Chapter 2 Review Literature Pharmaceutical Medicine 7 Jamia Hamdard 2.0 Review Literature 2.1. Pregabalin 2.1.1. History and place in therapy: Developed by Pfizer, Pregabalin, marketed under the brand name Lyrica. It is a 3- substituted analogue of gamma-amino butyric acid (GABA). It is a compound related to Pfizer's hugely successful antiepileptic drug gabapentin (Neurontin). In July 2004, Pfizer secured Europe-wide approval for Pregabalin for use in the management of peripheral neuropathic pain as well as an adjunctive therapy in the treatment of partial epileptic seizures. Subsequently, in December 2004 the company gained US Food and Drug Administration (FDA) approval for use of Pregabalin in neuropathic pain associated with diabetic peripheral neuropathy and postherpetic neuralgia; making it the first FDA-approved treatment for these neuropathic pain states. Pregabalin was also reviewed by the FDA as an adjunctive treatment for partial epileptic seizures in adults. In June 2005, the FDA granted approval to market Pregabalin for adjunctive treatment of partial epileptic seizures in adults. In June 2007, Pregabalin became the first drug to be approved by the FDA for the treatment of fibromyalgia. First marketed in 1983, gabapentin (Neurontin) has been one of Pfizer's top performing drugs. Pregabalin is seen as an important successor now that gabapentin is facing the threat of generic competition. Pregabalin is mechanistically similar to gabapentin and shares similar advantages, such as a lack of pharmacokinetic interactions with other medications or enzyme induction. But, there are several differences between the two drugs. According to preclinical studies, Pregabalin has an increased binding affinity for the α 2 -δ protein subunit of voltage-gated calcium channels, which is associated with analgesic and anticonvulsant activity, and has shown greater analgesic activity compared with gabapentin [Frampton et al 2004;
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Chapter 2 Review Literature
Pharmaceutical Medicine 7 Jamia Hamdard
2.0 Review Literature
2.1. Pregabalin
2.1.1. History and place in therapy:
Developed by Pfizer, Pregabalin, marketed under the brand name Lyrica. It is a 3-
substituted analogue of gamma-amino butyric acid (GABA). It is a compound related to
Pfizer's hugely successful antiepileptic drug gabapentin (Neurontin).
In July 2004, Pfizer secured Europe-wide approval for Pregabalin for use in the
management of peripheral neuropathic pain as well as an adjunctive therapy in the
treatment of partial epileptic seizures.
Subsequently, in December 2004 the company gained US Food and Drug Administration
(FDA) approval for use of Pregabalin in neuropathic pain associated with diabetic
peripheral neuropathy and postherpetic neuralgia; making it the first FDA-approved
treatment for these neuropathic pain states.
Pregabalin was also reviewed by the FDA as an adjunctive treatment for partial epileptic
seizures in adults. In June 2005, the FDA granted approval to market Pregabalin for
adjunctive treatment of partial epileptic seizures in adults. In June 2007, Pregabalin
became the first drug to be approved by the FDA for the treatment of fibromyalgia.
First marketed in 1983, gabapentin (Neurontin) has been one of Pfizer's top performing
drugs. Pregabalin is seen as an important successor now that gabapentin is facing the
threat of generic competition.
Pregabalin is mechanistically similar to gabapentin and shares similar advantages, such as
a lack of pharmacokinetic interactions with other medications or enzyme induction. But,
there are several differences between the two drugs. According to preclinical studies,
Pregabalin has an increased binding affinity for the α2-δ protein subunit of voltage-gated
calcium channels, which is associated with analgesic and anticonvulsant activity, and has
shown greater analgesic activity compared with gabapentin [Frampton et al 2004;
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Sabatowski et al. 2004; Frampton et al. 2005]. Despite these preclinical data, it is unclear
if Pregabalin has a clinical advantage over gabapentin. As the two drugs have not been
compared in clinical trials. Unlike gabapentin, Pregabalin exhibits linear
pharmocokinetics after oral administration, with low intersubject variability [Frampton et
al 2004; Sabatowski et al. 2004; Frampton et al. 2005]. This provides a more predictable
dose- response relationship, as plasma concentrations increase linearly with increasing
dose. Gabapentin on the other hand requires disproportionately larger dosage increases to
achieve increases in plasma concentrations. The large dosages required for some patients
receiving gabapentin could worsen dose-dependent adverse effects, such as dizziness and
somnolence. Thus, the linear pharmacokinetics of Pregabalin impart a better-defined
effective dosage range and may provide the basis for the efficacy of either fixed- or
flexible-dosage regimens. This property also accounts for the defined dose-dependent
adverse effects and benefits reported in clinical trials. Pregabalin's linear
pharmacokinetics and low intersubject variability allow it to be initiated at or adjusted to
the target dosage more rapidly. Whereas, gabapentin requires a long, slow adjustment to
the effective dosage [Bloomel ML, Bloomel AL 2007].
Pregabalin is comparable to gabapentin in terms of cost for all but the lowest doses of
Gabapentin. Pregabalin appears to be well tolerated; however, it does not have the proven
long term safety profile as gabapentin, which has been used in large numbers of patients
during years of clinical practice. Another limitation of Pregabalin is the potential for
abuse and dependence, necessitating the monitoring of patients for signs of Pregabalin
abuse [Bloomel ML, Bloomel AL 2007].
Despite the lack of long-term safety and efficacy information and the abuse potential for
Pregabalin, the initial clinical results, the comparable medication cost (especially at
higher doses), and the favorable pharmacokinetic profile of Pregabalin support the use of
Pregabalin as an alternative to gabapentin for Pregabalin's FDA-approved indications. As
Pregabalin and gabapentin have a similar mechanism of action, it is not expected that
Pregabalin would benefit a patient when used concomitantly with gabapentin; however,
these drugs could be given concomitantly with other medications used for diabetic
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peripheral neuropathy, postherpetic neuralgia, or seizures in refractory patients [Bloomel
ML, Bloomel AL 2007].
