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Bioavailability and Bioequivalence
299
Testing Models: For In Vitro Dissolution Technique A useful, effective and trustworthy in vitro dis-
solution technique should be able to predict the in vivo therapeutic (or pharmacological behavioural
pattern) to such a degree that the need for an in vivo bioavailability test may be ruled out completely.
Although the results duly obtained from the so-called standardized test performance stands by no
means a nearest perfect/comparable approach to the in vitro dissolution technique; the ultimate efforts
are only focused to the point of mimicking the closest environment as found in the ‘biologic system’.
Important Factors Governing the In Vitro Dissolution Technique There as three important experimental
parameters which predominantly govern the in vitro dissolution technique, namely:
1. The Dissolution Equipment: The dissolution equipment is duly based on six important char-
acteristic features, such as:
∑ size of the vessel (ranging between several millilitres capacity to several litres),
∑ design,
∑ shape of the vessel (fl at or round bottomed),
∑ agitation modality (oscillating, rotation, stirring, vortex mixing),
∑ variable speed of rotation,
∑ convenient and ease of performance and
∑ accuracy and precision of the ‘offi cial equipment’.
2. Dissolution Fluids: There are many variants of the ‘dissolution fl uids’ that are used abundantly in
in vitro dissolution assay methods, for instance:
∑ demineralized (Dm) water,
∑ 0.1 M hydrochloric acid (freshly prepared),
∑ phosphate buffer (as per IP, BP, USP),
∑ simulated gastric fl uid (or ‘artifi cial gastric juice),
9
∑ viscosity of the medium,
∑ operational ‘volume’,
10
∑ operational temperature (37 ± 2°C),
∑ maintaining the ‘sink parameters’
11
and
∑ maintaining the ‘non-sink conditions’.
12
Importantly, most of these aforesaid criteria pertaining to the nature and properties of the dissolu-
tion fl uids do play a vital role in the assay of drugs (dissolution test).
3. Process Parameters: The process parameters are solely responsible for the accurate determina-
tion in the in vitro dissolution technique, such as:
∑ timely incorporation of ‘drug product’ (or dosage form),
∑ typical sampling procedures (and replacing the requisite volume of fl uid with the dissolution
fl uid being used) and
∑ changing the ‘dissolution fl uid’ periodically.
9
That is, prepared strictly according to offi cial method in IP, BP, USP.
10
Volume: It is usually greater than that needed actually to complete the dissolution of the active drug under
investigation.
11
Sink Parameters: It refers to the drug concentration in solution constantly maintained at low level.
12
Non-sink Conditions: It means a gradual steady increase in the active drug concentration in the respective medium.
Essentials of Biopharmaceutics and Pharmacokinetics
300
The Drug Dissolution Estimation Apparatus With the increase in need and advent of skilled tech-
niques and wisdom, the ‘drug dissolution estimation apparatus’ has undergone a tremendous evolution
gradually from a simple beaker-type assembly to a completely automated and extremely versatile models.
Furthermore, these available devices may be duly categorized into several manners that are eventually
based upon the
∑ absence of sink conditions or
∑ presence of sink conditions,
wherein the specifi c dissolution apparatus has been duly designed on the following two distinct principles:
1. Open-Compartment Dissolution Apparatus: It specifi cally represents the ‘perfect sink con-
ditions’. In this particular instance, the ‘drug product’ is carefully contained in a column that is
continuously brought in direct contact with a fresh fl owing dissolution medium.
The aforesaid fl ow-through cell technique was initially developed by Dr. F. Langenbücher at the
Ciba-Geigy Laboratories (Switzerland).
13
The column-type dissolution method has been explicitly
illustrated in Figure 5.5(a) and (b).
Demerits of Open Flowthrough System: Importantly, the open fl owthrough system found in an
open-compartment dissolution apparatus [Figure 5.5(a) and (b)] possesses certain obvious demerits of its
own as given below:
1. The sieves (fi lters) S
1
and S
2
do have a tendency to get clogged because of the unidirectional fl ow
of the ‘dissolution fl uid’.
2. Besides, there is a great scope of pressure build-up almost near the end of the run. Therefore, in
majority of cases, it is absolutely necessary and important to have
∑ a build-in pressure transducers and
∑ an explicit feedback mechanism to enhance the pressure slowly so as to maintain a constant
fl ow rate.
