final project

Bioequivalence study of Rosuvastatin calcium 40mg Tablet under Fed Condition

TITLE OF THE PROJECT

“Bioequivalence Study of Rosuvastatin Calcium 40 mg Tablet in Healthy Human

Volunteers under Fed Condition”

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ABSTRACT

Purpose: To Establish Bioequivalence between two Rosuvastatin Calcium formulations (40

mg tablets) under fed condition.

Methods: The subjects received either 40 mg of the reference or of the test formulation in fed

(N=48) condition in each period. The study was conducted according to a single dose and

randomized crossover design. Blood samples were collected up to 96 hours after drug

administration. Plasma concentrations of Rosuvastatin were determined by LC-MS/MS

method. Pharmacokinetic parameters was calculated from the observed plasma concentration-

time profiles. Bioequivalence between the formulations was considering 90% confidence

interval for the ratio of means for lnCmax and lnAUC(0-t), LnAUC(0-inf) within 0.8-1.25.

Results: The 90% confidence interval for the ratio of the means for the lnCmax (92.90-

116.99), lnAUC(0-t) (95.56-116.32), LnAUC(0-inf) (95.49-114.17) was within the guideline

recommended range of bioequivalence 80-125 %

Conclusion: The results demonstrated that the test formulation is bioequivalent to the

reference formulation, as lnCmax and lnAUC fall within the recommended acceptable range.

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1. INTRODUCTION

1.1 Bioavailability and Bioequivalence

To exert an optimal therapeutic action an active moiety should be delivered to its site of

action in an effective concentration for the desired period.

Comparison of therapeutic performances of two medicinal products containing the same

active substance is a critical means of assessing the possibility of alternative use between the

innovator and any essentially similar medicinal product. Assuming that in the same subject an

essentially similar plasma concentration time course will result in essentially similar

concentrations at the site of action and thus in an essentially similar effect, pharmacokinetic

data instead of therapeutic results may be used to establish equivalence: bioequivalence.1

1.1.1 Bioavailability1,2

Bioavailability means 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 is understood to be the extent and the rate at which a substance or its active

moiety is delivered from a pharmaceutical form and becomes available in the general

circulation. It may be useful to distinguish between the "absolute bioavailability" of a given

dosage form as compared with that (100%) following intravenous administration (e.g. oral

solution vs. i.v), and the "relative bioavailability" as compared with another form

administered by the same or another non intravenous route (e.g. tablets vs. oral solution).

1.1.2 Bioequivalence

Bioequivalence means the absence of a significant difference in the rate and extent to which

the active ingredient or active moiety in pharmaceutical equivalents or pharmaceutical

alternatives becomes available at the site of drug action when administered at the same molar

dose under similar conditions in an appropriately designed study. Where there is an

intentional difference in rate (e.g., in certain extended release dosage forms), certain

pharmaceutical equivalents or alternatives may be considered bioequivalent if there is no

significant difference in the extent to which the active ingredient or moiety from each product

becomes available at the site of drug action. This applies only if the difference in the rate at

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which the active ingredient or moiety becomes available at the site of drug action is

intentional and is reflected in the proposed labeling, is not essential to the attainment of

effective body drug concentrations on chronic use, and is considered medically insignificant

for the drug. Two medicinal products are bioequivalent if they are pharmaceutically

equivalent or pharmaceutical alternatives and if their bioavailabilities after administration in

the same molar dose are similar to such degree that their effects, with respect to both efficacy

and safety, will be essentially the same.1,2

Studies to measure BA and/or establish BE of a product are important elements in support of

INDs, NDAs, ANDAs, and their supplements. As part of INDs and NDAs for orally

administered drug products, BA studies focus on determining the process by which a drug is

released from the oral dosage form and moves to the site of action. BA data provide an

estimate of the fraction of the drug absorbed, as well as its subsequent distribution and

elimination. BA can be generally documented by a systemic exposure profile obtained by

measuring drug and/or metabolite concentration in the systemic circulation over time. The

systemic exposure profile determined during clinical trials in the IND period can serve as a

benchmark for subsequent BE studies. BA and BE studies is generally applicable to non-

orally administered drug products where reliance on systemic exposure measures is suitable to

document BA and BE (e.g., transdermal delivery systems and certain rectal and nasal drug

products).

As part of INDs and NDAs for orally administered drug products, BA studies focus on

determining the process by which a drug is released from the oral dosage form and moves to

the site of action. BA data provide an estimate of the fraction of the drug absorbed, as well as

its subsequent distribution and elimination.BA can be generally documented by a systemic

exposure profile obtained by measuring drug and/or metabolite concentration in the systemic

circulation over time.

In BE studies, an applicant compares the systemic exposure profile of a test drug product to

that of a reference drug product. For two orally administered drug products to be

bioequivalent, the active drug ingredient or active moiety in the test product must exhibit the

same rate and extent of absorption as the reference drug product.3

If the test and reference products have the same galenic form and contain the same dose of the

same active ingredient(s), they are said to be bioequivalent when the profiles of the drug or

metabolite(s), or both are similar. The degree of similarity between the profiles needed to

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establish bioequivalence is determined by the appropriate statistical assessment and by

meeting standards established for the particular drug and formulations being compared.4

Bioavailability can be generally documented by a systemic exposure profile obtained by

measuring drug and/or metabolite concentration in the systemic circulation over time. The

systemic exposure profile determined during clinical trials in the early drug development can

serve as a benchmark for subsequent BE studies.

Bioequivalence studies should be conducted for the comparison of two medicinal products

containing the same active substance. The studies should provide an objective means of

critically assessing the possibility of alternative use of them. Two products marketed by

different licensees, containing same active ingredient(s), must be shown to be therapeutically

equivalent to one another in order to be considered interchangeable.5

Several test methods are available to assess equivalence, including:

Comparative bioavailability (bioequivalence) studies, in which the active drug substance

or one or more metabolites is measured in an accessible biological fluid such as plasma,

blood or urine

Comparative pharmacodynamic studies in humans

Comparative clinical trials

In-vitro dissolution tests

Both bioavailability and bioequivalence focus on the release of a drug substance from its

dosage form and subsequent absorption into the systemic circulation. For this reason, similar

approaches to measuring bioavailability should generally be followed in demonstrating

bioequivalence.5

For drugs approved elsewhere in the world and absorbed systemically, bioequivalence with

the reference formulation should be carried out wherever applicable.6

1.2 Importance of Bioequivalence Studies

In many parts of the world, medicines are protected by patents. This means no one else than

innovator (the company which originally discovered the medicine) can market the drug.

However patents are valid only for a limited period of time, the duration depends on the

country. If someone wants to sell the drug before the patent expires, they have to obtain

permission from the innovator company. But after the patent expires, anyone can market the

medicine. Such "copies" of innovator medicine is called Generic. A company that wishes to

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sell a generic version of a medicine has to prove that their formulation is as good as the

original innovator medicine. To prove this, two kinds of tests are done: First, some simple

chemical tests are done to prove the generic has the same substance in same quantity as the

innovator medicine. Second, it has to be proved that the generic medicine reaches the blood at

same extent as innovator after being administered. In other words, bioequivalence has to be

proven between the innovator medicine (called Reference formulation) and the Generic

medicine (called Test formulation). Governmental agencies carefully examine the results of

study. If they are satisfied (that the two formulations are bioequivalent), the Generic

Company may get permission to sell their formulation.

This elaborate procedure is meant to safeguard of patients using this type of medicine. Due to

this procedure, patients buying medicines can be confident that it will be effective without

regard to the company that manufactured it. The prices of these types of medicines are very

high because no one else is allowed to sell the medicine during the patent lifetime. Therefore,

bioequivalence studies benefiting mankind by lowering the overall cost of medicines.7

In bioequivalence studies, the plasma concentration time curve is used to assess the rate and

extent of absorption. Meaningful pharmacokinetic parameters and preset acceptance limits

allow the final decision on bioequivalence of the tested products. AUC, the area under the

concentration time curve, reflects the extent of exposure. Cmax, the maximum plasma

concentration or peak exposure, and the time to maximum plasma concentration, tmax, are

parameters that are influenced by absorption rate.8

Bioequivalence is an important part of an NDA in which formulation changes have been

made during and after pivotal clinical trials. Bioequivalence studies, as part of ANDA

submissions, in which a generic product is compared to a marketed, reference product, are

critical parts of the submission. Bioequivalence studies may also be necessary when

formulations for approved marketed products are modified. In general, most bioequivalence

studies depend on accumulation of pharmacokinetic (PK) data that provide concentrations of

drug in the bloodstream at specified time points following administration of the drug. These

studies are typically performed, using oral dosage forms, on volunteers who are incarcerated

(housed) during the study to ensure compliance with regard to dosing schedule as well as

other protocol requirements.9

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1.3 DyslipidemiaDyslipidemia is elevation of plasma cholesterol, triglycerides (TGs), or both, or a low HDL

level that contributes to the development of atherosclerosis. Causes may be primary (genetic)

or secondary. Diagnosis is by measuring plasma levels of total cholesterol, TGs, and

individual lipoproteins. Treatment is dietary changes, exercise, and lipid-lowering drugs.

1.4 Risk Factors: There are various risk factors which may cause Dyslipidemia as follows:

Epidemiologic, angiographic and postmortem studies have documented a causal relationship

between elevated serum cholesterol levels and the genesis of coronary heart disease.10,11

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RISK FACTORS OF DYSLIPIDEMIA

Emerging Risk Factors

Non Modifiable Risk Factors

Modifiable Risk Factors

A) Lipid risk factors Triglycerides Lipoprotein remnants Lipoprotein A Small LDL particles HDL subspecies Apolipoproteins

a) Apolipoprotein B b) Apolipoprotein A-I

B) Nonlipid risk factors Homocysteine Thrombogenic/

Hemostatic factors inflammatory markers Impaired fasting glucose

C) Subclinical atherosclerotic disease

Ankle-brachial blood pressure index (ABI)

Tests for myocardial ischemia

AgeMale SexFamily History of Premature CHD

A) Life Style Risk Factors Obesity Physical Inactivity Atherogenic Diet

B) HypertensionC) Cigarette SmokingD) Thrombogenic/ haemostatic StateE) Diabetes


Control of the modifiable risk factors is especially important in preventing premature CHD.