Other medications commonly used as first-line treatment of diabetic peripheral
neuropathy and postherpetic neuralgia include tricyclic antidepressants, opioids, and
topical lidocaine. The costs of tricyclic antidepressants and opioid analgesics are lower
than the cost of Pregabalin. Clinical trials comparing the efficacy of tricyclic
antidepressants and other clinical options with Pregabalin are lacking. A potentially
limiting adverse-effect profile (tricyclic antidepressants and opioids), lack of suitability
for long-term use (lidocaine), and slow onset of analgesic action (tricyclic
antidepressants) are some limitations of using these therapies for the treatment of diabetic
peripheral neuropathy and postherpetic neuralgia [Frampton et al. 2004; Frampton et al.
2005]. Until the results of comparison trials are available for Pregabalin and the other
accepted treatment options, Pregabalin should be considered as an alternative to tricyclic
antidepressants and opioids for the treatment of diabetic peripheral neuropathy or
postherpetic neuralgia in patients not responding to or tolerating the current treatment
regimen or in those patients who would not be suitable candidates for therapy with
tricyclic antidepressants or opioids.
2.1.2. Therapeutic indications and safety and efficacy of Pregabalin in various
indications:
2.1.2.1. Neuropathic pain
The International association for the study of pain defines neuropathic pain as “initiated
or caused by a primary lesion or dysfunction in the nervous system” and due to
disordered peripheral or central nerves. [Merskey et al.1994] This disorder can be caused
by compression, transection, infiltration, ischemia, or metabolic injury to neuronal cell
bodies, or in combination.
Neuropathic pain may be classified as either peripheral or central in origin [Dworkin
2002] Examples of the former include diabetic peripheral neuropathy (DPN),
postherpetic neuralgia (PHN), antineoplastic therapy–induced or HIV-induced sensory
The bioavailability (Cmax, Tmax/AUC) of formulation that is used in development of
IVIVC is predicted from its in vitro property using IVIVC. Comparison between
predicted bioavailability and observed bioavailability is done and % P.E is calculated.
According to FDA guidelines, the average absolute % P.E should be below 10% and %
P.E for individual formulation should be below 15% for establishment of IVIVC.
2.6.3.2. External predictability
The predicted bioavailability is compared with known bioavailability and % P.E is
calculated. The prediction error for external validation should be below 10% whereas
prediction error between 10-20% indicates inconclusive predictability and need of further
study using additional data set. Drugs with narrow therapeutic index, external validation
is required.
2.6.4. Reasons for poor in vitro-in vivo correlation [Aoyagi 1982]
A. Fundamentals – When in vivo dissolution is not the rate limiting pharmacokinetic
stage, and when no in vitro test can simulate the drug dissolution along the
gastrointestinal tract.
B. Study design – With inappropriate in vitro test conditions.
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C. Dosage form – When the drug release is not controlled by the dosage form or is
strongly affected by the stirring of synthetic liquid.
D. Drug substance – With a non- linear pharmacokinetics, for e.g, first - pass hepatic
effect, an absorption window, a chemical degradation and a large inter or intra subject
variability. All these factors are of vital concern and should be kept in mind, especially
the inter variability of patients’ response to a drug.
2.6.5. Biopharmaceutics classification system (BCS) [Mattok et al. 1972; Shaw et al.
1973; Chasseaud et al. 1983 ; Mathiowitz 1999; Dressman 2005]
Biopharmaceutics classification system is based on solubility, intestinal permeability and
dissolution rate, all of which governs the rate and extent of oral absorption from
immediate release solid oral dosage form. Based on solubility and permeability, there are
four classes of BCS as shown in table 2 solubility criteria defined in present regulatory
guidance for classifying an active pharmaceutical ingredients (API) as “highly soluble”
requires the highest strength to be soluble in 250 ml of water over the pH range of 1-7.5
at 37°C, otherwise it is considered as poorly soluble. The FDA and also EMEA Guidance
define “highly permeable” as having a fraction dose absorbed of not less than 90%. The
recently adopted WHO guidelines set a limit of not less than 85% of the fraction dose
absorbed, otherwise it is considered to be poorly permeable.
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Table LR 2: IVIVC expectations for immediate release products based on BCS
Class Solubility Permeability IVIVC expectations
I High High IVIVC expected, if dissolution rate is slower than gastric
emptying rate, otherwise limited or no correlations.
II Low High IVIVC expected, if in vitro dissolution rate is similar
to in vivo dissolution rate.
III High Low Absorption (permeability) is rate determining and limited
or no IVIVC with dissolution.
IV Low Low Limited or no IVIVC is expected.
A. Biowaiver for BCS Class I
On the basis of FDA guidelines, sponsor can request biowaiver for BCS Class I in
immediate release solid oral dosage form, if the drug is stable in GIT and having narrow
therapeutic index with no excipient interaction affecting absorption of drug in the oral
cavity. Once a drug enters in stomach; it gets solubilised in gastric fluid rapidly before
gastric emptying and the rate and extent of absorption is independent of drug dissolution
as in case of solution. Hence, the goal of biowaiver is achieved.
B. Biowaiver Extension Potential for BCS Class II
The rate and extent of absorption of BCS Class II drug depends on in vivo dissolution
behaviour of immediate release products. If in vivo dissolution can be predicted from in
vitro dissolution studies, in vivo bioequivalence study can be waived. In vitro dissolution
methods can mimic in vivo dissolution behaviour of BCS Class II drug and are appealing
but experimental methods can be difficult to design and validate because of number of
processes involved.
