Advances in Dissolution Tests in Pharmaceutical Analysis

Dissolution tests are one of the most important quality control tests in
pharmaceutical analysis. A direct relationship has been demonstrated between
in vitro dissolution rate of many drugs and their bioavailability and
is generally known as in vitro – in vivo correlation, IVIVC. For disintegrating
solid oral dosage forms, disintegration plays a vital role in the dissolution
process, but there is not always an automatic correlation between disintegration
and dissolution, especially for drugs with very low dissolution rates for which
dissolution may be the rate limiting step in the absorption process.

A variety of designs of apparatus for dissolution testing have been proposed
and tested.Different apparatus, procedures and techniques are required for
different dosage forms because of significant differences in formulation design
and the physicochemical properties of the drugs. Dissolution tests have therefore
been developed for various drug delivery systems including immediate release
solid dosage forms, several controlled release solid dosage forms and many novel
and special dosage forms. Most of the tests with recommended apparatus and other
specifications are now available as compendial standards in Pharmacopoeias and
are used in pharmaceutical analysis and drug development for the various drug
delivery systems. The dissolution test methods are also now designed to mimic
the general conditions encountered in the physiological environment of the GIT
and thus hold promise for the establishment of in vitro-in vivo correlation,
IVIVC, for many more pharmaceutical products. However, some dosage forms
still require more method development and refinement before standardized dissolution
test methods can be recommended.

Introduction

Dissolution is the process by which a solid solute enters a solution. In the
pharmaceutical industry, it may be defined as the amount of drug substance that
goes into solution per unit time under standardized conditions of liquid/solid
interface, temperature and solvent composition. Dissolution is considered one
of the most important quality control tests performed on pharmaceutical dosage
forms and is now developing into a tool for predicting bioavailability, and
in some cases, replacing clinical studies to determine bioequivalence. Dissolution
behaviour of drugs has a significant effect on their pharmacological activity.
In fact, a direct relationship between in vitro dissolution rate of many
drugs and their bioavailability has been demonstrated and is generally
referred to as in vitro-in vivo correlation, IVIVC¹.

In spite of IVIVC, dissolution is not really a predictor of therapeutic efficiency.
Rather, it is a qualitative and quantitative tool which can provide valuable
information about biological availability of a drug as well as batch-to-batch
consistency of products. Dissolution tests are therefore used to confirm compliance
with compendial specifications and are needed as part of a product license application.
Additionally, they are used during product development and stability testing
as part of the specifications for the product. However, no universal dissolution
test has been designed that gives the same in vitro dissolution and in
vivo bioavailability for different formulations and batches. Thus, different
compendial specifications have been developed for different formulations and
dosage forms.

{mospagebreak title=History of dissolution testing}

History of dissolution testing

The first reference to dissolution testing was made in a published paper by Noyes
and Whitney in 1897² and was referred to as the rate of solution of
solid substances in their own solution. The authors suggested that dissolution
rate was controlled by a layer of saturated solution that forms instantly around
a solid particle. A few years later in 1900, Brunner and Tolloczko proved that
dissolution rate depended on chemical and physical structures of the solid, the
surface area exposed to the medium, agitation speed, medium temperature and the
overall design of the dissolution apparatus. In 1904, Nernst and Brunner modified
the Noyes-Whitney equation by applying Fick’s law of diffusion to establish a
relationship between the dissolution rate and the diffusion coefficient. The
Food, Drug and Cosmetics Act made the first legislation that mandated drug manufacturers
to test their products for safety in 1938. This initiative was passed in the aftermath
of the elixir of sulfanilamide tragedy which killed 107 people, mostly infants
in 1938. The law forbade the sale of any drug unless the Food, Drug and Administration
(FDA) found it to be safe. In the 1950s, the emphasis moved from studying the
effects of physicochemical properties of drug on dissolution to correlation of
dissolution to bioavailability of dosage forms. The Swiss Pharmacopoeia Helvetica
was the first regulatory body to introduce a disintegration test for tablets in
1934. The disintegration test became an official United States Pharmacopoeia (USP)
method in 1950³.

The rotating bottle dissolution
method for extended release formulation was developed in 1958. In 1960, the
USP recognized a need for a standardized dissolution test and began experimenting
with a variety of basket and stirring devices. Levy and Hayes⁴ utilizing
a beaker blade stirrer at 30-60rpm, found significant differences in the in
vitro dissolution rates of different brands of aspirin tablets and linked
them to the incidence of gastric irritation caused by various brands due to
their slow dissolution rates. In 1970, USP 18 incorporated the first official
dissolution test for solid dosage forms using a rotating basket. In 1975, the
USP began the development of calibrators for dissolution testing and in 1978,
proposed three calibrator tablets – prednisone (disintegrating), salicylic acid
(non-disintegrating) and nitrofurantoin (disintegrating), but no predefined
calibration frequency was made. The paddle over disk and the rotating cylinder
were developed in 1990 while the reciprocating cylinder and the flow-through
cell were developed in 1995.