Observational studies suggest that modifiable risk factors account for 85% of excess risk (risk

over and above that of individuals with optimal risk-factor profiles) for premature CHD. The

presence of one or more conventional risk factors in 90% of patients with CHD belies claims

that a large percentage of CHD, perhaps as much as 50%, is not attributable to conventional

risk factors. Furthermore, these studies indicate that, when total cholesterol levels are below

160 mg/dl, CHD risk is markedly attenuated, even in the presence of additional risk factors.

This pivotal role of hypercholesterolemia in atherogenesis gave rise to the almost universally

accepted cholesterol-diet-CHD hypothesis:

Elevated plasma cholesterol levels cause CHD

Diets rich in saturated (animal) fat and cholesterol raise cholesterol levels

Lowering cholesterol levels reduces CHD risk.

Although the relationship between cholesterol, diet, and CHD was recognized nearly 50 years

ago, proof that cholesterol lowering was safe and prevented CHD death required extensive

epidemiological studies and clinical trials.12,13

Hyperlipidaemia is a major cause of atherosclerosis and atherosclerosis-associated conditions,

such as coronary heart disease (CHD), ischemic cerebrovascular disease, and peripheral

vascular disease (PVD). Although the incidence of these atherosclerosis-related events has

declined in the United States, these conditions still account for the majority of morbidity and

mortality among middle-aged and older adults. The incidence and absolute number of annual

events will likely increase over the next decade because of the epidemic of obesity and the

aging of the U.S. population. Dyslipidemias, including Hyperlipidaemia.

(Hypercholesterolemia) and low levels of high-density-lipoprotein cholesterol (HDL-C) are

major causes of increased atherogenic risk; both genetic disorders and lifestyle (sedentary

behavior and diets high in calories, saturated fat, and cholesterol) contribute to the

dyslipidemias seen in developed countries around the world.13

Hyperlipidemia (elevated levels of triglycerides or cholesterol) and reduced HDL-C levels

occur as a consequence of several interrelated factors that affect the concentrations of the

various plasma lipoproteins. These factors may be lifestyle or behavioral (e.g., diet or

exercise), genetic (e.g., mutations in a gene regulating lipoprotein levels), or metabolic (e.g.,

diabetes mellitus or other conditions that influence plasma lipoprotein metabolism).13

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Table:1 Adult Treatment Panel (ATP III) Classification of LDL, Total and HDL Cholesterol and Triglycerides (mg/dL)14

Total Cholesterol

<200 Desirable

200-239 Borderline High

≥240 High

LDL Cholesterol

<100 Optimal

100-129 Near or above optimal

130-159 Borderline High

160-189 High

≥190 Very high

HDL Cholesterol

<40 Low

≥60 High

Triglyceride

<150 Normal

150-199 Borderline high

200-499 High

≥500 Very high

The common adverse effects associated with statin therapy are relatively mild and often

transient (gastrointestinal symptoms, headache, rash). The most important adverse effects

associated with statins (atorvastatin, fluvastatin, lovastatin, pravastatin, Rosuvastatin, and

simvastatin) are asymptomatic increases in liver transaminases and myopathy.15

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2.0 OBJECTIVES OF THE STUDY

Primary objective was to assess the bioequivalence of Rosuvastatin calcium 40 mg tablet

[Torrent Pharmaceuticals Ltd., India] versus CRESTOR® 40 mg tablet containing 40 mg

Rosuvastatin Calcium [AstraZeneca LP, USA] in healthy human volunteers after

administration of single tablet of either test or reference formulation under fed condition in

each period.

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Secondary objective was to evaluate the safety of Rosuvastatin calcium 40 mg in healthy

human volunteers.

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3.0 RATIONALE OF DRUG

Rosuvastatin:

Statins are the drugs and possibly treatment of choice for LDL-cholesterol reduction; they

demonstrably reduce cardiovascular mortality. Statins inhibit hydroxymethylglutaryl CoA

(HMG-CoA) reductase, a key enzyme in cholesterol synthesis, leading to up-regulation of

LDL receptors and increased LDL clearance. They reduce LDL-cholesterol by up to 60% and

produce small increases in HDL and modest decreases in TGs. Statins also appear to decrease

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intra-arterial inflammation, systemic inflammation, or both by stimulating production of

endothelial nitric oxide and may have other beneficial effects.10

The introduction into clinical practice of statins has revolutionised the management of

dyslipidaemia and the treatment and prevention of CVD. These drugs competitively inhibit

HMG-CoA reductase, thereby reducing cholesterol synthesis in the liver, which leads to an

increased expression if hepatic LDL receptors and greater uptake of LDL cholesterol from

plasma. Production of very low density lipoprotein (VLDL), the precursor of LDL, is

decreased, the net effect being dose dependent reductions in LDL cholesterol of 20–60%,

accompanied by lesser reductions in plasma triglyceride and a small rise in HDL cholesterol.12

3.1 Clinical Efficacy16,17

The lowering of total cholesterol, LDL-cholesterol and apolipoprotein B has been shown to

reduce the risk of cardiovascular events and mortality. Mortality and morbidity studies with

Rosuvastatin have not yet been completed. Rosuvastatin is effective in adult patient

populations with hypercholesterolemia, with and without hypertriglyceridaemia, regardless of

race, sex, or age and in special populations such as diabetics, or patients with familial

hypercholesterolemia. In a large study of patients with heterozygous familial

hypercholesterolemia, 435 subjects were given Rosuvastatin from 20 mg to 80 mg in a force-

titration design. All doses of Rosuvastatin showed a beneficial effect on lipid parameters and

treatment to target goals. Following titration to 40 mg (12 weeks of treatment), LDL-C was

reduced by 53%. 33% of patients reached EAS guidelines for LDL-C levels (<3 mmol/l). In a

force-titration, open label trial, 42 patients with homozygous familial Hypercholesterolemia

were evaluated for their response to Rosuvastatin 20 - 40 mg. In the overall population, the

mean LDL-C reduction was 22%. In clinical studies with a limited number of patients,

Rosuvastatin has been shown to have additive efficacy in lowering triglycerides when used in

combination with fenofibrate and in increasing HDL-C levels when used in combination with

niacin.

According to American Heart Association /American College of Cardiology guidelines, In

the last few years, a great deal of effort has been expended to come to an agreement on

therapeutic strategies for coronary artery disease. In 1995, the American College of

Cardiology (ACC) and the American Heart Association (AHA) published a consensus

statement based on the National Cholesterol Education Program (NCEP) II 1995 guidelines.

This outlined pharmacologic approaches to therapy for patients with coronary and other

vascular diseases. Lipid targets were: LDL-C <100 mg/dL, high-density lipoprotein

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cholesterol (HDL-C) >35 mg/dL, and triglycerides <200 mg/dL. For patients with elevated

LDL-C (>130 mg/dL) and triglycerides <200 mg/dL, treatment with a statin, resin, or niacin

recommended. If triglyceride levels were 200–400 mg/dL, the recommendation was therapy

with either a statin or niacin. For those with triglycerides >400 mg/dL, a combination therapy

with niacin ± fibrate ± statin was to be considered. For patients with HDL-C <35 mg/dL, diet,

exercise, smoking cessation, and pharmacologic treatment were recommended.

Table: 2 Recommendations for drug Treatment of Dyslipidemia12

Type First choice If refractory

Hypercholesterolemia Statin Add cholesterol absorption inhibitor, Bile acid sequestrant, or nicotinic acid

Hypertriglyceridaemia Fibrate Add nicotinic acid or ω3 fatty acids

Mixed Hyperlipidaemia Statin Substitute or add fibrate (not gemfibrizil+ statin)

Low HDL cholesterol Statin Substitute or add fibrate or nicotinic acid

Until recently Atorvastatin was the most effective statin available for decreasing LDL when

given in doses of 10– 80 mg daily. However, Rosuvastatin, is even more effective than

atorvastatin in lowering LDL cholesterol over its licensed dose range of 10–40 mg, although

there was no significant difference between Rosuvastatin 40 mg and atorvastatin 80 mg in this

respect

Fig 1-Comparative LDL lowering efficacy of Rosuvastatin, atorvastatin, simvastatin, and pravastatin.12

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To reduce the systemic effects of statins, a high liver extraction, enterohepatic circulation and

a low degree of metabolism are considered to be preferable drug properties and rat studies

have shown that Rosuvastatin is selectively distributed into the liver. The absolute oral

bioavailability and hepatic extraction is estimated to be 20.1% and 63%, respectively, despite

a low degree of metabolism both in vitro and in vivo.18

The polar methyl sulfonamide moiety of Rosuvastatin confers relative hydrophilicity to the

molecule. Consistent with its relatively hydrophilic character, Rosuvastatin exhibits minimal

metabolism via the cytochrome P450 (CYP) system, including little or no metabolism via

CYP 3A4, the isoenzyme implicated in a wide variety of drug /drug interactions.

Although statins share a common HMG-like side chain, this statin ‘pharmacophore’ is linked

to additional groups that differ with respect to ring structure and substituents between the

different statin molecules. These differences affect pharmacologic properties of the

compounds, including

Affinity for the active site of HMG-CoA reductase

Rates of entry into hepatic and non-hepatic tissues

Availability in the systemic circulation for uptake into non-hepatic tissues

Routes and modes of metabolic transformation and elimination.

The pharmacology of an ‘ideal’ statin would include high affinity for the enzyme active site,

marked selectivity of uptake into hepatic compared with non-hepatic cells, low systemic

availability of active inhibitory equivalents, and relatively prolonged duration of effect. Thus,

the ideal statin would maximize the Pharmacodynamic activity in the liver and minimize the

inhibitory activity outside the liver, particularly in some vulnerable tissues, such as skeletal

muscle. An additional advantage would be a low risk of undesirable interactions with other

drugs as a result of utilization of common metabolic pathways. Such a combination of

pharmacologic properties should lead to a statin with an improved clinical profile. On the

basis of the above criteria, Rosuvastatin represents a step forward in efforts to optimize the

pharmacologic properties of the statin class. Consistent with the above, Rosuvastatin has been

shown to produce large reductions in low-density lipoprotein (LDL) cholesterol that exceed

those achieved with other available statins and marked improvements in other lipid measures

while maintaining a safety profile consistent with that of other available statins.

Rosuvastatin has the greatest number of binding interactions with HMG-CoA reductase. In

accord with these binding characteristics, Rosuvastatin exhibits a high affinity for the active

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site of HMG CoA reductase, with an inhibition constant (Ki) of approximately 0.1 nM.