C. Biowaiver Extension for BCS Class III
If excipient used in two pharmaceutically equivalent solid oral immediate release product
does not affect the drug absorption and the products dissolves very rapidly (>85% in 15
min.) in all relevant pH ranges, there is no reason to believe that these products would not
be bioequivalent.
2.6.6. Approaches for Development of Correlation [Sullivan et al. 1974; Di Santo et
al. 1975; Zaman et al. 1983; De Muth 1999]
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Basically, two methods are available for the development of correlations
2.6.6.1. Two stage deconvolution approach: This involve estimation of in vivo
absorption profile from plasma drug concentration - time profile using wagner nelson or
looe-riegelman method, subsequently the relationship with in vitro data is evaluated.
2.6.6.2 One stage convolution approach: It computes the in vivo absorption and
simultaneously models the in vitro – in vivo data.
Two stage methods allows for systematic model development while one stage obviates
the need for administration of an intravenous, oral solution or IV bolus dose. Mostly
IVIVC models developed are simple linear equation between in vitro drug released and in
vivo drug absorbed. But sometimes these data can be better fitted by using nonlinear
models like Sigmoid, Weibull, Higuchi or Hixon-crowell.
2.6.7. Parameters to be considered while developing IVIVC [Sullivan et al. 1974;
Dietrich et al. 1988; Vergnaud et al. 2005]
2.6.7.1. Metabolic factors
A drug must pass sequentially from the gastrointestinal lumen, through the gut wall, and
the liver, before entering in the systemic circulation. This sequence is an anatomic
requirement because blood perfusion virtually all gastrointestinal tissues drain into the
liver via the hepatic portal vein. Drug loss may occur in the GIT due to the instability of
the drug in the GIT and/or due to complexation of drug with the components of the GI
fluids, food, formulation excipients or other co-administered drugs. In addition, the drug
may undergo destruction within the walls of the GIT and/or liver.
2.6.7.2. Drug loss in GIT
Any reaction that completes with the absorption of a drug may reduce oral bioavailability
of a drug. Reaction can be both enzymatic and non-enzymatic. Acid hydrolysis is a
common non-enzymatic reaction. Enzymes in the intestinal epithelium and within the
intestinal microflora, which normally reside in the large bowel, metabolize some drug.
The reaction products are often inactive or less potent than the large molecule.
2.6.7.3. Stereochemistry
When one enantiomer has higher affinity towards receptors than other, the phenomenon
is termed as stereo selectivity which results in pharmacokinetics or pharmacodynamics. If
such stereoisomers in the form of racemate are administered orally, one form may have
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higher bioavailability than the other. Obviously use of in vitro dissolution data of
racemate will not be useful in the development of IVIVC and hence prediction of in vivo
availability of active enantiomer. So consideration of stereoisomerism in the development
of IVIVC may provide more meaningful relationship.
2.6.8. Parameters studied for IVIVC
Earlier disintegration was considered as the most important pertinent in vitro parameter
but recently, dissolution rate has been used as a manufacturing process standard and is
generally considered to be the in vitro parameter most likely to correlate with in vivo
bioavailability. In vivo bioavailability is described in terms of the rate and extent of drug
absorption. Rate of absorption is reflected in peak drug concentrations in plasma (Cmax)
and the terms at which they occur (Tmax). Other methods may be used to describe
absorption rate profile, for example, deconvolution and statistical moment theory.
However use of these approaches does not detract from the basic relationships between
absorption rate, Cmax and tmax. Extent of absorption is reflected in Cmax and also the
area under the plasma drug curve (AUC).
2.6.9. Attempts to establish in vitro – in vivo correlation [Benidikt et al. 1988; De
Muth 1999; Welling et al. 2006; Vergnaud et al. 2005]
Many attempts have been made to establish IVIVC for a variety of drugs. Some of these
are summarized in the table LR 3 which describes studies on a variety of dosage forms
for a broad spectrum of therapeutic indications, and provides a brief comment on the
results obtained.
Table LR 3: Investigations of In vitro Dissolution and In vivo Bioavailability Relationship
Drug Test Formulation Comments
Steroids and Hormones
Prednisolone 5mg tablets Products were bioequivalent despite difference in in vitro dissolution. Dissolution test modified to agree with in vivo data.
Prednisone 5mg and 50mg tablets In vitro dissolution rate not predictive of overall bioavailability
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Anti-inflammatory and analgesic agents
Aspirin Four 300mg tablets No IVIVC correlation
Ketoprofen 50mg conventional capsules and two 200mg sustained release capsules
Slower absorption and reduced systemic bioavailability from slower dissolving SR capsule.
Indomethacin Four Indomethacin preparations
All preparations were bioequivalent despite different dissolution rate of one preparation.
Respiratory Tract
Theophylline Four experimental controlled release formulations.
Correlations obtained between in vitro and in vivo data.
Central Nervous system Drugs
Promethazine Two 50mg tablets, one 25mg tablet and a solution.
No discrimination. No significant differences among products in in vitro or in vivo data.
Antibacterial and antifungal
Griseofulvin Four 100mg capsules compared in dog and humans
Good in vitro-in vivo correlation using specific sink condition dissolution method.
Doxycycline Three 100mg capsules compared with a suspension and a solution
Rank order correlation between dissolution rates and absorption rate constants, but no statistical significant difference in bioavailability of the three capsules products.
Hypoglycaemic Agents Glyburide Four marketed preparations Two dissolution tests yielded
different rank orders of dissolution rates. Neither test correlated with in vivo data.