Disintegration/dissolution process

Solid dosage forms may or may not disintegrate when they interact with gastrointestinal
fluid following oral administration depending on their design (Figure 1). For
disintegrating solid oral dosage forms, disintegration usually plays a vital
role in the dissolution process since it determines to a large extent the area
of contact between the solid and liquid. However it is well known that considerable
dissolution of the drug can take place before complete disintegration of the
dosage form, a phenomenon which depends largely on the mechanism of disintegration
and certain physicochemical properties of the drug, such as its solubility.
This could be important when considering the motility of the drug or dosage
form, and the release of the drug at specific sites, in the gastrointestinal
tract. Thus, correlations have been established between disintegration times
and dissolution rates for various pharmaceutical tablets^5-8. It should
be noted, however, that there is not always an automatic correlation between
disintegration and dissolution, especially for drugs with very low dissolution
rates.

For many drugs, particularly those that are poorly soluble in the gastric fluid,
the rate-limiting step in the absorption process is the dissolution rate and
a dissolution rate determination can therefore be a useful guide to comparative
bioavailability⁹.

Figure 1: Schematic diagram of the dissolution process

Thus in vitro dissolution
test should be considered where a drug displays poor aqueous solubility, low
intrinsic dissolution rate (e.g. when the particles are extremely hydrophobic),
where particle size has been shown or suspected to influence bioavailability,
where polymorphism is a possible problem, or if the tablet has a special coating
which must dissolve in vivo to release the drug. If the drug is poorly
absorbed or when there is significant first-pass metabolism, dissolution tests
can confirm or deny the importance of release of active ingredients from the
dosage form in the absorption process. Where there has been a deliberate attempt
to control rates of dissolution and release in specialized formulations, dissolution
tests should form an intrinsic part of the development process. However, even
if a drug has a high aqueous solubility, its formulations should be subjected
to a dissolution test at some stage during development to check the absence
of interactions which might adversely affect release.

The rate of solution of a solute
from a non disintegrating solid, in the absence of a chemical reaction between
solute and solvent, is given by the Noyes-Whitney equation²:

d_w/d_t = K (C_s – C)
(1)

where w is the weight of
drug in solution, C is the concentration of drug in solution at time
t and C_s is the saturation solubility of the solute
(drug) at equilibrium. K is given by

K = DA /h
(2)

where D is the diffusion
coefficient of the solute, A is the surface area of the dissolving solid
and h the diffusion layer thickness. Under sink conditions, where C
< 0.1C_s, equation (1) reduces to

d_w/d_t
= KC_s (3)

 

 Dissolution testing Equipment

A variety of designs of apparatus
for dissolution testing have been proposed and tested, varying from simple beaker
with stirrer to complex systems with lipid phases and lipid barrier where an
attempt is made to mimic the biological milieu. The choice of the apparatus
to be used depends largely on the physicochemical properties of the dosage form.
All parts of the apparatus that may come in contact with the preparation being
examined or with the dissolution medium should be chemically inert and should
not adsorb, react with or interfere with the preparation being examined. The
metal parts should be made from stainless steel or coated with a suitable material
to ensure that such product do not react or interfere with the preparation being
examined and the dissolution medium. An apparatus that permits observation of
the preparation being examined and the stirrer during the test is preferable.

In general, compendial apparatus
and methods used in drug development for the various drug delivery systems
are available in the United State Pharmacopoeia¹⁰ and variants of
these apparatus are available as well, in some other Pharmacopoeias including
the British Pharmacopoeia (BP) ¹¹ and the European Pharmacopoeia
(PhEur)¹².

Dissolution tests for various dosage forms

Dissolution testing was initially
developed for immediate release solid dosage forms and then extended to controlled/modified
release solid oral dosage forms and has recently widened to a variety of “novel”
or “special” dosage forms such as suspensions, orally disintegrating tablets,
dissolve-in-the-mouth dosage forms, chewable tablets, chewing gums, powders,
granules, solid solution and solid dispersion, transdermal patches, semisolid
topical preparations, suppositories, liquid-filled capsules, implant and microparticulate
formulations. For orally administered immediate release solid drug products,
the test is referred to as a “dissolution” test, since the drug is intended
to dissolve rapidly in the test medium. For non-oral dosage forms such as topical
and transdermal delivery systems and suppositories, the test is referred to
as a “drug release” or as an “in vitro release” test procedure. Because
of significant differences in formulation design among these novel/special dosage
forms, which afford very different physicochemical and release characteristics,
it is not possible to devise a single system that could study the drug release
properties for all products. Rather, different apparatus, procedures and techniques
are employed in each case. The method may be specific to the dosage form category,
the formulation type or the particular product.

Dosage forms for which a specific
method can be recommended include the following:

Oral suspensions (for systemic use):

In general, the rotating paddle
method using an adequate dissolution medium is the recommended method for dissolution.
To obtain representative samples, product preparation should follow standardized
procedures based on shaking or mixing. Method parameters such as sample introduction
and agitation rate should be established on the basis of the viscosity and composition
of the suspension matrix. The sample introduction technique must be accurate,
precise and reproducible. Even though oral suspensions of any viscosity would
be exposed to similar ranges of shearing forces after administration in vivo,
the in vitro agitation rate should be selected to facilitate discrimination
between batches with different release properties.