Studies in a purified cloned catalytic fragment of human HMG-CoA reductase showed that

Rosuvastatin had a numerically lower 50% inhibitory concentration (IC50) value (5 nM) than

Atorvastatin (8 nM), Cerivastatin (10 nM), Simvastatin (11 nM), Fluvastatin (28 nM) and

Pravastatin (44 nM) statistically significant compared with Simvastatin, Fluvastatin, and

Pravastatin. In particular, Rosuvastatin is about 8-fold more potent than Pravastatin.

A prolonged elimination half-life can constitute a relative advantage for a statin, in that it can

ensure maintained inhibition of the liver enzyme during the dosing interval and maximal

associated upregulation of hepatic LDL receptors. Of available statins, Rosuvastatin has the

longest elimination half-life, approximately 20 h compared with 14 h for Atorvastatin and 1-2

h for Fluvastatin, Pravastatin, and Simvastatin (2-3 h for Cerivastatin). In clinical trials in

hypercholesterolemic patients, Rosuvastatin has been shown to reduce LDL cholesterol and

improve other elements of the lipid profile significantly more than atorvastatin, pravastatin,

and simvastatin and to exhibit a safety profile similar to that of other available statins. 19

The efficacy of Rosuvastatin across its dose range of 10 to 40 mg is superior to that of other

statins across their dose range, although the safety is similar, and thus, Rosuvastatin has a

favorable benefit–risk profile across this dose range.20

Table: 3 Outcomes of LDL-Cholesterol Lowering Therapy in CHD21

Intervention No. of Trials

No. Treated

Mean Cholesterol

reduction(%)

CHD incidence (%) change

CHD Mortality

(%)change

Surgery 1 421 22 -43 -30

Sequestrants 3 1,992 9 -21 -32

Diet 6 1,200 11 -24 -21

Statins 12 17,405 20 -30 -29

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4.0 REVIEW OF LITERATURE

4.1 Biopharmaceutics and Pharmacokinetics

4.1.1 Biopharmaceutics

Biopharmaceutics is defined as the study of factors influencing the rate and amount of drug

that reaches the systemic circulation and the use of this information to optimize the

therapeutic efficacy of the drug products. The process of movement of drug from its site of

administration to the systemic circulation is called as absorption.

After a drug is introduced into a biological system, it is subjected to a number of processes

whose rates control the concentration of drug in the elusive region known as the “site of

action,” thus affecting its onset, its duration of action and the intensity of the biological

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response. Some knowledge of these rate processes governing the fate of a drug is necessary

for a full understanding of the observed pharmacological activity of the drug.23

The Biopharmaceutics Classification System (BCS) is a scientific framework for classifying

drug substances based on their aqueous solubility and intestinal permeability. When combined

with the dissolution of the drug product, the BCS takes into account three major factors that

govern the rate and extent of drug absorption from IR solid oral dosage forms: dissolution,

solubility, and intestinal permeability. According to the BCS, drug substances are classified as

follows:36

Class 1: High Solubility – High Permeability

Class 2: Low Solubility – High Permeability

Class 3: High Solubility – Low Permeability

4.1.1.1 SolubilityThe solubility class boundary is based on the highest dose strength of an IR product that is the

subject of a biowaiver request. A drug substance is considered highly soluble when the

highest dose strength is soluble in 250 ml or less of aqueous media over the pH range of 1-

7.5. The volume estimate of 250 ml is derived from typical BE study protocols that prescribe

administration of a drug product to fasting human volunteers with a glass (about 8 ounces) of

water.36

4.1.1.2 PermeabilityThe permeability class boundary is based indirectly on the extent of absorption (fraction of

dose absorbed, not systemic BA) of a drug substance in humans and directly on

measurements of the rate of mass transfer across human intestinal membrane. Alternatively,

nonhuman systems capable of predicting the extent of drug absorption in humans can be used

(e.g., in vitro epithelial cell culture methods). In the absence of evidence suggesting instability

in the gastrointestinal tract, a drug substance is considered to be highly permeable when the

extent of absorption in humans is determined to be 90% or more of an administered dose

based on a mass balance determination or in comparison to an intravenous reference dose.36

4.1.1.3 A Waiver of In Vivo BA/BE StudiesThe drug substance for which a waiver is being requested should be highly soluble and highly

permeable.

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An immediate release drug product should be rapidly dissolving. Excipients used in the

dosage form should have been used previously in FDA approved IR solid dosage forms. The

quantity of excipients in the IR product should be consistent with their intended function.

Large quantities of certain excipients, such as surfactants like sodium lauryl sulfate, may be

problematic.

Additional considerations for requesting a waiver are:

Stability of the drug in gastrointestinal tract

Evaluation of excipients

Exceptions like sublingual or buccal tablets, for which a waiver can not be requested.

Waiver is not applicable for narrow therapeutic index drug.36

4.1.2 Pharmacokinetics

Pharmacokinetics is defined as the study of time courses of the drug ADME (Absorption,

Distribution, Metabolism and Excretion) and their relation to its therapeutic and toxic effects

of the drug. The use of pharmacokinetic principles in optimizing the drug dosage to suit

individual patient needs and achieving maximum therapeutic utility is called as “clinical

pharmacokinetics”.23

4.1.2.1 Plasma drug concentration Time profile22

A direct relationship exists between the concentrations of drug at the biophase (site of action)

and the concentration of drug in plasma.

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Fig 2-Pharmacokinetic profile for different routes of drug administration

A typical plasma drug concentration time curve obtained after a single oral dose and showing

various pharmacokinetic and pharmacodynamic parameters is depicted in Figure 3. Such a

profile can be obtained by measuring the concentration of drug in plasma samples taken at

various intervals of time after administration of a dosage form and plotting the concentration

of drug in plasma (Y-axis) versus the corresponding time at which the plasma sample was

collected (X-axis).

Fig 3: A typical plasma drug concentration time curve obtained after a single oral dose

20

Rosuvastatin Calcium 40 mg


4.1.2.2 Pharmacokinetic Parameters

The three important pharmacokinetic parameters that describe the plasma level-time curve

and useful in assessing the bioavailability of a drug from its formulation are;22,23

1) Peak plasma concentration (Cmax)

The point of maximum concentration of drug in plasma is called as the peak and the

concentration of drug at peak is known as peak plasma concentration. It is also called as peak

height concentration and maximum drug concentration. Cmax is expressed in mcg/mL. The

peak level depends upon the administered dose and rate of absorption and elimination. The

peak represents the point of time when administration rate equals elimination rate of drug.

The portion of curve to the left of peak represents absorption phase. i.e. when the rate of

absorption is greater than the rate of elimination. The section of right of peak generally

represents elimination phase i.e. when the rate of elimination exceeds rate of absorption. Peak

concentration is often related to the intensity of pharmacologic response and should ideally be

above minimum effective concentration (MEC) but less than the maximum safe concentration

(MSC).

2) Time of peak concentration (Tmax)

The time for drug to reach peak concentration in plasma (after extravascular administration) is

called as the time of peak concentration. It is expressed in hours and is useful in estimating

the rate of absorption. Onset time and onset of action are dependent upon Tmax. The

parameter is of particular importance in assessing the efficacy of drugs used to treat acute

conditions like pain and insomnia which can be treated by a single dose.

3) Area under the curve (AUC)

It represent the total integrated area under the plasma level-time profile and expresses the total

amount of drug that comes into the systemic circulation after its administration. AUC is

expressed in mcg/mL×hours. It is the most important parameter in evaluating the

bioavailability of a drug from its dosage form as it represents the extent of absorption. AUC is

also important for drugs that are administered repetitively for the treatment of chronic

conditions like asthma or epilepsy. The concentration of the drug in plasma and hence the

onset of action, and the intensity and duration of response depend upon the bioavailability of

the drug from its dosage form.

4.2 Bioavailability

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Bioavailability is defined as the rate and extent of drug absorption.

The therapeutic effectiveness of a drug depends upon the ability of the dosage form to deliver

the medicament to its site of action at a rate and amount sufficient to elicit the desired

pharmacologic response. This attribute of the dosage form is referred to as physiological

availability, biologic availability or simply bioavailability. For most drugs, the pharmacologic

response can be related directly to the plasma levels. Thus, the term bioavailability is defined

as the rate and extent (amount) of absorption of unchanged drug from its dosage form. It is an

absolute term.

The rate or rapidity with which a drug is absorbed is an important consideration when a rapid

onset of action is desired as in the treatment of acute conditions such as asthma attack, pain,

etc. A slower absorption rate is however desired when the aim is to prolong the duration of

action or to avoid the adverse effects. On the other hand, extent of absorption is of special

significance in the treatment of chronic conditions like hypertension, epilepsy, etc

If the size of the dose to be administered is same, then bioavailability of a drug from its

dosage form depends upon 3 major factors:

1. Pharmaceutical factors related to physicochemical properties of the drug and characteristics

of the dosage form.

2. Patient related factors

3. Route of administration

The influence of route of administration on drug’s bioavailability is generally in the following

order: parenteral >oral >rectal >topical with few exceptions. Within the parenteral route,

intravenous injection of a drug results in 100% bioavailability as the absorption process is

bypassed. However, for reasons of stability and convenience, most drugs are administered

orally. In such cases, the dose available to the patient, called as the bioavailable dose, is often

less than the administered dose. The amount of drug that reaches the systemic circulation (i.e.

extent of absorption) is called as systemic availability or simply availability. The term

bioavailable fraction F refers to the fraction of administered dose that enters the systemic

circulation.23

4.2.1 Absolute bioavailability25

22

F = Bioavailable Dose

Administered Dose


Absolute bioavailability compares the bioavailability (estimated as area under the curve, or

AUC) of the active drug in systemic circulation following non-intravenous administration

(i.e., after oral, rectal, transdermal, subcutaneous administration), with the bioavailability of

the same drug following intravenous administration. It is the fraction of the drug absorbed

through non-intravenous administration compared with the corresponding intravenous

administration of the same drug. The comparison must be dose normalized if different doses

are used; consequently, each AUC is corrected by dividing the corresponding dose

administered.

In order to determine absolute bioavailability of a drug, a pharmacokinetic study must be

done to obtain a plasma drug concentration vs. time plot for the drug after both intravenous

(IV) and non-intravenous administration. The absolute bioavailability is the dose-corrected

area under curve (AUC) non-intravenous divided by AUC intravenous. For example, the

formula for calculating F for a drug administered by the oral route (po) is given below.