Cardiovascular Isosorbide dinitrate Two experimental 40mg
tablets Products were bioequivalent despite different in vitro release
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rates. Digoxin Seven 0.25mg tablets Close correlation between
dissolution rate and bioavailability.
2.6.10. Applications [Chalk et al. 1986 ; Leon Shargel et al. 1999; Mathiowitz 1999;
Vergnaud et al. 2005]
The most vital application of IVIVC is to use in vitro dissolution study in lieu of human
bioequivalence studies which will reduce the number of human bioequivalence studies
during initial approval process as well as certain scale up and post approval changes.
2.6.10.1. Manufacturing Control
The extended release products are distinguished through their input rate to the absorption
site. Therefore, the rate of drug release from these products is an important feature and
should be carefully controlled and evaluated. The in vitro dissolution/release test is
meaningful only when the test results are correlated to the products’ in vivo
performances.
2.6.10.2. Process Change Assurance
The manufacturing processes of approved products are regulated by the regulatory
agencies. The manufacturers are required to demonstrate that kind of change, even an
engineering improvement, does not cause changes in the finished product’s in vivo
performance. Consequently, many changes have to be supported by a bioequivalence
study. With the availability of an in vitro test with one-to-one correlation to the product’s
in vivo performance, a bioequivalence study should no longer be necessary. In such
cases,
the scientists and regulatory agencies may consider a pilot pharmacokinetic study as an
assurance that the new excipient does not inadvertently affect the absorption.
2.6.10.3. Dissolution/Release Rate Specifications
Without a correlation, the specifications of an in vitro test can be established only
empirically. This approach is data driven but is valid only if all the batches have been
extensively evaluated in clinical trials; furthermore, it probably can detect only relatively
large differences between different batches. It is therefore more precise to set up the
specification using the correlation to evaluate the in vivo consequences of the range.
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Clearly, the pharmacokinetic consequences alone are not sufficient to set up the
specifications. The pharmacodynamic knowledge is the key to make the specification
clinically meaningful. In the absence of the information, some scientists may be willing
to rely on the empirical bioequivalence range of ±20% as the first guidance. In case of a
one-to-one correlation, this automatically translates in a dissolution rate change of ±20%.
It is empirically derived dissolution range is much wider than ±20%, and then the
companies invariably believe that the products have been punished by the presence of
one-to-one correlation.
2.6.10.4. Early development of Drug Product and Optimization
In the early stages of drug product development drug products are characterised by some
in vitro systems and some in vivo studies in animal models to find out toxicity and
efficacy issues.
2.6.10.5. Biowaiver for Minor Formulation and Process Changes
After the evaluation of critical manufacturing variables and in vitro dissolution rate for
controlled release formulation an IVIVC has been established. In vitro dissolution data is
used to justify minor formulation and process changes. The changes may include minor
change in shape, size, amount and composition of materials, colours, flavours, procedure,
and coating, source of inactive and active ingredients, equipment or site of
manufacturing.
2.7 Bioavailability
2.7.1. Definition: As per US-FDA, Bioavailability is defined as the rate and extent to
which the active ingredient or active moiety is absorbed from a drug product and
becomes available at the site of action. For drug products that are not intended to be
absorbed into the bloodstream, bioavailability may be assessed by measurements
intended to reflect the rate and extent to which the active ingredient or active moiety
becomes available at the site of action [Bioavailability and Bioequivalence Studies for
Orally Administered Drug Products--General Considerations. CDER, 2003].
The EMEA guidance defines Bioavailability as the rate and extent to which the active
substance or active moiety is absorbed from a pharmaceutical form and becomes
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available at the site of action [Note for guidance on the investigation of bioavailability
and bioequivalence, EMEA].
The CDSCO, India, defines Bioavailability as the relative amount of drug from an
administered dosage form which enters the systemic circulation and the rate at which the
drug appears in the systemic circulation [Guidelines for bioavailability & bioequivalence
studies, CDSCO, 2005].
2.7.2. The need for Bioavailability and Bioequivalence Studies:
1. Bioavailability studies provide an estimate of the fraction of the orally
administered dose that is absorbed into the systemic circulation when compared to
the bioavailability for a solution, suspension, or intravenous dosage form that is
completely available.
2. Bioavailability studies provide other useful information that is important to
establish dosage regimens and to support drug labeling, such as distribution and
elimination characteristics of the drug.
3. Bioavailability studies provide indirect information regarding the presystemic and
systemic metabolism of the drug and the role of transporters such as p-
glycoproteins.
4. Bioavailability studies designed to study the food effect provide information on
the effect of food and other nutrients on the absorption of the drug substance.
5. Such studies when designed appropriately provide information on the linearity or
nonlinearity in the pharmacokinetics of the drug and the dose proportionality.
6. Bioavailability studies provide information regarding the performance of the
formulation and subsequently are a means to document product quality.
7. Bioequivalence studies provide a link between the pivotal and early clinical trial
formulation, a link between formulations used in the pivotal clinical trial and the
stability studies, the pivotal clinical trial and the to-be-marketed drug product, and
other comparisons as appropriate.
8. Bioequivalence studies are the basis for determination of the therapeutic
equivalence between a pharmaceutically equivalent generic drug product and a
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corresponding reference listed drug. This list is provided in the orange book
[Approved drug products with therapeutic equivalence evaluations, 1999].
9. Bioequivalence studies provide information on product quality and performance
when there are changes in components, composition, and method of manufacture
after approval of the drug product. The FDA has provided guidance for the
industry, such as SUPAC-IR [Scale up and post approval changes: Immediate
release forms: FDA guidance, 1995] and SUPAC-MR [Scale up and post approval
changes: Modified release forms: FDA guidance, 1997], to determine when
changes in components and composition and/or method of manufacture of the
drug product suggests a need to perform further in vitro/in vivo studies.