For low viscosity suspensions,
an accurate dose can be delivered to the bottom of the dissolution vessel using
a volumetric pipette. A slow agitation of 25rpm is generally recommended for
less viscous suspensions¹⁰. For high viscosity samples, the dose
may be determined by weight with a quantitative sample transfer to the dissolution
vessel to ensure accuracy of the sample size introduced. High-viscosity suspensions
may also require a faster agitation rate such as 50 or 75rpm to prevent sample
mounding at the bottom of the vessel. Ideally, sample weight/volume should reflect
a typical dose of the product. However, testing a partial dose – for instance
≥10% to 20% of the usual product dose - is recommended rather than using
a surfactant to obtain sink conditions.

Orally disintegrating tablets:

Orally disintegrating tablets (ODTs)
create an in situ suspension by rapidly disintegrating, typically within
1 minute or less. Administration of ODTs may not inherently result in a faster
therapeutic onset but can circumvent problems such as difficulty in swallowing
traditional oral dosage forms like tablets and capsules. Taste masking (drug
coating) is very often an essential feature of ODTs and thus can be the rate-determining
mechanism for dissolution/release.

In vitro dissolution testing
should follow the principles of solid oral dosage forms (tablets) or suspension.
The rotating paddle would be the method of first choice, with an agitation rate
of 50 rpm. Higher agitation rates may be necessary in the case of sample mounding.
A potential difficulty for in vitro dissolution testing may arise from
sample floating. For ODTs that dissolve very quickly, a disintegration test
may be used in lieu of a dissolution test if it is shown to be a good discriminating
method. If taste masking (using polymer coating) is a key aspect of the dosage
form, a multipoint profile in a neutral medium with early points of analysis
(e.g. ≤5minutes) may be recommended¹³.

Dissolve-in-the-mouth dosage forms :

Over the last few years, there
has been a great increase in interest for dissolve-in-the-mouth dosage forms.
This interest is spurred by the improved patient compliance compared to tablets
and capsules that have to be swallowed whole. Unfortunately, such dosage forms
bring about new problems – taste and characterization. The characterization
of dissolution in the mouth is not easily done by current methodologies, such
as variants of the USP dissolution tests. The reason for this is the speed of
disintegration/dissolution compared to typical dosage forms designed to be swallowed
whole. It appears that there is a dearth of in vitro systems to evaluate
the buccal dissolution of such dosage forms.

Recently, Hughes and Gehris¹⁴
described a novel dissolution testing system that is capable of characterizing
buccal dissolution. This system was demonstrated to correlate with human taste
perception. The method was found to be rapid, repeatable and can be applied
to all common liquids and dissolve-in-the-mouth dosage forms. There are a number
of factors that are unique to characterizing buccal dissolution that do not
apply to gastrointestinal dissolution. They include small volume, short residence
time, solids transfer, composition and incomplete dissolution. Most of the USP
dissolution tests use large volumes of solution defined as ‘sink conditions’.
For buccal dissolution, the volume of saliva is very small compared to that
of the stomach and the residence time in the mouth is also very short, the bulk
of the dosage form being swallowed within a minute (with the exception of lozenges).
In addition, complete dissolution is not usually required or desirable. For
example, for fast-melt tablets, what is required is actually complete disintegration
not dissolution. This means that the removal of finely divided solids from the
test vessel is absolutely critical to get any type of correlation to bioavailability.
The system developed by Hughes and Gehris¹⁴ has been able to address
all these factors. The system is illustrated in Figure 2.

Figure 2: Schematic diagram of Buccal Dissolution Apparatus

It comprises a single, stirred, continuous flow-through filtration cell that includes a dip tube designed to remove finely divided solid particles. Filtered solution is removed continuously and used to analyze for dissolved drug. The volume of liquid in the cell is approximately 10ml and fluid is pumped through it at about 6ml per minute. This gives a residence time in the cell of approximately 100sec for 63% of the dosage form and gives almost complete removal in about 8minutes. Approximately two third of the flow exits via the dip-tube and the other third exits through the filter for analysis. In use, the cell is filled and flows as set up first and allowed to reach steady state before the dosage form (solid, liquid, suspension or powder) is introduced. The filtered sample is either analyzed in-line e.g. by UV flow-through cell, or samples are collected in a fraction collector for later analysis. In order for this test to give meaningful results it is necessary to use a dissolution fluid that simulates saliva. The composition of simulate saliva used in the study is given in Table 1.

