issues related to drug instability in final formulation Because the drug–excipient compatibility testing is conducted at an early drug development stage when a fully validated HPLC method is not available, the same GLP HPLC method as for forced degradation studies can be used for this test as well... thermodynamically unstable forms during preformulation stage of drug development is important Typical methods used for solid-state characterization of polymorphism are DSC, POLYMORPHISM 595 FT/IR, microscopy, and X-ray powder diffraction [49, 50] HPLC is used to evaluate chemical stability of different polymorphic forms as well as for solubility determination, and this parameter is very critical for drug development,... 5 A Avdeef, Absorption and Drug Development, Wiley Interscience, New York, 2003 6 J I Wells, Pharmaceutical Preformulation, Ellis Horwood Chichester, UK, 1988 7 J T Carstensen, Pharmaceutical Preformulation, Technomic Publishing Company, Lancaster, PA, 1998 8 M Gibson (ed.), Pharmaceutical Preformulation and Formulation, Interpharm/ CRC Press, Boca Raton, Florida, 2004, pp 585 9 H van de Waterbeemd, . specific class for the drug candidate is
critical for future development of dosage forms.
Different platforms are used for solubility measurements: UV; HPLC with
UV. H
+
↔+
+
580 ROLE OF HPLC IN PREFORMULATION
(12-3)
T
his allows for the estimation of the prevailing drug form at a particular pH.
Ionic form of any organic
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