3. ‘Pump’ Effect: It represents one of the most vital limitations of an open fl owthrough system. It
has been duly demonstrated that different kinds of ‘pumps’ used, e.g.,
∑ peristaltic pump and
∑ centrifugal type pump variants,
invariably give rise to altogether dissimilar dissolution results.
4. Finally, one requires relatively large volumes of the dissolution media (or fl uid) essentially required
to perform long-duration testing procedures.
(a) Closed-Compartment Dissolution Apparatus: The closed-compartment dissolution
apparatus is fundamentally a limited-volume apparatus usually working under the ‘non-sink
parameters’. Interestingly, in this particular instance, the respective ‘dissolution fl uid’ is strictly
restrained to the size of the container, such as:
∑ rotating basket-type dissolution apparatus and
∑ rotating paddle-type dissolution apparatus,
which shall be discussed briefly, with appropriate diagrammatic presentation, in the follow-
ing sections.
13
Langenbücher F: J. Pharm. Sci., 58: 1265, 1969; USP23/NF 18, 5th Supplement, p. 3470.
Bioavailability and Bioequivalence
301
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Essentials of Biopharmaceutics and Pharmacokinetics
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Figure 5.6 (a) Diagram of basket-type dissolution apparatus. (b) The detailed cross-section of the stirring
basket element.
(b) Rotating Basket-Type Dissolution Apparatus: In the rotating basket-type dissolution appara-
tus [Figure 5.6(a) and (b)],
14
the stirring element is a ‘basket’.
Modus Operandi: The various steps involved are as follows:
1. The metallic shaft rotates with a constant smooth motion and without causing any signifi cant
wobbling.
2. ‘Basket’ has two distinct segments:
∑ Top part with a vint is duly attached to the shaft ‘C’ and is fi tted with three spring clips or
other suitable means which would allow the lower part being removed, as and when required
for needful introduction of the ‘sample’ under investigation; and also holds the lower part of the
basket concentric fi rmly with the axis of the vessel during rotation.
14
Indian Pharmacopoeia, Vol. 1: The Indian Pharmacopoeia Commission, Ghaziabad, India, 2007, pp. 179–180.
Bioavailability and Bioequivalence
303
∑ Lower part is a detachable component of the ‘basket’ and is usually made up of welded-steam
cloth having
_ wire thickness of 0.254 nm diameter and
_ 0.381 mm square openings, duly formed into a ‘cylinder’ with a narrow rim of sheet metal
around the top and bottom.
Note
The entire ‘basket’ may be appropriately gold plated with a 2.5 mm coating for its use in an acidic
environment freely.
3. The actual distance between the ‘inside bottom’ of the vessel and the ‘basket’ is duly maintained
between 23 and 27 mm during the performance of the ‘dissolution test’.
4. Rotating Paddle-Type Dissolution Apparatus: The rotating paddle-type dissolution appara-
tus
15
is crafted and designed in such a manner so as to determine the compliance with the required
dissolution requirements for the solid drug products (or dosage forms, viz. tablets or capsules), as
illustrated in Figure 5.7(a) and (b):
15
Indian Pharmacopoeia, Vol. 1: The Indian Pharmacopoeia Commission, Ghaziabad, India, 2007, pp. 179–180.
Figure 5.7 (a) Diagram of a paddle-type dissolution apparatus. (b) The detailed cross-section of the paddle
stirring element.
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Essentials of Biopharmaceutics and Pharmacokinetics
304
Description of the apparatus: The following are the various aspects of the apparatus:
1. All contact parts of the dissolution apparatus with the formulation (drug product) under examina-
tion or with the dissolution fl uid (medium) must be
∑ chemically inert,
∑ non-absorbable,
∑ non-reactive and/or
∑ non-interfering with the ‘formulation’ being examined.
2. All metallic contact parts of the dissolution apparatus either with the formulation (or dosage form)
or with the respective dissolution fl uid (or medium) should always be made from stainless steel (SS)
type 316 or equivalent or coated with an appropriate substance to ensure that such components do
not interfere or react with the formulation being studied or the ensuring dissolution fl uid.
3. Invariably, a dissolution apparatus that critically allows observation of the formulation (or dosage form)
under examination and the stirring process during the entire dissolution test is indeed most preferable.
4. A cylindrical vessel, A, made up of either borosilicate glass or any other suitable transparent polymeric
material (e.g., polyacrylic sheet), has a hemispherical bottom. It should have a normal capacity
of 1 L and an internal diameter of 98–106 mm, as shown in Figure 5.7(a). Besides, the cylindrical
vessel has a fl anged upper rim duly fi tted, with a lid which essentially possesses a ‘number of openings’,
the main one of which is strategically located in the centre.