Table 1: Composition of simulated saliva

KH2PO4	12 mM
NaCl	40 mM
CaCl2	1.5 mM
NaOH	to pH 6.2

In essence, the method characterizes
the amount of a drug that dissolves during passage through the mouth. The data
from this method allows the prediction of the intensity of the taste of a dosage
form relative to another dosage form or of a performance target. The method
is particularly suited for evaluating taste masking. It is rapid taking only
about 20minutes per test and it is repeatable. The method could be used as a
quality control test to ensure dosage uniformity and as a development tool to
optimize formulation before human testing, thus reducing the amount of human
testing needed.

A feature of the concentration-time curve for the dissolution of dissolve-in-the-mouth
dosage forms is the characteristic peak in the curve, which has been found to
correlate with taste¹⁴. The time to reach the peak is generally not
as relevant to taste as the peak itself. For these dosage forms, poor dissolution
is not a “bad” result. However, the amount that dissolves will directly affect
the taste of the products. Figure 3 shows the result of the dissolution of three
different types of Drug X formulations and a control.

Figure 3: The concentration-time curve for Drug X formulations.

Formulation A- 5mg drug X – taste masked

Formulation B – 5mg Drug X – taste
masked

Formulation C – 0.1mg Drug X –
not taste masked

The taste of the drug was directly
proportional to the peak concentration of the drug. The results also showed
excellent correlation with the evaluation of the taste panel employed (Table
2).

Table 2: Taste Panel evaluation
of Drug X formulations

Formulation	Taste-masked	Taste panel
A	Yes	Not acceptable, worst than B
B	Yes	Not acceptable, better than A
C	No	Maximum acceptability

Chewable tablets

In principle, the procedure employed for chewable tablets should be the same
as that used for regular tablets. This concept is based on the possibility that
a patient might swallow the dosage form without proper chewing, in which case
the drug would still need to be released to ensure the desired pharmacological
action ¹⁵. Where applicable, test conditions would preferably be
the same as used for conventional tablets of the same active pharmaceutical
ingredient, but because of the non-disintegrating nature of the dosage form,
it may be necessary to alter the test conditions (e.g. increase the agitation
rate) and specifications (eg. increase the test duration). The reciprocating
cylinder (USP apparatus 3) with the addition of glass beads may provide more
“intensive” agitation for in vitro dissolution testing of chewable tablets.
As another option, mechanical breaking of chewable tablets prior to exposing
the specimen to dissolution testing could be considered. While this option would
more closely reflect the administration of the product and the corresponding
formulation and manufacturing features, no approach validating such method has
apparently been reported in the literature¹³.

Chewing gums

In case of chewing gums, the intensity and frequency of shearing forces/activities
(i.e. chewing action) can have a large influence on the release rate. The European
Pharmacopoeia¹² provides a description of a stainless steel 3-piston-apparatus
that is required for testing of “Medicated chewing gums” (PhEur 2.9.25). The
test is typically operated at 37^oC and at 60 cycles/minute. Test
media with a pH of 6 are commonly used, since this corresponds to reported saliva
pH. During development, it is recommended to keep the “chewing residue” for
later analysis/assay. However, to date there has been insufficient international
experience with this apparatus to draw a firm conclusion about its suitability¹².
However, more method development and refinement will be required before a final
recommendation of a standardized drug release method can be made.

Transdermal patches

Although several apparatus and
procedures have been used to study in vitro release characteristics of
transdermal patches, it is desirable to avoid unnecessary proliferation of dissolution
test equipment. Current compendial apparatus include the paddle over disk/disk
assembly (USP apparatus 5/PhEur 2.9.4.1), the rotating cylinder (USP apparatus
6/PhEur 2.9.4.3), the reciprocating disk (USP apparatus 7), and a paddle over
extraction cell method (PhEur 2.9.4.2).

The paddle over disk procedure
with a watch glass-patch-screen sandwich assembly is considered to be the method
of choice, as it has been shown experimentally that this procedure results in
almost the same release profile as other, more complicated apparatus for all
US-marketed transdermal patches¹⁶. An example of such apparatus is
the Franz diffusion cell assembly (Figure 4).

The pH of the medium ideally should
be adjusted to pH 5 to 6 and the test temperature typically set at 32^oC,
reflecting physiological skin conditions. The European Pharmacopoeia considers
100rpm a typical agitation rate and also allows for testing an aliquot patch
section.

Figure 4: Franz Diffusion Cell

Semisolid topical dosage forms

Semisolid topical dosage forms
include creams, ointments and gels. In vitro release from semisolid topical
dosage forms has been extensively investigated using the Franz cell diffusion
system¹⁶ with a synthetic membrane and to some extent using the enhancer
cell¹⁷. Depending on the solubility of the drug substance, the receptor
medium may contain alcohol and/or surfactant. Deaeration is critical to avoid
bubble formation at the interface with the membrane. Depending on the characteristics
of the drug product, it may be possible to conduct the in vitro test
without a synthetic membrane¹⁸. As with transdermal products, the
test temperature is typically 32^oC to reflect skin temperature except
for products for specific sites of action. For example, vaginal creams are tested
at 37^oC.