F =[AUC]po

[AUC]i.v

[D]i.v

[D]po

Therefore, a drug given by the intravenous route will have an absolute bioavailability of 1

(F=1) while drugs given by other routes usually have an absolute bioavailability of less than1.

4.2.2 Relative bioavailability25

This measures the bioavailability (estimated as area under the curve, or AUC) of a certain

drug when compared with another formulation of the same drug, usually an established

standard, or through administration via a different route. When the standard consists of

intravenously administered drug, this is known as absolute bioavailability.

Relative Bioavailability =[AUC]A

[AUC]B

[D]B

[D]A

4.2.3 Objective of Bioavailability studies23

1. Bioavailability studies are important in a suitable dosage form for a new drug entity.

2. Determination of influence of Excipients, patient related factors and possible interaction

with other drugs on the efficiency of absorption.

3. Development of new formulations of the existing drugs.

23


4. Control of quality of a drug product during the early stages of marketing in order to

determine the influence of processing factors, storage and stability on drug absorption.

4.2.4 Types of Bioavailability-Bioequivalence Studies22,23,24

1. Study to evaluate the absolute bioavailability of an oral, topical, intramuscular, or any

other dosage form. Ideally, the test dosage form should be compared with an intravenous

reference dose. In reality, however, a suitable intravenous form may not be readily available,

and the test dosage form is usually compared, instead, with an oral solution or suspension to

determine if the former would be adequate for subsequent clinical studies. Normally, the

study is conducted in 6-12 subjects using a single dose crossover design.

2. Dose proportionality study to determine if bioavailability parameters [i.e., peak

concentration (Cmax) and area under concentration-time curve (AUC)] are linear over the

proposed dose range to be used in medical practice. Oral doses usually are given as a solution

or suspension covering the therapeutic range for a single dose and tested using a three-way

crossover design (low, mid, and high dose) in 12-18 subjects.

3. Intra/intersubject variability study to determine what the variability of bioavailability

parameters are at any one dose level. Oral doses at one dose level are usually given as a

solution or suspension in a mock three-way crossover design.

4. Dosage form(s) study to determine if that used during clinical trials is bioequivalent to that

proposed for marketing. This is normally a single dose crossover study evaluating the highest

strength of the proposed marketed dosage form. The number of subjects to be used is

dependent on available information on dose proportionality and inter- and intrasubject

variability.

5. Dosage form proportionality study to determine if equipotent drug treatments

administered as different dose strengths of the market form produce equivalent drug

bioavailability. Normally, multiple strengths are evaluated by bracketing (i.e., studying the

lowest and highest strengths at the same dose level in a single dose crossover design). The

number of subjects again is based on dose proportionality and inter- and intrasubject

variability of the drug.

6. Effect of various type of intervention studies to examine the effects of, for example, food

and concomitant medication on bioavailability parameters. These are normally single and

multiple dose studies conducted using the dosage form proposed for marketing.

24


7. Bioequivalence study needed as a result of changes in the formulation or manufacturing

process (i.e., to show that the old and the new product are bioequivalent).

8. ANDA bioequivalence studies conducted for the purpose of filing an abbreviated new

drug application (ANDA). The goal is to show that a generic drug is bioequivalent to the

innovator's product in order to make claims of therapeutic equivalence. The three important

pharmacokinetic parameters that describe the plasma level-time curve and useful in assessing

the bioavailability of a drug from its formulation are:

1. Peak Plasma Concentration (Cmax)

2. Time of Peak Concentration (tmax)

3. Area Under the Curve (AUC)

4.2.5 Assessment of Bioequivalence5

Several test methods are available to assess equivalence, including:

Comparative bioavailability (bioequivalence) studies, in which the active drug substance

or one or more metabolites is measured in an accessible biological fluid such as plasma,

blood or urine

Comparative pharmacodynamic studies in humans

Comparative clinical trials

In-vitro dissolution tests

4.2.6 Estimation of Bioavailability23

The methods useful in quantitative evaluation of bioavailability can be broadly divided into

two categories-pharmacokinetic methods and pharmacodynamic methods.

A. Pharmacokinetic methods

These are very widely used and based on the assumption that the pharmacokinetic profile

reflects the therapeutic effectiveness of a drug.

Thus, these are indirect methods. The two major pharmacokinetic methods are:

I. Plasma level-time studies.

II. Urinary excretion studies.

Plasma level- time studies

25


Unless determination of plasma drug concentration is difficult or impossible, it is the most

reliable method and method of choice in comparison to urine data. The method is based on

the assumption that two dosage forms that exhibit superimposable plasma level time profiles

in a group of subjects should result in identical therapeutic activity. With single dose study,

the method requires collection of serial blood samples for a period of 2 to 3 biological half-

lives after drug administration, their analysis for drug concentration and making a plot of

concentration versus corresponding time of sample collection to obtain the plasma level-time

profile. With i.v. dose, sampling should start within 5 minutes of drug administration and

subsequent samples taken at 15 minute intervals. To adequately describe the disposition

phase, at least 3 samples points should be taken if the drug follows one-compartment kinetics

and 5 to 6 points if it fits two-compartment model. For oral dose at least 3 point should be

taken on the ascending part of the curve for accurate determination of Ka. The points for

disposition or descending phase of the cure must be taken in a manner similar to that for i.v.

dose. The three parameters of plasma level-time studies which are considered important for

determining bioavailability are:

1. Cmax: The peak plasma concentration that gives an indication whether the drug is

sufficiently absorbed systemically to provide a therapeutic response.

2. Tmax: The peak time that gives an indication of the rate of absorption.

3. AUC: The area under the plasma level-time curve that gives a measure of the extent of

absorption or the amount of drug that reaches the systemic circulation.

The extent of bioavailability can be determined by following equation:

F =[AUC]oral

[AUC]i.v

[D]i.v

[D]oral

F =[AUC]test

[AUC]std

[D]std

[D]test

Where D stands for dose administered and subscripts i.v and oral indicates the route of

administration. Subscripts test and std. indicate the test and the standard dose of the same

drug to determine relative availability.

Urinary excretion studies

26


This method of assessing bioavailability is based on the principle that the urinary excretion of

unchanged drug is directly proportional to the plasma concentration of drug. Thus, even if a

drug is excreted to some extent (at least 10 to 20%) in the urine, bioavailability can be

determined. The study is particularly useful for drugs extensively excreted unchanged in the

urine – for example, certain thiazide diuretics and sulfonamides and for drugs that have urine

as the site of action. - For example, urinary antiseptics such as nitrofurantoin and hexamine.

Concentration of metabolites excreted in urine is never taken into account in calculations

since a drug may undergo presystemic metabolism at different stages before being absorbed.

The method involves collection of urine at regular intervals for a time span equal to 7

biological half lives, analysis of unchanged drug in the collected sample and determination of

the amount of drug excretion in each interval and cumulative amount excreted. At each

sample collection, total emptying of the bladder is necessary to avoid errors resulting from

addition of residual amount to the next urine sample. Frequent sampling is also essential in

the beginning in order to compute correctly the rate of absorption. The three major parameters

examined in urinary excretion rate obtained with a single oral study are:

1. (dXu/dt) max: The maximum urinary excretion rate, it is obtained from the peak of plot

between rate of excretion versus midpoint time of urine collection period. It is analogous to

the Cmax derived from plasma level studies since the rate of appearance of drug in the urine

is proportional to its concentration in systemic circulations. Its value increases as the rate of

and /or extent of absorption increases.

2. (tu)max: The time for maximum excretion rate, it is analogous to the tmax of plasma level

data. Its value decreases as the absorption rate increases.

3. Xu: The cumulative amount of drug excreted in the urine, it is related to the AUC of

plasma level data and increases as the extent of absorption increases. The extent of

bioavailability is calculated from equations given below:

F =[Xinf]oral

[Xinf]i.v

[D]i.v

[D]oralF =

[Xinf]test

[Xinf]std

[D]std

[D]test

B. Pharmacodynamic methods

These methods are complementary to pharmacokinetic approaches and involve direct

measurement of drug effect on a pathophysiologic process as a function of time. The two

pharmacodynamic methods involve determination of bioavailability from:

27


Acute pharmacologic response.

Therapeutic response.

Acute pharmacological response

When bioavailability measurement by pharmacokinetic methods is difficult, inaccurate or

nonreproducible, an acute pharmacologic effect such as change in ECG or EEG readings,

pupil diameter, etc. is related to the time course of a given drug. Bioavailability can then be

determined by construction of pharmacologic effect-time curve as well as dose response

graphs. The method requires measurement of responses for at least 3 biological half-lives of

the drug in order to obtain a good estimate of AUC (Area under Curve). A disadvantage of

this method is that the pharmacologic response tends to be more variable and accurate

correlation between measured response and drug available from the formulation is difficult.

Moreover, the observed response may be due to an active metabolite whose concentration is

not proportional to the concentration of parent drug responsible for the pharmacologic effect.

Therapeutic response

Theoretically the most definite, this method is based on observing the clinical response to a

drug formulation given to patients suffering from disease for which it is intended to be used.

A major drawback of this method is that quantitation of observed response is too improper to

allow for reasonable assessment of relative bioavailability between two dosage forms of the

same drug.

4.3 Various Equivalence Studies23

4.3.1 Equivalence: It is the relative term that compared drug products with respect to a

specific characteristic or function or to a defined set of standards. There are several types of

equivalences.

4.3.2 Bioequivalence: The absence of a significant difference in the rate and extent to

which the active ingredient or active moiety in pharmaceutical equivalents or pharmaceutical

alternatives become available at the site of drug action when administered at the same molar

dose under similar conditions in an appropriately designed study.2

It is commonly observed that there are several formulations of the same drug, in the same

dose, in a similar dosage form and meant to be given by the same route. Substitution of one

product for another can be made provided they are equally effective therapeutically as the

28


standard accepted. In order to ensure clinical performance of such drug products,

bioequivalence studies should be performed.

When statistically differences are observed in the two or more drug products,

bioinequivalence is indicated

4.3.3 Chemical equivalence: It indicates that two or more drug products contain the

same labeled chemical substance as an active ingredient in the same amount.

4.3.4 Pharmaceutical Equivalence: This term implies that two or more drug products

are identical in strength, quality, purity, content, uniformity and disintegration and dissolution

characteristics; they may however differ in containing different excipients.