2.7.3. Bioavailability and Bioequivalence testing recommended by the FDA:
Some of the situations when bioavailability and bioequivalence testing is essential for a
drug are mentioned below:
• For all new molecular entities.
• For new formulations of active drug ingredients.
• For a new dosage form of a drug.
• For a new dosage strength or dosage regimen.
• For a new salt or ester of a drug.
• For a new indication
• For the administration in special patient populations, e.g., Pediatrics.
• For a change in the manufacturing process of the drug or the drug product
that produces variabilities beyond the specifications of approved
applications.
2.7.4. Bioavailability assessment methods:
Bioavailability is the measurement of the rate and extent of drug that is systemically
available. Hence, pharmacokinetic parameters that give information on the amount of
drug reaching the systemic circulation (extent) and the time taken to reach the systemic
circulation (rate) are used as measures for assessing bioavailability. Bioavailability can be
measured by direct and indirect methods mentioned below.
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2.7.4.1. Direct measures of Bioavailability
1. Based on plasma drug concentrations:
Drug concentrations in the blood and plasma are the most direct methods of determining
the systemic availability of a drug. The pharmacokinetic parameters that describe the rate
and extent of absorption and systemic exposure based on plasma drug concentration data
are summarized below.
a.) The area under the plasma drug concentration and time curve (AUCt, AUC∞, or AUCtau) (units = ng.h/ml): AUC is the measure of the extent of drug bioavailability. This gives a measure of the total systemic exposure. AUC can be obtained by a numerical integration method such as the trapezoidal rule. A recent recommendation by the FDA has been the use of early exposure as a measure of rate of systemic exposure [Chen, 1992]. This can be calculated as a partial AUC, where the area can be truncated at the population median of the tmax values. Measurement of early exposure may be useful when rapid onset of action is desirable (e.g., an analgesic effect) or if a slow input is required to achieve efficacy or safety. The FDA has recently proposed a shift away from the focus on rate and extent of absorption to the measurement of systemic exposure, which can be determined as total, peak, or early exposure (if needed). This is based on the understanding that these measures better reflect the rate and extent of absorption [Chen 1992; Bois et al.1994; Tozer et al. 1996]. b.) The peak plasma drug concentration (Cmax) (units = ng/ml): The Cmax is also a measure of the extent of bioavailability or peak exposure and indicates concentration required for a therapeutic or toxic response. It relates to peak exposure of the drug. Cmax is obtained directly from the plasma concentration time profile. c.) The time to peak plasma drug concentration (tmax) (units=hours, minutes, etc.): The
tmax is a measure of the rate of drug absorption and is the time required to reach the
maximum drug concentration after drug administration. The tmax is obtained directly
from the plasma concentration time profile.
2.7.4.2. Indirect measures of Bioavailability:
1. Based on urinary excretion data
This method can be used only if urinary excretion of unchanged drug is the main
mechanism of elimination of the drug and urine samples have been collected in intervals
as short as possible to measure the rate and amount of excretion as accurately as possible.
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Limitations of using urinary data:
• There is a high degree of variability associated with the cumulative amount of
drug excreted in the urine, and the method is less reliable compared with the
estimation of bioavailability from plasma concentration time profiles.
• Urinary data should be collected for a period of time equal to five times the half
life corresponding to the terminal phase of the drug concentration- time profile to
achieve 97% recovery after a single dose.
• Urinary data are valid only if the excretion of the drug or metabolite is related to
the bioavailable dose of the drug.
• Urinary data cannot be reliably used to determine bioequivalence, Cmax, tmax,
absorption rate, and duration. Theoretically data could be used for determination
of bioequivalence, but practically they will not be reliable because of the high
degree of variability that could be associated with the parameter estimation.
2. Based on acute pharmacodynamic effect: This approach may be applicable when the
drug is not intended to be delivered into the bloodstream for systemic availability. It is an
indirect measure of bioavailability in cases where the analytical method for assessing
drug concentrations in the plasma or other biological fluids cannot be developed. In such
cases a dose-response relationship must be established. This method can be used only if
the method is sensitive, accurate, and reproducible. The pharmacodynamic parameters
evaluated to assess bioavailability are the following:
a. Total area under the pharmacodynamic effect-time curve
b. Peak pharmacodynamic effect
c. Time to peak pharmacodynamic effect
3. From Well-Controlled Clinical Trials: Well-controlled clinical trials that establish
safety and efficacy of a drug product, for purposes of establishing bioavailability can be
used. However, this approach is the least accurate, sensitive, sensitive, and reproducible
approach. This approach can be used when analytical methods cannot be developed for a
particular drug.
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4. From dissolution Studies: In vitro dissolution studies are used to assess product
quality. In ideal circumstances in vitro dissolution rate should correlate with in vivo
bioavailability. A dosage form with a rapid dissolution rate is likely to have a rapid rate
of drug bioavailability in vivo. However, bioavailability is not only dependent on the
dissolution of the drug product, but also the permeability and solubility of the drug
substance. When an in vitro-in vivo correlation is available, the in vitro test can serve as
an indicator of how the product will perform in vivo.
2.7.5. Absolute and Relative Bioavailability
2.7.5.1. Absolute Bioavailability: Absolute bioavailability of a drug is the systemic
availability of the drug after extravascular administration of the drug and is measured by
comparing the area under the drug concentration-time curve after extravascular
administration to that after IV administration, provided the Kel and Vd are independent
of the route of administration. Extravascular administration of the drug comprises routes
such as oral, rectal, subcutaneous, transdermal, nasal, etc.