No compendial apparatus, procedures,
or requirements for in vitro release testing of semisolid topical dosage
forms have been described in relevant pharmacopoeias to date. Because of the
value and importance of release rate, it is highly desirable to determine the
release data of semisolid dosage forms¹⁹. There is therefore a need
to develop compendial test methods.

Suppositories

In principle, for hydrophilic suppositories that release the drug by dissolving
in the rectal fluids, the basket, paddle, or flow through cell, can all be used.
Lipophilic suppositories release the drug after melting in the rectal cavity
and are significantly affected by the rectal temperature, reported as typically
36 to 37.5^oC^20,21. After melting, the drug will have to
partition between the lipophilic base and the receptor fluid. This may lead
to distribution equilibrium between the two phases rather than complete dissolution.
For this reason, sink conditions during the test are essential in order to simulate
the in vivo conditions, where absorption across the rectal membrane is
continuously reducing the concentration of the drug in the rectal fluids. For
lipophilic suppositories, a modified basket method, a paddle method with a wired
screen and a sinker, and modified flow-through cell with specific dual chamber
suppository cell (PhEur 2.9.3-6), has all been recommended. To achieve the specified
temperature in the test cell, the temperature in the water bath may have to
be set up to 5^oC higher. Experience with the compendial flow-through
cell has shown that it may generate highly variable data because of the behaviour
of the suppository in the cell especially with formulations containing spreading
agent²¹. No single test method will be suitable for all suppository
formulations. However, when starting the development of an in vitro dissolution/release
test, it might be advantageous to begin with the basket or paddle in the case
of hydrophilic, and with modified flow-through cell in the case of lipophilic
suppositories.

Liquid-filled capsules

Liquid-filled capsules can consist
of either hydrophilic or lipophilic formulations. In the case of lipophilic
formulations, they may or may not include a surfactant for self emulsifying
purposes. The USP recommends a dissolution test procedure using the rotating
paddle method (Apparatus 2) with minimum amount of surfactant, if needed (e.g.
dissolution of valproic acid or methoxasalen capsules). If the liquid-filled
capsule contains a water-soluble base, then the addition of surfactant is generally
not needed. However, this is a function of the solubility of the active ingredient
as well as the formulation itself. The rotating paddle can have disadvantages
for some liquid-filled capsule formulations, as it may be difficult to keep
the formulation immersed. Also, emulsified formulations might separate at the
liquid-vessel-air interface, and/or formulations could adhere to the paddle
or beaker walls¹³.

The modified dual chamber flow-through
cell as recommended for lipophilic suppositories (PhEur 2.9.3-6) is considered
an appropriate apparatus for liquid-filled capsules. One potential disadvantage
is that screens might be blocked during the test. Other apparatus have also
been successfully used, such as the rotating basket (which keeps the formulation
immersed but might also result in blocked meshes) and the reciprocating cylinder
(which offers good mechanical agitation but a limited media volume).

A range of test media should also
be used to characterize and understand the formulation characteristics especially
during the development phase. In the case of lipid-filled capsules, enzymes
in addition to surfactants may be necessary to simulate digestion in vivo.
The advantage of using lipases is that they more closely reflect physiological
conditions. The disadvantages are that the method can be expensive and labour
intensive when used as a routine test and it typically leads to higher variability¹³.

 Powders, granules, solid solutions and solid dispersions

The flow-through apparatus offers
specific sample cells for studying drug release from powder and granular dosage
forms. However, it is important to note that the dissolution behaviour of these
dosage forms may be greatly influenced by their wettability, surface area and
particle size distribution. For powders, especially when exhibiting poor wettability,
it may be necessary to add a surfactant to the dissolution medium to obtain
reproducible dissolution results. Care should be taken to use a level of surfactant
that does not increase the solubility of the drug to the extent where the test
is no longer discriminatory. In certain cases, a physical mixture of the powder
with glass beads and/or other substances that encouraged wetting may be used.

Solid solutions and dispersions
may be presented in oral dosage forms such as capsules and tablets. If this
is the case, their in vitro release characteristics can be determined
using the same methods typically used to characterize the release from solid
oral dosage forms. Solid solutions and dispersions often lead to super-saturation
of the medium. Therefore, for these types of formulations, dissolution tests
under non-sink conditions can be a predictive tool during formulation development
as well as for batch to batch quality control.

However, more method development
and refinement will also be required before a final recommendation of (a) standardized
drug release method(s) can be made¹³.

Parenterals: Implants and microparticulate formulations

The compendial and the modified
flow-through cell have been used successfully for implants and microparticulate
formulations. The compendial flow-through method is modified with regard to
the inner diameter to suit the special properties for testing parenterals i.e
a low volume of the fluid is used in the acceptor compartment and the flow rate
is set very slow. Use of High Pressure Liquid Chromatography (HPLC) pumps may
be considered to provide the necessary accuracy and precision at very low flow
rates. Static or rotating bottles have also been used for in vitro testing.