5. A separate electric motor is provided with a ‘speed regulator’ duly capable of maintaining the
speed of rotation (rpm) of the paddle within a limit of 4% of the stipulated speed mentioned in
the individual monograph (IP, 2007). The aforesaid electric motor is carefully fi tted with a ‘stirring
element’ that essentially comprises [see Figure 5.7(b)]
∑ a ‘drive shaft’ and
∑ a ‘blade forming a paddle’.
6. The ‘blade’ passes via the diameter of the ‘drive shaft’ in order that the bottom of the blade is fl ush
with the bottom of the shaft. Now, the ‘drive shaft’ is positioned strategically so that its axis falls
very much within 2 mm of the axis of the vessel, whereas the lower-edge of the blade is nearly
23–27 mm away from the inside bottom of the cylindrical vessel.
Finally, the rotating paddle-type dissolution apparatus functions in such a manner that the pad-
dle rotates smoothly and without signifi cant noticeable wobbling.
7. Electric Water Bath: An electric water bath is set to maintain the dissolution fl uid between
36.5°C and 37.5°C. However, the bath liquid is usually kept in constant and smooth motion dur-
ing the test. The cylindrical vessel is securely clamped in the water bath in such a fashion that the
overall displacement vibration from the rest of the equipment, viz. water-circulating device, is
minimized to the least extent.
Closed Column-Type System: Abdou et al. (1978)
16
developed an altogether newer technique based
on a variation of the fl ow-through concept. It is essentially a closed column-type system that represents
a combination of a miniaturized rotating basket having a closed fl ow-through dissolution apparatus to
maintain and sustain the ensuing concentration of the drug substance at an acceptable and workable
range for the required quantitative assay, as illustrated in Figure 5.8(a) and (b).
It may be worthwhile to state here that the aforesaid closed column-type system happens to be
∑ a semi-automated one in conjunction with HPLC
17
and
∑ employed for the accurate estimation of the dissolution rate of 0.1 mg fl udrocortisone acetate tablets.
16
Abdou HM et al.: J. Pharm. Sci., 67: 1397, 1978.
17
HPLC: High performance liquid chromatography.
Bioavailability and Bioequivalence
305
Figure 5.8(a) explicitly depicts the miniaturized USP
18
basket apparatus, duly combined with a fl ow-
through mechanism.
Figure 5.8(b) shows a comparison between the observed dissolution rate of fl udrocortisone acetate
duly recorded at different rotation speeds of the miniature rotating basket [see Figure 5.8(a)]. Thus, each
point is an average of six drug tablets taken carefully from the same lot:
{ = at 50 rpm;
= at 100 rpm;
= at 125 rpm.
18
USP 23/NF 18, 5th Suppl. pp. 3469, 3470; Ibid, 6th Suppl. p. 3794.
Figure 5.8(a) Diagram of a closed fl ow-through dissolution apparatus for the determination of dissolution rate
of dosages with extremely small quantum of active ingredients.
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Essentials of Biopharmaceutics and Pharmacokinetics
306
2.6. Statistical Methods for Bioavailability Studies
Preamble: It has been duly established that after having conducted
∑ bioequivalence evaluation and
∑ determining appropriate conditionalities,
one should critically examine the obtained pharmacokinetic data as per the set of predetermined char-
acteristic features so as to either confi rm or refute the relevant ‘bioequivalency’ of the ‘test sample’ and
‘reference drug products’.
In other words, it is always necessary to determine whether the ‘test sample’ and ‘reference drug prod-
ucts’ actually show a difference very much within a predefi ned level of statistical signifi cance.
Importance of Statistical Methods: As the ‘statistical results’ duly obtained from an elaborated
‘bioequivalence study’ form the ultimate primary basis of the decision either for or against the so-
called therapeutic equivalence of two dosage forms, it becomes absolutely important that the generated
‘experimental data’ may be analyzed with the help of a suitable statistical test.
Selection of Statistical Methods: The various available statistical methods intended to be employed
in bioavailability studies must be selected with utmost care with respect to the following critical aspects:
USP
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Figure 5.8(b) A comparison between the dissolution rate of fl udrocortisone acetate at variable rotation
speeds, viz. 50, 100 and 125 rpm.