Absolute bioavailability is denoted as F, which is also the fraction of the dose that is
absorbed. After IV administration, the entire dose is placed into systemic circulation;
therefore, the fraction of the dose absorbed (f) or the absolute bioavailability is equal to
unity. For routes other than IV administration F≤1, absolute bioavailability is most
commonly expressed as a percentage, where an F of 1 is 100% bioavailable or an F of 0.8
is 80% bioavailable.
Absolute bioavailability can be calculated from the following equations:
Absolute bioavailability = AUC extravascular x dose i.v. / AUC i.v. x dose extravascular
2.7.5.2. Relative bioavailability: The relative bioavailability is the systemic availability
of a drug from one drug product (A) compared to another drug product (B). Relative
bioavailbilty can be calculated from the following equations:
Relative bioavailability = AUC of A / AUC of B
2.7.6. Factors affecting Bioavailability
Some of the important factors that affect bioavailability are outlined as follows:
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2.7.6.1. Gastric emptying: Although not true in all cases, increased gastric emptying
generally enhances bioavailability of orally administered drugs. Gastric emptying
depends on the following factors:
• Volume of liquid intake
• Volume of solid food intake and its fat content
• Viscosity of stomach content
• pH of the stomach
• Intake of other drugs
• Age and weight of the patients
• Physical activity of the patients taking drug
• Emotional state of the patient
• Various disease states
The variability seen in the absorption of orally administered drugs is mainly due to
different rates of gastric emptying, which are affected by the various factors listed above.
Hence, to minimize variability, bioavailability studies may be conducted under controlled
conditions, such as healthy individuals of controlled weight and age under fasted
conditions or with a controlled diet. The use of healthy subjects minimizes both inter and
intrasubject variability.
2.7.6.2. Presystemic and systemic metabolism: Presystemic metabolism, which occurs
during first- pass metabolism are commonly seen:
• First-pass metabolism: First – pass metabolism occurs when an absorbed drug
passes directly through the liver before reaching systemic circulation after oral
administration.
• Intestinal metabolism: Drug metabolizes in the intestine itself or during the
passage through the intestinal wall.
• Hydrolysis of the drug in the stomach fluids.
• Transporters such as p-glycoprotein may influence the bioavailability of a
drug.
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2.7.6.3. Complexation with other agents in the gastrointestinal tract: Formulation
factors, such as may occur with inert ingredients, the manufacturing process and /or use
of surfactants, etc.
2.7.7. Design of Bioavailability and bioequivalence studies
Both bioavailability and bioequivalence focus on measuring the absorption of the drug
into systemic circulation; hence, similar study design approaches are used to establish
bioavailability of a drug or to assess bioequivalence. Bioavailability is a comparison of
the drug product to an intravenous formulation, a solution, or a suspension, whereas
bioequivalence is a more formal comparative test that uses specified criteria for
comparisons with predetermined bioequivalence limits for evaluation.
The study design for bioequivalence mainly depends on the criteria for evaluation. Since
July 1992, the center for drug evaluation and research (CDER) has recommended the use
of the average bioequivalence criterion as published in the guidance [Statistical
procedures for bioequivalence using a standard two-treatment crossover design: FDA
guidance, 1992]. This criterion calls for a conventional nonreplicate crossover study for
evaluating bioequivalence. Recently, two new approaches have been described for
evaluating bioequivalence, which are termed the individual Bioequivalence criterion and
the population Bioequivalence criterion. The individual Bioequivalence criterion calls for
a replicate study design, wheraeas the population Bioequivalence criterion does not
involve a replicate study design, but a replicate cross-over design or parallel design,
which can also be used for this criterion [Statistical Approaches to establishing
bioequivalence, FDA guidance, 2001]. A replicate study design is one in which both the
test and the reference drug products are administered to the same individuals on two
separate occasions. The general study design considerations for conducting
bioavailability or bioequivalence studies are as follows:
• An initial pilot study with a smaller number of subjects to assess variability,
optimize sample collection time (as suitable for the immediate release and
modified release dosage forms), and other useful information.
• A conventional two-formulation, two-period, two-sequence non replicate
crossover design. This design is used for the average bioequivalence criterion. It
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is also used if a population criterion is chosen for bioequivalence comparisons.
This study is recommended for a single-dose study. A single-dose bioequivalence
study is generally more sensitive in assessing release of the drug substance from
the drug product into systemic circulation for both conventional and modified
release products. The non-replicate design is recommended b the FDA for most
orally administered immediate-release dosage forms [Bioavailability and
Bioequivalence studies of orally administered drug products-general
considerations, FDA guidance, 2000].
• A replicate-crossover study design with four periods, two sequences, and two
formulations. The FDA recommends the use of the average bioequivalence
criterion for this study design as well, [Bioavailability and Bioequivalence studies
of orally administered drug products-general considerations, FDA guidance,
2000] although this study design is not necessary when an average approach is
used to establish bioequivalence. Replicate crossover designs allow for estimation
of within-subject variances for the test (T) and Reference (R) measures and the
subject-by-formulation interaction component. The same lot of the test and
reference formulation should be used for the replicated administration. This
design is desirable for modified-release dosage forms or highly variable drug
products, and is suitable for an individual bioequivalence approach.
• A parallel design could also be used under special circumstances, for example, a
drug with a long half-life.
• The reference standard in a bioequivalence study is a formulation currently
marketed with an approved full NDA, for which there are valid scientific safety
and efficacy data. The list of reference products is provided in the orange book
[Approved drug products with therapeutic equivalence evaluations, 1999].