As tests are often run over a long
time period (e.g. several weeks to months), measures have to be taken to compensate
against evaporation. Suitable preservatives (e.g. paraben, benzalkonium chloride)
may be added to prevent microbial contamination. The selection of the preservative
has to be based on criteria such as compatibility with the active ingredient
as well as other formulation ingredients and the pH of the test medium.

The composition of the medium should
take into consideration the osmolality, pH and buffer capacity of the fluid
at the site of administration, which are usually assumed to resemble those of
the plasma (or muscle) but with lower buffer capacity. However, the main challenges
with this type of dosage form are to determine the appropriate duration of the
test and the times at which the samples are drawn in order to characterize the
release profile adequately²³.

Modified release dosage forms

FDA guidelines recommend USP dissolution
apparatus 1, 2, 3 or 4 for modified release dosage forms. However, sometimes
current dissolution equipment may require modifications or completely new designs
to accommodate the release mechanism. For example, non-disintegrating dosage
forms requiring a delivery orifice for drug release may dictate a special design
or modification of the dissolution apparatus so that the orifice is not blocked.
In contrast, disintegrating or eroding delivery systems pose the challenge of
transferring the dosage form to different media without losing any of the pieces.
In general, methods of agitation, changing the media, and holding the dosage
form in the media without obstructing the release mechanism are relevant to
drug release and require careful planning. Simulating the in vivo release
of the delivery system on a computer often helps in designing an appropriate
dissolution test. It is important, however, to take into consideration certain
physical, chemical and physiological parameters such as variability in gastric
emptying and GI transit time and changes in the environment. Knowing the solubility
and permeability characteristics of the compound, in combination with release
rate from the delivery system, helps to predict whether drug release is occurring
under sink or non-sink conditions.

A challenging component of a dissolution
test for a modified release delivery system is changing the media to obtain
a pH gradient or simulate fed and fasted conditions. The ability to easily change
the media is the focus of commercially available dissolution equipment targetted
for modified-release delivery systems and several equipment designs are available.
The USP Apparatus 3, reciprocating cylinder, dips a transparent cylinder containing
the dosage form at a rate determined by the operator ^22,24. The tubes
have a mesh base to allow the media to drain into a sampling reservoir as the
tube moves up and down thus creating convective forces for dissolution. The
cylinders can also be transferred to different media at specified times, automatically.
A second design is the rotating bottle apparatus, which also allows for changing
of media to simulate a pH gradient or fed and fasted conditions. The USP Apparatus
4 i.e the flow-through cell containing the dosage form is fed with dissolution
media from a reservoir. Directing the fluid through a porous glass plate or
a bed of beads produces a dispersed flow of media. Turbulent or laminar flow
can be achieved by changing the bottom barrier. As with USP Apparatus 3, the
media can be changed to provide a pH gradient, surfactants, etc. For controlled
release systems, the ability of the dosage form to remain intact in the physiological
conditions of the stomach and small intestine is generally assessed by conducting
drug release studies in 0.1M HCl for 2 hours (mean gastric emptying time) and
in phosphate buffer (pH 7.4) for the rest of the experiment^25,26.

For colon specific drug delivery
systems, the in vitro test conditions should closely mimic in vivo
conditions with regards to pH, bacteria, types of enzyme, enzymatic activity,
volume, stirring, plus all the components found in vivo from food ingestion
and other conditions. The ability of the tablets to remain intact in the physiological
conditions of the stomach and small intestine is generally assessed by conducting
drug release studies in 0.1M HCl for 2 hours (mean gastric emptying time) and
in phosphate buffer (pH 7.4) for 3 hours (mean small intestinal transit time)
using USP dissolution rates test apparatus or flow-through dissolution apparatus.
The ability of the delivery system to release the drug in the colon is tested
in vitro by incubating it in a buffer medium in the presence of enzymes
(e.g pectinase, dextranase) or rat, guinea pig or rabbit ceacal content^27-30.
The amount of drug release at different time intervals during the incubation
is estimated to find out the degradation of the carrier under study. Another
in vitro method involves incubation of the drug delivery system in a
fermenter with commonly found colonic bacteria, such as Streptococcus feacium³¹
and Bacillus ovatus³² in a suitable medium under anaerobic
conditions in which the amount of drug released at different intervals is determined.
Unlike purified enzymes preparation, the procedure is considered to be a more
complex and natural method of evaluating stability and degradation of different
colon-specific drug delivery systems³³.

Herbal Medicinal Products (HMPs)

In contrast to chemically defined
medicinal products, the biopharmaceutical quality and behaviour of Herbal Medicinal
Products (HMPs) are often not well documented³⁴. In most cases, an
in vitro/in vivo biopharmaceutical characterization is complicated by
the complex composition of herbal drug preparations, the extensive metabolism
of their constituents and the resulting analytical difficulties. The active
substance of HMPs is defined to be the whole herbal drug preparation (e.g. the
extract) in its entirety. In case of extracts, the European Pharmacopoeia defines
the various types as

A.    
Standardized extracts containing constituents (single or in groups) that are
solely responsible for the acknowledged and documented therapeutic activity.
Standardization (adjustment) to a defined content is acceptable (e.g. standardized
Senna leaf dry extract) using inert excipients or preparations of higher or
lower content.