Bioavailability and Bioequivalence
307
∑ Variations observed among individuals
∑ Variations seen among the batches of nominally identical produced ‘dosage forms’
∑ Skilful knowledge and wisdom duly solicited for planning, analysis and interpretations of these
experimental results
2.6.1. Methods of Data Analysis
The duly planned and executed ‘bioequivalency studies’ invariably emerge with a copious volume of
extremely useful experimental results that may be analyzed statistically in several available different
means and ways.
Notari (1987)
19
and McDonald et al. (1969)
20
suggested and proved generously that the statistical
methods of analysis solely depend on the typical statistical model of the ‘concentration–time curve’.
Nevertheless, they observed various sources of variation in statistical model, such as:
∑ person-to-person variations,
∑ non-independent measurements
21
and
∑ detailed experimental layout plans.
Importantly, the most preferred and easiest way to make use of the concentration–time curves generally
for the comparison of two dosage forms may be precisely accomplished by
∑ not simply comparing the whole curves and/or
∑ carrying out a comparison of the characteristic features of the ‘curves’
with respect to such vital and important criteria:
∑ Area under the curves (AUCs),
∑ Peak heights and
∑ Rates of absorption.
Paired Comparisons: In a specifi c situation when only a ‘single characteristic feature’ is involved pre-
dominantly, one may use an appropriate technique of analysis invariably referred to as ‘paired compari-
sons’. Interestingly, here each and every individual gives rise to a typical ‘paired difference’.
2.6.2. Statistical Analysis
The following important aspects related to the statistical methods used in ‘bioequivalence study’ have
been duly discussed in Section 4(3.5). Besides, the corresponding statistical method variants, such as Latin-
square cross-over design and balanced incomplete block design (BIBD), have also been discussed under
Section 4(3.5.1).
Enough dependable ‘statistical methods’ that are usually employed for the critical comparison of two
or more sets of variables may fi nd their utility in the study involving more than two dosage forms.
Note
19
Notari RE: Biopharmaceutics and Pharmacokinetics: An Introduction, 4th edn., Marcell Dekker, New York, 1987.
20
McDonald H et al.: Clin. Med. (Dec.): 30–33, 1969.
21
Barr WH et al.: Clin. Pharmacol. Ther. 13(1): 97–108, 1972.
Essentials of Biopharmaceutics and Pharmacokinetics
308
2.6.3. Statistical Interpretation of Bioequivalence Data
The ‘analysis of variance’ (ANOVA) refers to a statistical procedure largely used to compare the means of
two or more groups. Obviously, whenever a statistically signifi cant difference is observed, it is absolutely
necessary and important to establish whether it is ‘really clinically signifi cant’. It has been found invariably
that when the statistically signifi cant difference stands at only 10%, it clearly indicates that the extent of
absorption prevailing between the two dosage forms (or drug products) is almost insignifi cant clinically.
Universally Acceptable Generalized Rules: In usual practice, one may confi dently make use of
the universally acceptable generalized rule that if the observed relative bioavailability of the ‘test’ dosage
form and ‘reference standard’ falls within a range between 80% and 120% (of reference sample), it should
be considered as ‘bioequivalent’.
In addition to the above dictum, one may also rely upon the critical observation that the actual dif-
ference existing between the bioavailabilities of the ‘test drug products’ must appear to be greater than
±20% average of the ‘reference standard’.
2.7. Factors that Decrease Bioavailability
A survey of literature reveals that there exists three crucial factors that decrease bioavailability in a pro-
gressive manner, namely:
∑ Presystemic metabolism,
∑ Instability and
∑ Complexation,
which would be discussed separately in the following sections.
2.7.1. Presystemic Metabolism
Immediately after the oral administration of a solid dosage form, the ‘absorbed drug’ undergoes fi rst-
pass metabolism which critically implies that the latter directly gains entry to the liver before ultimately
reaching the systemic circulation. In fact, this kind of an approach defi nitely enhances the degree of
metabolism with regard to an IV dose.
Peripheral vein vs. portal vein: In laboratory model ‘dog’, the AUC values duly obtained for
lidocaine (a local anaesthetic) and aspirin (an analgesic) varies signifi cantly, i.e., found to be
∑ greater—when infused directly into a peripheral vein and
∑ lower—when infused into the portal vein.
Examples
However, the administration of drug(s) directly into the portal vein is very much similar to the ‘oral
absorption’.
Cause of decrease bioavailability may be expatiated due to the relevant exposure of drugs to the liver
before approaching the circulating blood that eventually facilitates
∑ reasonable dilution and
∑ effective distribution
of the available drugs to other sites.