• The reference product is usually the innovator’s brand-name product. The total
content of the active drug substance in the test product must be within 5% of the
reference product. Usually similar routes of administration are used for the test
and reference products unless an alternative route is needed to answer specific
pharmacokinetic questions. In some cases the reference material could be a
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solution, suspension, IV product, or the clinical trial material containing the same
quantity of active drug ingredient.
• Healthy subjects are preferred as the study population and should be ≥18 years of
age. In some cases it may be useful to conduct the study in patients. A
heterogenous population would be preferable that includes males and females,
young and elderly people, and subjects from different racial groups or the targeted
age and gender if the drug product is to be specifically used in those populations.
• An adequate number of subjects should be enrolled to allow for dropouts. It is not
desirable to replace dropouts. At least 12 subjects should be included in a study.
• The highest marketed strength should be used for evaluating bioequivalence.
• The test and reference product should be administered with 240 ml of water.
• The test and reference drug products should be administered under fasting
conditions (overnight) and the fast should continue for up to 4 h after dosing.
Subjects should abstain from alcohol for 48 h prior to each study period and until
after the last sample from each period is collected. Subjects can be allowed water
as desired except for 1 h before and after drug administration.
• An adequate washout period should separate each treatment.
• Plasma and blood samples are preferred over urine and other tissue samples for
evaluating drug/ metabolite concentrations. An adequate number of samples
should be taken to characterize the absorption, distribution, and elimination
phases of the drug/metabolite accurately.
• For bioavailability studies, the parent compound or the active moiety and the
active metabolites should be measured if analytically feasible. For bioequivalence
studies, the measurement of the parent compound is desirable, unless the parent
drug levels in the plasma or serum are too low to allow reliable measurements. In
addition to measuring the parent, the measurement of the metabolite is important
when it contributes to either safety or efficacy of the drug product. The
bioequivalence criterion is applied to the parent with supportive evidence from
the metabolite measurements. Similarly, measurement of enanatiomers or
racemate may be necessary as appropriate.
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2.7.8. Pharmacokinetic information for evaluation of Bioequivalence studies: The
following pharmacokinetic information for the drug should be obtained for the
evaluation of bioequivalence studies:
• Area under the plasma concentration-time curve from zero to time t and its
log transformation.
• Area under the plasma concentration-time curve from zero to time infinity
and its log transformation.
• Peak drug concentration and its log transformation and time to peak drug
concentration.
• Elimination rate constant and half-life of the drug.
• Cmin, Cavg, and degree of fluctuation, swing and evidence of attainment of
steady state, if steady state studies are used.
2.7.9. Bioequivalence evaluation criteria
In the past 20 years the evaluation criteria for bioequivalence studies recommended
by the FDA have evolved and been revised several times. For bioequivalence
comparisons the new formulation or method of manufacture is the test product (T)
and the prior formulation or method of manufacture is the reference (R) product. To
establish bioequivalence, the difference between the bioavailability of the test product
and the reference product must be within the prespecified bioequivalence limit as
governed by the approach taken to assess bioequivalence.
The first approach that was used by the FDA to evaluate bioequivalence was the
75/75/125 Rule, which required that a test and reference ratio for 75% of the subjects
should fall between the interval of 75 to 125%. In subsequent years this approach was
replaced by the power approach, which utilized a standard t-test for testing
equivalence. The power approach consisted of testing the hypothesis of no difference
at a 0.05 level with an estimated power of 0.80 to detect a 20% difference in the
means of the test and reference.
The current evaluation criteria are based on the two one-sided test approach, also
commonly referred to as the confidence interval approach or average bioequivalence,
which determines whether the average values for the pharmacokinetic parameters
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measured after the administration of test and reference products are comparable. This
approach involves the calculation of a 90% confidence interval about the ratio of the
averages of T and R products for AUC and Cmax values. To establish
bioequivalence, the AUC and Cmax of the T product should not be less than 0.80
(80%) or greater than 1.25 (125%) of the R product based on log transformed data
(i.e., a bioequivalence limit of 80 to 125%). For some time prior to the use of log-
transformed data, the non transformed data were used to assess bioequivalence. In
1989, it was realized that log transformation of the data enables a comparison based
on the ratio of the two averages rather than the difference between the averages in an
additive manner [Schuirmann, 1989]. Moreover, most biological data correspond to a
log-normal distribution rather than to a normal distribution.
More recent proposals discussed for evaluating bioequivalence are based on
approaches termed individual Bioequivalence and population Bioequivalence. The
average bioequivalence approach focuses only on the comparison of population
averages (µT, µR) of a bioequivalence metric of interest and not on the variances of
the metric for the T and R products. The individual bioequivalence approach not only
compares the population averages (µT, µR), but also assess the within-subject
variability (σ2WT, σ2
WR) as well as the subject-by-formulation interaction (σ2D). The
population bioequivalence approach is designed to assess the total variability i.e.,
within- and between- subject variability (σ2TT, σ2
TR) of the pharmacokinetic
parameter (metric) in the population. The individual and population bioequivalence
approach allow the use of mixed scaling, which takes into account the variability of
the R product (termed as reference scaling). Reference scaling is used when the R
product is highly variable; otherwise constant scaling is used.