B1. Quantified extracts containing chemically defined constituents (single
or groups) possessing relevant pharmacological properties (active markers).
These substances are likely to contribute to the clinical efficacy. However,
evidence that they are solely responsible for the clinical efficacy is not yet
available (e.g. extracts of Gingko, St. John’s Wort).The characterization of
these extracts should take into consideration, as far as possible, the particular
state of knowledge concerning the documented efficacy, quality and safety of
an extract. Standardization by blending different lots of an herbal drug before
extraction or by mixing different lots of herbal drug preparation is appropriate
and acceptable. Adjustment/standardization using excipients is not acceptable.

B2. Other extracts containing no constituents documented as being determinant
or relevant for efficacy, or as having pharmacological or clinical relevance.
In these cases, chemically defined constituents (markers) without known therapeutic
activity may be used for control purposes (e.g. Valerian or Stinging nettle
root extract). These markers may be used to monitor good manufacturing practice
or as an indicator for the assay of the drug product.

In contrast to synthetic substances,
the composition of an herbal drug preparation is determined by the manufacturing
process (product by process) and the quality of the herbal drug. For HMPs corresponding
to Type A, the in vitro release of the constituents with known therapeutic
activity should be compared with the reference product. Pharmaceutical equivalence
may be accepted if in both products the in vitro release of the constituents
of known therapeutic activity exceeds 90%. In the case of a low in vitro
release or if the constituents have a low solubility, bioequivalence studies
may be necessary.

For HMPs corresponding to Type
B1, the in vitro release of “active marker” and solubility of the extract
should be compared with reference product. Pharmaceutical equivalence may be
accepted if both parameters in both products exceed 90%. In the case of a low
in vitro release or low solubility, additional clinical safety studies
or bioequivalence studies may be necessary.

For HMPs corresponding to Type
B2, the solubility should be compared with the reference product. Pharmaceutical
equivalence may be accepted if solubility of both products exceeds 90%. If the
solubility of the extract cannot be tested, the release of (an) appropriate
marker (s) should be compared. The appropriateness of the marker for the biopharmaceutical
characterization must be justified. If no appropriate marker can be found, a
comparison of disintegration may be acceptable.

Physiological conditions that can affect drug release

Dissolution tests are now designed
to mimic the general conditions encountered in the physiological environment
of the GIT. The dissolution of drugs from orally administered solid dosage forms
in vivo and in vitro is influenced by variations in the natural
or simulated gastrointestinal fluid (e.g. pH, surfactants) and physical variables
such as hydrodynamic flow^35,36, and mechanical stress^23,37.
The physiological conditions that can affect drug release include the following:

Intestinal transit time, gastric
emptying and variable pH

The effect of gastric emptying
on drug release from a modified release delivery system generally occurs when
the dosage form is non-disintegrating and can be due to the variability of retention
times in the stomach usually between the fed and fasted states, but can occur
within each condition as well. For example, if the patient is in the fasted
state, gastric emptying generally occurs within two hours. However, in the fed
state, a non-disintegrating delivery system will remain in the stomach, either
floating on top of the stomach contents or sinking to the bottom depending on
the density³⁸. When this occurs, gastric emptying rather than the
dosage form controls drug release. In addition, if the dosage form has a delayed
release component, the drug may not be sufficiently protected for residence
time greater than 2 hours in the gastric pH of 1.2. Low pH may also alter the
performance by causing chemical reactions of the materials used in the dosage
for modifying the release of drug. Therefore, while the final dissolution test
may only require a 1-2 hour presoak at gastric pH, the dosage form should be
thoroughly evaluated at gastric pH if there is potential for long gastric residence
times.

If the goal of the dosage form
is to release the drug in the duodenum, e.g., target transport through tight
junctions, then the dissolution test should reflect the possibility of a short
residence time. This is especially true if the mechanism for targeting the release
is enteric coating. Further hampering of drug release can occur if the enteric
coating erodes at pH 6.5, since the pH at the proximal duodenum is closer to
5.5 than 6.5. Therefore, an appropriate dissolution test for pH sensitive release
mechanism such as enteric-coated dosage forms may require several pHs simultaneously
taking into consideration the potential in vivo residence time at each pH.

Coated particles/beads currently
used in both extended release and delayed release dosage forms offer advantages
over larger, non-disintegrating delivery systems. Depending on the design of
the delivery system, dissolution tests for bead formulations may consist of
2-3 hours in simulated gastric fluid at pH 1.2, followed by 15-30 minutes in
simulated intestinal fluid at pH 5.5, and then simulated intestinal fluid at
pH 6.8 or pH 8.0.