4.4 General recommendations for a standard BE study based on pharmacokinetic

measurements is provided as mentioned below:3

For both replicate and non-replicate, in vivo pharmacokinetic BE studies, the following

general approaches are recommended, recognizing that the elements can be adjusted for

certain drug substances and drug products.

4.4.1 Regulatory requirements

The test or reference products can be administered with about 8 ounces (240 milliliters) of

water to an appropriate number of subjects under fasting conditions, unless the study is a

food-effect BA and BE study.

Generally, the highest marketed strength can be administered as a single unit. If warranted

for analytical reasons, multiple units of the highest strength can be administered,

providing the total single-dose remains within the labeled dose range.

An adequate washout period (e.g., more than 5 half lives of the moieties to be measured)

would separate each treatment.

The lot numbers of both test and reference listed products and the expiration date for the

reference product would be stated. The drug content of the test product cannot differ from

that of the reference listed product by more than 5 percent. The sponsor can include a

statement of the composition of the test product and, if possible, a side-by-side

comparison of the compositions of test and reference listed products. Samples of the test

and reference listed product must be retained for 5 years.

Before and during each study phase, it is recommended that subjects

29


o Be allowed water as desired except for 1 hour before and after drug administration.

o Be provided standard meals not less than 4 hours after drug administration.

o Abstain from alcohol for 24 hours before each study period and until after the

sample from each period is collected.

4.4.2 Sample collection and sampling times

It is recommended that under normal circumstances, blood, rather than urine or tissue, be

used. In most cases, drug, or metabolites are measured in serum or plasma. However, in

certain cases, whole blood may be more appropriate for analysis. It is recommended that

blood samples be drawn at appropriate times to describe the absorption, distribution, and

elimination phases of the drug.

For most drugs, it is recommended that 12 to 18 samples, including a pre-dose sample, be

collected per subject per dose. This sampling can continue for at least three or more

terminal half lives of the drug. The exact timing for sample collection depends on the

nature of the drug and the input from the administered dosage form.

The sample collection can be spaced in such a way that the maximum concentration of the

drug in the blood (Cmax) and terminal elimination rate constant (λz) can be estimated

accurately. At least three to four samples can be obtained during the terminal log-linear

phase to obtain an accurate estimate of (λz) from linear regression. It is recommended that

the actual clock time when samples are drawn as well as the elapsed time related to drug

administration be recorded.

4.4.3 Subjects with predose plasma concentrations

If the predose concentration is ≤ 5 percent of Cmax value in that subject, the subject’s data

without any adjustments can be included in all pharmacokinetic measurements and

calculations. It is recommended that if the pre-dose value is > than 5 percent of Cmax, the

subject be dropped from all BE study evaluations.

4.4.4 Data deletion due to vomiting

It is recommended that data from subjects who experience emesis during the course of

a BE study for immediate-release products be deleted from statistical analysis if

vomiting occurs at or before 2 times median Tmax. In the case of modified-release

30


products, the data from subjects who experience emesis any time during the labeled

dosing interval can be deleted.

The following pharmacokinetic information is recommended for submission:

Plasma concentrations and time points

Subject, period, sequence, treatment

AUC0-t, AUC0-inf., Cmax, Tmax, λz, and t1/2

Intersubject, intrasubject, and/or total variability, if available

Cmin (concentration at the end of a dosing interval), Cav (average concentration

during a dosing interval), degree of fluctuation [(Cmax-Cmin)/Cav], and swing

[(Cmax-Cmin)/Cmin] if steady-state studies are employed.

In addition, it is recommended that the following statistical information be provided

for AUC0-t, AUC0-inf., and Cmax:

Geometric mean

Arithmetic mean

Ratio of means

Confidence intervals

It also recommends that logarithmic transformation be provided for measures used for

BE demonstration.

4.4.5 Rounding off of confidence interval values

It is recommended that confidence interval (CI) values not be rounded off; therefore, to

pass a CI limit of 80 to 125, the value would be at least 80.00 and not more than 125.00.

4.4.6 Study Population

It is recommended that, unless otherwise indicated by a specific guidance, subjects

recruited for in vivo BE studies be 18 years of age or older and capable of giving

informed consent. It is recommended that if the drug product is intended for use in both

sexes, the sponsor should attempt to include similar proportions of males and females in

the study.

4.5 Types of Study Design2626

31


I. Single-dose study Design

Single-dose study should be crossover in design, unless a parallel design or other design is

more appropriate for valid scientific reasons, and should provide for a drug elimination

period.2

II. Multiple-dose study Design2,5

In selected circumstances it may be necessary for the test product and the reference material

to be compared after repeated administration to determine steady-state levels of the active

drug ingredient or therapeutic moiety in the body.

A multiple-dose study may be required to determine the bioavailability of a drug product in

the following circumstances:

There is a difference in the rate of absorption but not in the extent of absorption.

There is excessive variability in bioavailability from subject to subject.

The concentration of the active drug ingredient or therapeutic moiety, or its

metabolite(s), in the blood resulting from a single dose is too low for accurate

determination by the analytical method.

Where the drug has a long terminal elimination half-life and blood concentrations after

a single dose cannot be followed for a sufficient time.

Where assay sensitivity is inadequate to follow the terminal elimination phase for an

adequate period of time.

For drugs, which are so toxic that ethically they should only be administered to

patients for whom they are a necessary part of therapy, but where multiple dose

therapy is required, e.g. many cytotoxics.

For modified-release products where it is necessary to assess the fluctuation in plasma

concentration over a dosage interval at steady state.

For those drugs which induce their own metabolism or show large intra- individual

variability.

The drug product is an extended release dosage form.

32


For combination products where the ratio of plasma concentration of the individual

drugs is important.

For drugs that exhibit non-linear (i.e., dose- or time- dependent) pharmacokinetics.

Where the drug is likely to accumulate in the body.

I. Non-replicated Designs

A conventional non-replicated design, such as the standard two-formulation, two-period,

two-sequence crossover design, can be used to generate data where an average or

population approach is chosen for BE comparisons. Under certain circumstances, parallel

designs can also be used.

II. Replicated Crossover Designs

Replicated crossover designs can be used irrespective of which approach is selected to

establish BE, although they are not necessary when an average or population approach is

used. Replicated crossover designs are critical when an individual BE approach is used to

allow estimation of within-subject variances for the T and R measures and the subject-by

formulation interaction variance component. The following four-period, two-sequence,

two-formulation design is recommended for replicated BE studies.

Periods

1 2 3 4

Sequence 1 T R T R

2 R T R T

For this design, the same lots of the T and R formulations should be used for the replicated

administration. Each period should be separated by an adequate washout period.

Other replicated crossover designs are possible. For example, a three-period design, as shown

below, could be used. A greater number of subjects would be encouraged for the three-period

design compared to the recommended four-period design to achieve the same statistical power

to conclude BE.

Periods

1 2 3

33


Sequence 1 T R T

2 R T R

III. Food Effect Study Design:5,37

Food-effect BA studies:

Randomized, balanced, single-dose, two-treatment (Fed vs. Fasting), two-period, two

sequence crossover design for studying the effects of food on the BA of either an immediate

release or a modified-release drug product. The formulation to be tested should be

administered on an empty stomach (fasting condition) in one period and following a test meal

(fed condition) in the other period.

Fed BE Studies:

A similar, two-treatment, two-period, two sequence crossover design for a fed BE study

except that the treatments should consist of both test and reference formulations administered

following a test meal (fed condition). An adequate washout period should separate the two

treatments in food-effect BA and fed BE studies.

Generally a single dose study should be conducted after an overnight fast (at least 10 hrs),

with subsequent fast of 4 hrs following dosing.

For multiple dose fasting state studies, when an evening dose must be given, two hours of

fasting before and after the dose is considered acceptable. However, when it is recommended

that the study drug be given with food or where the dosage form is a modified release product,

fed state studies need to be carried out in addition to the fasting state studies. Fed state studies

are also required when fasting state studies make assessment of Cmax and Tmax difficult.

Studies in the fed state require the consumption of a high-fat breakfast before dosing. Such a

break fast must be designed to provide 950 to 1000 KCals. At least 50% of these calories

must come from fat, 15 to 20% from proteins and the rest from carbohydrates. The vast ethnic

and cultural variations of the Indian subcontinent preclude the recommendation of any single

standard high fat break fast.

4.5.2 Sample Size and Dropouts:

34


A minimum number of specified evaluable subjects should be included in any BE study.

When an average BE approach is selected using either nonreplicated or replicated designs,

methods appropriate to the study design should be used to estimate sample size. The number

of subjects for BE studies based on either the population or individual BE approach can be

estimated by simulation if analytical approaches for estimation are not available. Sponsors

should enter a sufficient number of subjects in the study to allow for dropouts.

Because replacement of subjects during the study could complicate the statistical model and

analysis, dropouts generally should not be replaced. Sponsors who wish to replace dropouts

during the study should indicate this intention in the protocol. The protocol should also state

whether samples from replacement subjects, if not used, will be assayed. If the dropout rate is

high and sponsors wish to add more subjects, a modification of the statistical analysis may be

recommended. Additional subjects should not be included after data analysis unless the trial

was designed from the beginning as a sequential or group sequential design.

4.6 Data Analysis26

4.6.1 Average Bioequivalence:

a. Overview

Parametric (normal-theory) methods are recommended for the analysis of log transformed BE

measures. Due to the nature of normal-theory confidence intervals, this is equivalent to

carrying out two one-sided tests of hypothesis at the 5% level of significance. The 90%

confidence interval for the difference in the means of the log-transformed data should be

calculated using methods appropriate to the experimental design. The antilog of the

confidence limits obtained constitute the 90% confidence interval for the ratio of the

geometric means between the T and R products.

b. Non-replicated Crossover Designs

For non-replicated crossover designs, this guidance recommends parametric (normal theory)

procedures to analyze log-transformed BA measures. General linear model procedures

available in PROC GLM in SAS or equivalent software are preferred, although linear mixed-

effects model procedures can also be indicated for analysis of nonreplicated crossover studies.

For example, for a conventional two-treatment, two-period, two-sequence (2 x 2) randomized

crossover design, the statistical model typically includes factors accounting for the following

sources of variation: sequence, subjects nested in sequences, period, and treatment.

35


c. Replicated Crossover Designs

Linear mixed-effects model procedures, available in PROC MIXED in SAS or equivalent

software should be used for the analysis of replicated crossover studies for average BE.

d. Parallel Designs

For parallel designs, the confidence interval for the difference of means in the log scale can be

computed using the total between-subject variance.