The bioequivalence or the evaluation of bioequivalence (BE) based on the average,
individual, and population approaches are given in Equations 1 through 3. This
criteria should be ≤ BE limit (θA, θI, θP for average, individual, and populaton
approaches, respectively) for each approach:
Average BE
(µT - µR)2≤ θA2 (1)
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Individual BE
[(µT - µR)2+ σ2D + (σ2
WT - σ2WR) ]/ σ2
WR ≤ -θI (2)
Population BE
[(µT - µR)2 + (σ2TT - σ2
TR)]/ σ2TR ≤θP (3)
2.7.10. Statistical models to assess bioequivalance
Log-transformed data are used for comparisons to represent a normal distribution of
the data. For AUC and Cmax, the log of ratio (In T/R or log T/R) between the test and
reference are used for comparisons. The arithmetic mean for the test and reference
products, geometric means, means of the logs, standard deviation of he logs, or
coefficients of variation should be calculated for individual bioequivalence approach
the subject-by –formulation interaction variance and the within-subject variance for
the T and R product should be determined
General linear model or mixed effect model procedures are performed on the
pharmacokinetics parameters AUC and Cmax to test the data for difference within
and between test and reference groups. For a general linear model, the statistical
model should include factors accounting for various sources of variability, such as
sequence, subjects, study period, and treatment or formulation depending on the study
design.
For the average bioequivalence approach, two one sided test of hypothesis at the 5%
level of significance are carried to construct 90% confidence intervals. For the
population and individual bioequivalence approach, an upper 95% confidence bound
for the population or individual criterion is estimated, which should be less than or
equal to the bioequivalence limit (i.e. θI, θP ).
2.7.11. Criteria for waiver of evidence of in vivo bioavailability or bioequivalence
studies
Under the following circumstances at the applicants request, the FDA [CFR Vol.21,
part 320; 2000] may wave the requirement for in vivo bioavailability studies for a
drug product if the drug product meets any of the following provisions:
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The following drug products that also meet the condition of containing he same active
and inactive ingredients in the same concentrations as a drug product that is the
subject of a full approved NDA can receive a waiver for in vivo evidence of
demonstrating bioavailability of the drug product:
• The drug product is a parenteral solution intended solely for administration by
injection.
• The drug product is an ophthalmic or otic solution.
The following drug products that also meet the condition of containing the same
active ingredients in the same dosage form as a drug product that is the subject of a
full approved NDA can receive a waiver for in vivo evidence of demonstrating
bioavailability of the drug product:
• The drug product is administered by inhalation as a gas, e.g., a medicinal or
inhalation anesthetic.
• The drug product is a solution for application to the skin.
• The drug product is an oral solution, elixir, syrup, tincture, or a similar solubilized
form. These products should not contain any inactive ingredient that is known to
significantly affect absorption of the active drug ingredient.
If the drug product is in the same dosage form, but in a different lower strength and
the following conditions have been met:
• The drug product is proportionally similar in its active and inactive ingredients to
another product for which the same manufacturer has obtained approval by
meeting the bioavailability requirements for a submission.
• Both drug products meet an appropriate in vitro test approved by the FDA.
• An in vivo study has been conducted on the highest strength.
These criteria could be used for immediate release tablets or capsules.
a. If the drug product is in the same dosage form, but in a higher strength, the waiver for
in vivo bioavailability will depend on:
• Clinical safety or efficacy data.
• Linear elimination kinetics over the dose range.
• Higher strength being proportionally similar to the lower strength.
• Similar dissolution profiles.
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These criteria could be used for immediate release tablets or capsules.
b. If the drug product is a modified-release dosage form, a lower strength could be
waived if the following conditions are met:
• For beaded capsules, the difference should only be in the amount of beads present
and the lower-strength capsules should have similar dissolution profiles.
• For tablets, the lower-strength tablet should be compositionally proportional to
the higher strength both should have the same drug release mechanism and similar
dissolution profiles.
c. The drug product shows an in vitro-in vivo correlation.
d. The drug product is a reformulated product that is identical, except for a different
color, flavor, or preservative that could not affect the bioavailability of the reformulated
drug product, to another drug product for which the same manufacture has demonstrated
bioavailability and obtained approval and that has an FDA approved in vitro test.
e. In vivo bioavailability requirements may be waived for a good cause that is compatible
with the protection of public health.
f. For a drug product that was approved prior to 1962 and is determined to be effective in
at least one indication in a drug efficacy study implementation (DESI) notice and is listed
not to have a potential bioequivalence problem.
g. Recently the FDA has proposed the waiver of bioequivalence studies for immediate-
release solid oral dosage forms for a class I drug substance based on the
Biopharmaceutics Classification System (highly soluble and highly permeable) and for a
rapidly dissolving product [Waiver of in vivo bioequivalence studies for immediate
release forms based on BCS, FDA guidance, 2000].
2.7.12. Limitations of Bioavailability and Bioequivalence studies
1. A crossover design may be difficult for drugs with a long elimination half-life. Three
to four elimination half-lives may extensively prolong the duration of the study in a
crossover design. In this situation a parallel design can be used for bioequivalence
studies.
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2. Highly variable drugs may require a far greater number of subjects to meet the FDA
bioequivalence criteria [Shah et al. 1996]. The variability seen in the performance of
certain drug products may be due to the inherent characteristics of the drug or due to the
drug formulation or both.
3. Certain characteristics in the biotransformation of drugs make it difficult to evaluate
the bioequivalence of such drugs. For example, for drugs that are stereoisomers with a
different rate of biotransformation and a different pharmacodynamic response, the
measurement of independent isomers may be difficult for analytical reasons.
4. Drugs that are administered by routes other than the oral route or drugs/dosage forms
that are intended for local effects have minimal systemic bioavailability. Some examples
of such drug classes are the ophthalmics, dermals, intranasal, and inhltion drug products.
Bioequivalence assessment of drugs that are insignificantly absorbed into the systemic
circulation are difficult. In some cases, for such drugs a biological marker has been
established for the assessment of bioequivalence. Examples of biological markers used
are skin blanching in the case of hydrocorticosteroids and neutralization of stomach acid
for antacids. For certain cases a pharmacodynamic end point may be more appropriate for