Food Effects

The extent of food effect on the
performance of a dosage form is very difficult to establish, and in many cases
just as difficult to mimic with a dissolution test. As discussed above, food
can affect gastric emptying, but may also alter the release of the drug from
the dosage form, the solubilization of the drug, and the transport of the drug
across the intestinal wall. For lipophilic, water-insoluble drugs, fatty meals
can do one, or both, of two things. First, a fatty meal can increase gastric
residence time thereby increasing the time available for solubilization. Second,
fatty meals may enhance the solubilization of the drugs by the lipids contained
in the meal, or by increasing the amount of bile salts released into the intestine,
or both. Dissolution media for water insoluble drugs generally contains a surfactant
to aid the dissolution. Previous dissolution tests consisted of hydroalcoholic
mixtures; however this combination was abandoned for the more physiologically
relevant surfactants^39-41. Formulas of dissolution media with mixed
micelles designed to mimic the fed state are available in the literature, but
can be very costly due to the bile salts and lecithin required^42,43.
These formulas generally consist of mixtures of sodium taurocholate and egg
lecithin. One study in particular showed that a 4:1 ratio of bile salt to lecithin
could be used as a dissolution medium for water insoluble drugs⁴³.
The same authors compared the effect of bile salt concentration and lecithin/bile
salts mixtures on the dissolution of several poorly soluble drug salts and developed
a correlation between the log of the octanol:water partition coefficient and
the solubility of several poorly soluble steroids in 15 mM sodium taurocholate.
Also important from this work was the finding that no significant increase in
solubility was observed at "fasted" concentrations of the bile salt.
Routine use of such media for determining batch to batch variability during
manufacturing may be costly; however the benefit may outweigh the cost when
establishing IVIVC's for scale-up and post approval changes (SUPAC).

The use of oil/water emulsions
as dissolution media to mimic a fatty meal has also been considered. However,
these systems can be difficult to work with⁴⁴. Agitation and elevated
temperatures, i.e. 37°C, can affect the stability of the emulsion thus limiting
the length of time available for dissolution. In addition, extraction of the
drug from the oil phase may require several steps thereby lengthening the analytical
time and cost of operation. However, when food effects are observed in vivo,
dissolution media with higher lipid content may be necessary if an IVIVC is
the desired endpoint. As with the micellar systems, current efforts have focused
on studying the solubilization and dissolution of water insoluble drugs into
emulsions formulated with different synthetic surfactants.

Studies have shown that foods may
also alter the permeability of the intestine⁴⁰. While this may have
little relevance in designing the dissolution media, it can impact on the IVIVC.
For example, lipids may increase the fluidity of the intestinal wall thereby
increasing the permeability or high concentrations of glucose may increase the
leakiness of the tight junctions.

Effect of metabolism

Intestinal metabolism of drugs
has received much attention over the last few years and enzymes have been routinely
added to both simulated gastric and simulated intestinal fluid. A thorough understanding
of the stability of the drug and dosage form in the presence of gastrointestinal
enzymes is important when determining the need for enzymes in the media. As
with the use of lecithin and bile salts, lumenal enzymes can be costly but necessary
if the goal is an IVIVC⁴⁴.

Conclusion

Dissolution test continues to increase in importance as a quality control test
in pharmaceutical analysis and drug development. This is largely due to the
fact that specific dissolution test methods have been developed for various
drug delivery systems and are available as compendial standards. The methods
are also designed to mimic the physiological environment of the GIT and have
increased the likelihood of establishing in vitro – in vivo correlation,
IVIVC for more pharmaceutical products. However, some dosage forms still
require more method development and refinement before standardized dissolution
test methods can be recommended.

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Oluwatoyin A. Odeku and Oludele A. Itiola

Department of Pharmaceutics and Industrial Pharmacy, Faculty
of Pharmacy, University of Ibadan, Ibadan,

*Corresponding author Oluwatoyin A. Odeku is a Senior
lecturer in the Department of Pharmaceutics and Industrial Pharmacy, Faculty
of Pharmacy, University of Ibadan, Ibadan, Nigeria, where she obtained her
Ph.D degree. Her research interests include drug delivery systems, formulation
studies and the development of materials obtained from local sources in Nigeria
as pharmaceutical excipients. She teaches undergraduate and postgraduate courses
in Pharmaceutics. She is a registered Pharmacist and a Member of the Pharmaceutical
Society of Nigeria. She has authored several publications in learned journals
and presented research findings at scientific conferences.

Contact info:

O. A. Odeku ,Department of Pharmaceutics & Industrial Pharmacy, Faculty
of Pharmacy, University of Ibadan, Ibadan, Nigeria.Phone : +234-803-3235828
and Fax 234-2-8106403. E-mail: pejuodeku@yahoo.com, odeku@skannet.com.

Oludele A. Itiola obtained his Ph.D degree from the University
of London. He is a Professor of Pharmaceutics and current Dean of the Faculty
of Pharmacy, University of Ibadan, Ibadan, Nigeria. His research interests
include drug delivery systems, formulation studies and drug dosage form design.
He teaches undergraduate and postgraduate courses in Pharmaceutics. He is a
registered Pharmacist and a Member of the Pharmaceutical
Society of Nigeria. He has authored several publications in learned journals
and presented research findings at scientific conferences.