4.6.2 Population Bioequivalence:

a. Overview:

Analysis of BE data using the population approach should focus first on estimation of the

mean difference between the T and R for the log-transformed BA measure and estimation of

the total variance for each of the two formulations. This can be done using relatively simple

unbiased estimators such as the method of moments (MM). After the estimation of the mean

difference and the variances has been completed, a 95% upper confidence bound for the

population BE criterion can be obtained, or equivalently a 95% upper confidence bound for a

linearized form of the population BE criterion can be obtained. To obtain the 95% upper

confidence bound of the criterion, intervals based on validated approaches can be used.

b. Non-replicated Crossover Designs:

For non-replicated crossover studies, any available method (e.g., SAS PROC GLM or

equivalent software) can be used to obtain an unbiased estimate of the mean difference in log-

transformed BA measures between the T and R products. The total variance for each

formulation should be estimated by the usual sample variance, computed separately in each

sequence and then pooled across sequences.

c. Replicated Crossover Designs:

For replicated crossover studies, the approach should be the same as for non-replicated

crossover designs, but care should be taken to obtain proper estimates of the total variances.

One approach is to estimate the within- and between-subject components separately, as for

individual BE and then sum them to obtain the total variance.

d. Parallel Designs:

The estimate of the means and variances from parallel designs should be the same as for non-

replicated crossover designs.

36


4.6.3 Individual Bioequivalence:

Analysis of BE data using an individual BE approach should focus on estimation of the mean

difference between T and R for the log-transformed BA measure, the subject-by formulation

interaction variance, and the within-subject variance for each of the two formulations. For this

purpose, the MM (method of moments) approach has been recommended. To obtain the 95%

upper confidence bound of a linearized form of the individual BE criterion, intervals based on

validated approaches can be used. After the estimation of the mean difference and the

variances has been completed, a 95% upper confidence bound for the individual BE criterion

can be obtained.

The restricted maximum likelihood (REML) method may be useful to estimate mean

differences and variances when subjects with some missing data are included in the statistical

analysis.

4.7 Dyslipidemia:Dyslipidemias are disorders of lipoprotein metabolism, including lipoprotein overproduction

or deficiency. These disorders may be manifested by elevation of the serum total cholesterol,

low-density lipoprotein (LDL) cholesterol and triglyceride concentrations, and a decrease in

the high-density lipoprotein (HDL) cholesterol concentration.11

Table:4 Fredrickson Classification of the Dyslipidemia11

Phenotype Lipoprotein(s)

Elevated

Serum

Cholesterol level

Serum

Triglyceride

level

Atherogenicity

I ChylomicronsNormal to↑ ↑↑↑↑

None seen

IIa LDL ↑↑ Normal +++

IIb LDL and VLDL ↑↑ ↑↑↑ +++

III IDL ↑↑ ↑↑ +++

37


IV VLDLNormal to ↑ ↑↑ +

V VLDL&

chylomicronsNormal to ↑ ↑↑↑↑ +

LDL=low-density lipoprotein; IDL=intermediate-density lipoprotein; VLDL=very-low-density lipoprotein;

HDL=high-density lipoprotein; ↑=mildly increased; ↑↑=moderately increased; ↑↑↑ =severely increased; ↑↑↑↑

=very severely increased; + =mild to moderate Atherogenicity; +++ =severe atherogenicity.

4.7.1 Etiology

Primary (Genetic) Dyslipidemias: Several monogenic disorders (hereditary condition

involving single gene) have been defined that lead to different type of dyslipidemia, but for

many cases, the etiology is polygenic.14

Primary causes are single or multiple gene mutations that result in either overproduction or

defective clearance of TG and LDL cholesterol, or in underproduction or excessive clearance

of HDL10

Table: 5 Primary Hyperlipoproteinemia Caused by Known Single Gene Mutation29

Genetic Disorder Gene

Defects

Lipoprotein

Elevated

Clinical Findings Genetic

Transmission

Lipoprotein Lipase

Deficiency

LPL Chylomicron Eruptive xanthomas,

Hepatospleenomegaly

Pancreatitis

AR

Familial

Hypercholesterolemia

LDL

receptor

LDL Tendon

Xanthomas,CHD

AD

Familial Hepatic Lipase

Deficiency

Hepatic

Lipase

(LIPC)

VLDL

remnants

Premature

Atherosclerosis

AR

Familial

Dysbetalipoproteinemia

ApoE Chylomicron

& VLDL

remnants

Palmar &

tuberoeruptive

Xanthomas,CHD,PVD

AR

AD

Familial apolipoprotein

C-II deficiency

ApoC-II Chylomicron Eruptive xanthomas,

Hepatospleenomegaly

AR

38


Pancreatitis

Sitosterolemia ABCG5or

ABCG8

LDL Tendon

Xanthomas,CHD

AR

AR, autosomal recessive; AD, autosomal dominant; VLDL, very low density lipoprotein; CHD, coronary heart disease; PVD, peripheral vascular disease; LDL, low-density lipoprotein

Secondary Dyslipidemia: The medical conditions associated with mild or even severe

dyslipidemia even in the absence of underlying genetic disorder.14

Table:6 Selected Causes of Secondary Dyslipidemia11

Increased LDL Cholesterol

Level

Increased Triglyceride

Level

Decreased HDL Cholesterol

Level

Diabetes mellitus

Hypothyroidism

Nephrotic syndrome

Obstructive liver disease

Drugs:

Anabolic steroids

Progestins

Beta-adrenergic blockers

(without intrinsic

Sympathomimetic

action)

Thiazides

Renal insufficiency

Alcoholism

Diabetes mellitus

Hypothyroidism

Obesity

Drugs:

Beta-adrenergic

blockers (without

intrinsic sympatho-

mimetic action)

Bile acid binding

resins

Estrogens

Ticlopidine

Cigarette smoking

Diabetes mellitus

Hypertriglyceridaemia

Menopause

Obesity

Puberty (in males)

Uremia

Drugs:

Anabolic steroids

Beta-adrenergic blockers

(without intrinsic

Sympatho mimetic

action)

Progestins

LDL=low-density lipoprotein; HDL=high-density lipoprotein

Atherogenic dyslipidemia: Atherogenic dyslipidemia is defined by elevation of serum

triglycerides, presence of small LDL particles, and low HDL-cholesterol levels. For clinical

purposes, elevated triglyceride (≥150 mg/dL) plus low HDL cholesterol (< 40 mg/dL) define

atherogenic dyslipidemia.14

39


4.7.2 Symptoms and Signs:10

Dyslipidemia itself usually causes no symptoms but can lead to symptomatic vascular disease,

including coronary artery disease (CAD) and peripheral arterial disease. High levels of TGs

(> 1000 mg/dL [> 11.3 mmol/L]) can cause acute pancreatitis. High levels of LDL can cause

eyelid xanthelasmas; arcus corneae; and tendinous xanthomas at the Achilles, elbow, and

knee tendons and over metacarpophalangeal joints. Patients with the homozygous form of

familial hypercholesterolemia may have the above findings plus planar or cutaneous

xanthomas. Patients with severe elevations of TGs can have eruptive xanthomas over the

trunk, back, elbows, buttocks, knees, hands, and feet. Patients with the rare

Dysbetalipoproteinemia can have palmar and tuberous xanthomas.

Severe hypertriglyceridemia (> 2000 mg/dL [> 22.6 mmol/L]) can give retinal arteries and

veins a creamy white appearance (lipemia retinalis). Extremely high lipid levels also give a

lactescent (milky) appearance to blood plasma. Symptoms can include paresthesias, dypsnea,

and confusion.

4.8 Plasma Lipoprotein Metabolism13

Lipoproteins are macromolecular assemblies that contain lipids and proteins. The lipid

constituents include free and esterified cholesterol, triglycerides, and phospholipids. The

protein components, known as apolipoproteins or apoproteins, provide structural stability to

the lipoproteins, and also may function as ligands in lipoprotein-receptor interactions or as

cofactors in enzymatic processes that regulate lipoprotein metabolism. In all spherical

lipoproteins, the most water-insoluble lipids (cholesteryl esters and triglycerides) are core

components, and the more polar, water-soluble components (apoproteins, phospholipids, and

unesterified cholesterol) are located on the surface. The major classes of lipoproteins and a

number of their properties are presented in Table 7.

40


Table:7 Molecular Properties of Lipoproteins

Lipoprotein class

Density ofFlotation,

g/ml

Major lipidconstituent

Tg: cholratio

Site ofSynthesis

Mechanism(s) ofcatabolism

Chylomicrons and remnants

<<1.006 Dietary triglycerides

and cholesterol

10:1 Intestine Triglyceride hydrolysis by

LPL,

ApoE-mediated remnant uptake

by liverVLDL <1.006 "Endogenous"

or hepatictriglycerides

5:1 Liver Triglyceride hydrolysis by

LPLIDL 1.006-1.019 Cholesteryl

esters and "endogenous" triglycerides

1:1 Product of VLDL

catabolism

50% converted to LDL mediated

by HL,50% apoE-

mediated uptake by Liver.

LDL 1.019-1.063 Cholesteryl esters

NS Product of VLDL

catabolism

ApoB-100-mediated uptake by LDL receptor (~75% in liver)

HDL 1.063-1.21 Phospholipid, cholesteryl

esters

NS Intestine, liver, plasma

Transfer ofcholesteryl ester

to VLDL andLDL,

Uptake of HDL cholesterol byHepatocytes

Lp(a) 1.05-1.09 Cholesteryl esters

NS Liver Unknown

41


Abbreviations: apo, apolipoprotein; CHOL, cholesterol; HDL, high-density lipoproteins; IDL, intermediate-density lipoproteins; Lp(a), lipoprotein(a); LDL, low-density lipoproteins; NS, not significant (triglyceride is less than 5% of LDL and HDL); TG, triglyceride; VLDL, very-low-density lipoproteins; HL, hepatic lipase; LPL, lipoprotein lipase

Apoproteins14,29

Apoproteins that have well-defined roles in plasma lipoprotein metabolism. These

apolipoproteins include apolipoprotein (apo) A-I, apoA-II, apoA-IV, apoA-V, apoB-100,

apoB-48, apoC-I, apoC-II, apoC-III, apoE, and apo(a). Except for apo(a), the lipid-binding

regions of all apoproteins contain structural features called amphipathic helices that interact

with the polar, hydrophilic lipids (such as surface phospholipids) and with the aqueous plasma

environment in which the lipoproteins circulate. Differences in the non-lipid-binding regions

determine the functional specificities of the apolipoproteins.(Table 8)

42


Fig 4-Lipid Synthesis, Metabolism and Transport14

Table:8 Types of Apoproteins

43


Apolipoprotein Primary Source Lipoprotein Association

Function

ApoA-I Intestine, liver HDL, chylomicrons Structural protein for HDL; activates

LCAT

ApoA-II liver HDL, chylomicrons Structural protein for HDL

ApoA-IV Intestine HDL, chylomicrons Unknown

ApoA-V liver VLDL Unknown

ApoB-48 Intestine chylomicrons Structural protein for

chylomicrons

ApoB-100, Liver VLDL, IDL, LDL, Lp(a)

Structural protein for VLDL, LDL, IDL, Lp(a); ligand for binding to LDL

receptor

ApoC-I Liver Chylomicrons, VLDL, HDL

Unknown

ApoC-II Liver Chylomicrons, VLDL, HDL

Cofactor for LPL

ApoC-III Liver Chylomicrons, VLDL, HDL

Inhibits lipoprotein binding to receptors

ApoD Spleen, brain,

testes, adrenals

HDL Unknown

ApoE Liver Chylomicron remnants, IDL, HDL

Ligand for binding to LDL receptor

ApoH Liver Chylomicrons, VLDL, LDL, HDL

B2 glycoprotein I

Note: HDL, high-density lipoprotein; LCAT, lecithin-cholesterol acyltransferase; VLDL, very low density lipoprotein; IDL, intermediate-density lipoprotein; LDL, low-density lipoprotein; Lp(a), lipoprotein A; LPL, lipoprotein lipase.

44


Cholesterol is a fat-like substance (lipid) that is present in cell membranes and is a precursor

of bile acids and steroid hormones. Cholesterol travels in the blood in distinct particles

containing both lipid and proteins (lipoproteins). Three major classes of lipoproteins are

found in the serum of a fasting individual: low density lipoproteins (LDL), high density

lipoproteins (HDL), and very low density lipoproteins (VLDL). Another lipoprotein class,

intermediate density lipoprotein (IDL), resides between VLDL and LDL.21

Triglycerides represent a form of energy store and cholesterol is a basic building block of

biological membranes. Both lipids are water insoluble and require appropriate transport

vehicles in the aqueous media of lymph and blood. Small amounts of lipid are coated with a

layer of phospholipids, embedded in which are additional proteins—the apolipoproteins(A).

According to the amount and the composition of stored lipids, as well as the type of

apolipoprotein, one distinguishes 4 transport forms:30

LDL cholesterol typically makes up 60–70 percent of the total serum cholesterol. It contains

a single apolipoprotein, namely apo B-100 (apo B). LDL is the major atherogenic lipoprotein

and is the primary target of cholesterol- lowering therapy. This focus on LDL has been

strongly validated by recent clinical trials, which show the efficacy of LDL-lowering therapy

for reducing risk for CHD. 21

HDL cholesterol normally makes up 20–30 percent of the total serum cholesterol. HDL

cholesterol levels are inversely correlated with risk for CHD. HDL protects against the

development of atherosclerosis, although a low HDL level often reflects the presence of other

atherogenic factors. 21

VLDL are triglyceride-rich lipoproteins, but contain 10–15 percent of the total serum

cholesterol. VLDL are produced by the liver and are precursors of LDL; some forms of

VLDL, particularly VLDL remnants, appear to promote atherosclerosis, similar to LDL. 21

Chylomicrons are also triglyceride-rich lipoproteins; they are formed in the intestine from

dietary fat and appear in the blood after a fat-containing meal. 21

4.9 TREATMENT OF DYSLIPIDEMIA29

4.9.1 Non pharmacological Treatment

Diet: Dietary modification is an important component in the management of hyperlipidemia.

In the hypercholesterolemic patient, dietary saturated fat and cholesterol should be restricted.

45


For patients who are hypertriglyceridemic, the intake of simple sugars should also be

curtailed. For severe hypertriglyceridaemia [>11.3 mmol/L (>1000 mg/dL)], restriction of

total fat intake is critical.

Foods and Additives: Certain foods and dietary additives are associated with modest

reductions in plasma cholesterol levels. Plant stanol and sterol esters are available in a variety

of foods such as spreads, salad dressings, and snack bars. They interfere with cholesterol

absorption and reduce plasma LDL-C levels by 10 to 15% when taken three times per day.

The addition to the diet of psyllium, soy protein, or Chinese red yeast rice (which contains

lovastatin) can have modest cholesterol-lowering effects. Other herbal approaches such as

guggulipid require further study to assess their effectiveness.

Weight Loss and Exercise: The treatment of obesity, if present, can have a favorable

impact on plasma lipid levels and should be actively encouraged. Plasma triglyceride and

LDL-C levels tend to fall and HDLC levels tend to increase in obese persons who lose

weight.

46


4.9.2 Pharmacological TreatmentTable: 9 Summary of the Major Drugs Used for the Treatment of Hyperlipidemia

DrugMajor

IndicationsStarting

DoseMaximal

Dose Mechanism Common Side Effects

HMG-CoA reductase inhibitors:LovastatinPravastatinSimvastatinFluvastatinAtorvastatinRosuvastatin

Elevated LDL20 mg daily40 mg qhs20 mg qhs20 mg qhs10 mg qhs10 mg qhs

80 mg daily80 mg qhs80 mg qhs80 mg qhs80 mg qhs40 mg qhs

↓Cholesterol synthesis,

↓hepatic LDLreceptors ↓VLDL

production

Myalgias, arthralgias,

elevated transaminases,

dyspepsia

Bile acid sequestrants:

CholestyramineColestipol

ColesevelamElevated LDL

4 g daily5 g daily3750 mg

daily

32 g daily40 g daily4375 mg

daily

↓Bile acid excretion

And ↑LDL receptors

Bloating, constipation,

elevated triglycerides

Nicotinic acid:IRSRER

Elevated LDL and TG,Low HDL,

100 mg tid250 mg bid500 mgqhs

2 g tid1.5 g bid2 g q hs

↓VLDL hepatic

synthesis

Cutaneous flushing; GI

upset; elevated glucose,

uric acid, and liver

function tests

Fibric acid Derivatives:GemfibrozilFenofibrate

Elevated TG, elevatedremnants

600 mg bid160 mg qd

600 mg bid160 mg qd

↑LPL, ↓VLDLsynthesis

Dyspepsia, myalgia,

gallstones, elevated

transaminases

Fish oils Severely elevated TG 3 g daily 12 g daily

↓Chylomicron and

VLDL production

Dyspepsia, diarrhea, fishyodor to breath

Cholesterol absorption inhibitors:Ezetimibe

Elevated LDL 10 mg daily 10 mg daily↓ Intestinal cholesterolabsorption

Elevated transaminases

47


HMG-CoA reductase inhibitors (statins): By inhibiting cholesterol biosynthesis, HMG-

CoA reductase inhibitors (statins) lead to increased hepatic LDL receptor activity and

accelerated clearance of circulating LDL, resulting in a dose-dependent reduction in plasma

LDL-C. HMG-CoA reductase inhibitors also reduce plasma triglycerides in a dose-dependent

fashion, which is proportional to their LDL-C lowering effects. HMG-CoA reductase

inhibitors are well tolerated and can be taken in tablet form once a day. Potential side effects

include dyspepsia, headaches, fatigue, and muscle or joint pains. Severe myopathy and even

rhabdomyolysis occurs rarely. The risk of myopathy is increased by the presence of renal

insufficiency and by coadministration of drugs that interfere with the metabolism of HMG-

CoA reductase inhibitors, such as erythromycin and related antibiotics, antifungal agents,

immunosuppressive drugs, and fibric acid derivatives.

Bile Acid Sequestrants (Resins): Bile acid sequestrants bind bile acids in the intestine and

promote their excretion in the stool. In order to maintain an adequate bile acid pool, the liver

diverts cholesterol to bile acid synthesis. The decreased hepatic intracellular cholesterol

content upregulates the LDL receptor and enhances LDL clearance from the plasma. Bile acid

Sequestrants, including cholestyramine, colestipol, and colesevelam primarily reduce plasma

LDL-C levels but can increase plasma triglycerides. Therefore, patients with

hypertriglyceridaemia should not be treated with bile acid–binding resins.

Nicotinic Acid (Niacin): Nicotinic acid, or niacin, is a B-complex vitamin that reduces

plasma triglyceride and LDL-C levels and raises the plasma HDL-C in high doses. Niacin is

the only currently available lipid-lowering drug that significantly reduces plasma levels of

Lipoprotein A. If properly prescribed and monitored, niacin is a safe and effective lipid-

lowering agent.

Fibric Acid Derivatives (Fibrates): Fibric acid derivatives, or fibrates, are agonists of

PPARα , a nuclear receptor involved in the regulation of carbohydrate and lipid metabolism.

Fibrates stimulate LPL activity (enhancing triglyceride hydrolysis), reduce apoC-III synthesis

(enhancing lipoprotein remnant clearance), and may reduce VLDL production.

Fibrates are the most effective drugs available for reducing triglyceride levels, and they also

raise HDL-C levels.

48


Omega-3 Fatty Acids (Fish Oils): N-3 polyunsaturated fatty acids (PUFAs) are present in

high concentration in fish and in flax seeds. The most widely used n-3 PUFAs for the

treatment of hyperlipidemia are the two active molecules in fish oil, eicosapentanoic acid

(EPA) and decohexanoic acid (DHA). Fish oil supplements can be used in combination with

fibrates, niacin, or statins to treat hypertriglyceridemia.

4.10 HMG-COA reductase inhibitors30

Fig 5-Regulation by cellular cholesterol concentration of HMG-CoA reductase and LDL-receptors

inhibition of HMG CoA reductase, hepatic cholesterol content does not fall, because

hepatocytes compensate any drop in cholesterol levels by increasing the synthesis of LDL

receptor protein (along with the reductase) Because the newly formed reductase is inhibited,

too, the hepatocyte must meet its cholesterol demand by uptake of LDL from the blood.

Triglyceride Reduction by Statins: Triglyceride levels >250 mg/dl are reduced

substantially by statins, and the percent reduction achieved is similar to the percent reduction

in LDL-C. Accordingly, hypertriglyceridemic patients taking the highest doses of the most

potent statins (simvastatin and atorvastatin, 80 mg/day; Rosuvastatin, 40 mg/day) experience

a 35% to 45% reduction in LDL-C and a similar reduction in fasting triglyceride levels.13

Effect of Statins on HDL-C Levels: Most studies of patients treated with statins have

systematically excluded patients with low HDL-C levels. In studies of patients with elevated

49


LDL-C levels and gender-appropriate HDL-C levels (40 to 50 mg/dl for men; 50 to 60 mg/dl

for women), an increase in HDL-C of 5% to 10% was observed, irrespective of the dose or

statin employed. However, in patients with reduced HDL-C levels (35 mg/dl), statins may

differ in their effects on HDL-C levels. Simvastatin, at its highest dose of 80 mg, increases

HDL-C and apoA-I levels more than a comparable dose of atorvastatin.In preliminary studies

of patients with hypertriglyceridaemia and low HDL-C, Rosuvastatin appears to raise HDL-C

levels by as much as 15% to 20%.13

Effects of Statins on LDL-C Levels: Statins lower LDL-C by 20% to 55%, depending

on the dose and statin used. In large trials comparing the effects of the various statins,

equivalent doses appear to be

5 mg of simvastatin = ~15 mg of lovastatin = ~15 mg of pravastatin = ~40 mg of

fluvastatin

20 mg of simvastatin = ~10 mg of atorvastatin

20 mg of atorvastatin = 10 mg of Rosuvastatin.

Analysis of dose-response relationships for all statins demonstrates that the efficacy of LDL-

C lowering is log-linear; LDL-C is reduced by ~6% (from baseline) with each doubling of the

dose. Maximal effects on plasma cholesterol levels are achieved within 7 to 10 days.13

Table 10. Doses (mg) of Statins Required to Achieve Various Reductions in Low-Density-Lipoprotein Cholesterol (LDL-C) from Baseline.13

20 -25(%)

26 -30(%)

31 -35(%)

36 -40(%)

41 -50(%)

51 -55(%)

Atorvastatin — — 10 20 40 80

Fluvastatin 20 40 80

Lovastatin 10 20 40 80

Pravastatin 10 20 40

Rosuvastatin — — — 5 10 20, 40

Simvastatin — 10 20 40 80

4.11 Extra cardiac Effects of Rosuvastatin:

50


Statins and Apoptosis: Rosuvastatin has the potential to prevent damage to and apoptosis of

HUVECs (human umbilical vein endothelial cells) induced by high glucose exposure, by

reducing oxidative stress. The action of Rosuvastatin on antioxidant pathways is related to the

inhibition of the overexpression of components of NAD(P)H oxidase induced by the two

conditions (constant and intermittent) of high glucose.31

Statins and GFR: It is reported that long-term treatment with Rosuvastatin (≥ 96 weeks)

results in an increase of approximately 4 ml/min/1.73 m2 in the estimated glomerular filtration

rate (eGFR) compared with baseline.32

Statins and Endothelial Functions: A variety of studies have established that the vascular

endothelium plays a dynamic role in vasoconstriction/relaxation. Hypercholesterolemia

adversely affects the processes by which the endothelium modulates arterial tone. Statin

therapy enhances endothelial production of the vasodilator nitric oxide, leading to improved

endothelial function after a month of therapy.13

Statins and Plaque Stability: The vulnerability of plaques to rupture and thrombosis is of

greater clinical relevance than the degree of stenosis they cause. Statins may affect plaque

stability in a variety of ways.13

Statins and Inflammation: Appreciation of the importance of inflammatory processes in

atherogenesis is growing , and statins may have an anti-inflammatory role. Statins decreased

the risk of CHD and levels of C-reactive protein (CRP, an independent marker for

inflammation and high CHD risk) independently of cholesterol lowering.13

Statins and Coenzyme Q (Ubiquinone): The inhibition of the mevalonate pathway

restricts the biosynthesis of other nonsteroidal products of this loop, including coenzyme Q10.

Statin therapy with CoQ10 to support the deficient cellular bioenergetic state and ameliorate

oxidative stress.

Mevalonate is the precursor not only of cholesterol, but also of many nonsteroidal isoprenoid

compounds vital to diverse cellular functions, including cell proliferation. These isoprenoids

include dolichols, required for glycoprotein synthesis and coenzyme Q, involved in

intracellular electron transport and energy generation. Thus, while inhibition of the

mevalonate pathway suppresses cholesterol production; it also reduces CoQ bioavailability,

causing CoQ deficiency.

In humans, the fat-soluble nutrient coenzyme Q10, also known as CoQ10 or ubiquinone (2,3-

dimethoxy-5-methyl-6-decaprenyl-1,4-benzoquinone), is a major participant in electron

51


transfer during oxidative phosphorylation in the mitochondria. In addition to its role in

intracellular energy generation, CoQ10is a potent antioxidant and free radical scavenger, and is

a membrane stabilizer that preserves cellular integrity.33

4.12 DRUG PROFILE18,18,34

Rosuvastatin Calcium

Pharmacological Class: HMG CoA reductase inhibitor.

Chemical Name: bis [(E)-7- [4-(4-fluorophenyl)-6- isopropyl-2-[methyl (methylsulfonyl)

amino] pyrimidin-5 yl] (3R, 5S)-3, 5 -dihydroxyhept-6-enoic acid] calcium salt

Empirical Formula: (C22H27FN3O6S) 2 Ca.

Structural formula:

Molecular Weight: 1001.14

Physical Appearance: white amorphous powder.

Solubility: Sparingly soluble in water and methanol, and slightly soluble in ethanol.

Storage: Store at controlled room temperature, 20-25°C (68-77°F), Protect from moisture.

4.12.1 Clinical Pharmacology

4.12.2 Mechanism of Action:

Rosuvastatin is a selective and competitive inhibitor of HMG-CoA reductase, the rate-limiting

enzyme that converts 3-hydroxy-3- methylglutaryl coenzyme A to mevalonate, a precursor of

cholesterol. In vivo studies in animals and in vitro studies in cultured animal and human cells

have shown Rosuvastatin to have a high uptake into, and selectivity for, action in the liver, the

target organ for cholesterol lowering. In in vivo and in vitro studies, Rosuvastatin produces its

lipid-modifying effects in two ways. First, it increases the number of hepatic LDL receptors

52


on the cell-surface to enhance uptake and catabolism of LDL. Second, Rosuvastatin inhibits

hepatic synthesis of VLDL, which reduces the total number of VLDL and LDL particles.

Rosuvastatin reduces total cholesterol (total-C), LDL-C, ApoB, and nonHDL-C (total

cholesterol minus HDL-C) in patients with homozygous and heterozygous familial

hypercholesterolemia (FH), nonfamilial forms of hypercholesterolemia, and mixed

dyslipidemia. Rosuvastatin also reduces TG and produces increases in HDL-C. Rosuvastatin

reduces total-C, LDL-C, VLDL-cholesterol (VLDL-C), ApoB, nonHDL-C and TG, and

increases HDL-C in patients with isolated hypertriglyceridaemia. The effect of Rosuvastatin

on cardiovascular morbidity and mortality has not been determine.

4.12.3 Pharmacokinetics and Metabolism

Absorption:

The peak plasma concentration of Rosuvastatin reaches in 3 to 5 hours following oral dosing.

Both peak concentrations (Cmax) and Area under the curve (AUC) increased in approximate

portion to Rosuvastatin dose. The absolute bioavailability of Rosuvastatin is 20%.

Administration of Rosuvastatin with food decrease the rate of absorption by 20% as assessed

by Cmax, but there was no effect on extent of absorption as assessed by AUC.

Distribution:Mean volume of distribution at steady state of Rosuvastatin is approximately 134 litres. Drug

is 88 % bound to plasma protein, mostly albumin. This binding is reversible and independent

of plasma concentration.

Metabolism:

Rosuvastatin is not extensively metabolized; only 10% of a radiolabelled dose is recovered as

metabolite. The major metabolite is N-desmethyl Rosuvastatin, which is formed principally

by cytochrome P450 2C9 and in vitro studies have demonstrated that N-desmethyl

Rosuvastatin has approximately one-sixth to one-half the HMG-CoA reductase inhibitory

activity of Rosuvastatin. Overall greater than 90% of active plasma HMG-CoA reductase

inhibitory activity is accounted for by Rosuvastatin.34

Two metabolites of Rosuvastatin have been previously identified in human plasma, urine and

faeces, Rosuvastatin lactone and N-desmethyl Rosuvastatin. Hence, reports based on human

microsomes have shown that the metabolic clearance of Rosuvastatin is low and studies show

53


discrepancies as to which CYP450 enzymes are involved .Research with human hepatocytes

has shown that Rosuvastatin and other Statins are metabolized by UDP glucuronosyl

transferases (UGTs) UGT1A1 and UGT1A3 to form β-1-O-acyl glucuronide.

Excretion:Following oral administration, Rosuvastatin and its metabolites are primarily excreted in the

feces (90%). The elimination half life (t1/2) of Rosuvastatin is approximately 19 hours. After

an i.v dose approximately 28% of total body clearance was via the renal route, and 72% by

the hepatic route.

Rosuvastatin is excreted via the biliary route in humans, and the transport and accumulation

of Rosuvastatin in bile compared to that in plasma is rapid and extensive.

4.12.4 Pharmacokinetics in Special Populations:

Race: A population pharmacokinetic analysis revealed no clinically relevant differences in

pharmacokinetics among Caucasian, Hispanic, and Black or Afro-Caribbean groups.

However, pharmacokinetic studies, including one conducted in the US, have demonstrated an

approximate 2-fold elevation in median exposure (AUC and Cmax) in Asian subjects when

compared with a Caucasian control group.

Gender: There were no differences in plasma concentrations of Rosuvastatin between men

and women.

Geriatric: There were no differences in plasma concentrations of Rosuvastatin between the

nonelderly and elderly populations (age ≥65 years).

Pediatric: In a pharmacokinetic study, 18 patients (9 boys and 9 girls) 10 to 17 years of age

with heterozygous FH received single and multiple oral doses of Rosuvastatin. Both Cmax

and AUC of Rosuvastatin were similar to values observed in adult subjects administered the

same doses.

Renal Insufficiency: Mild to moderate renal impairment (creatinine clearance ≥30 mL/min/

1.73 m2) had no influence on plasma concentrations of Rosuvastatin when oral doses of 20 mg

Rosuvastatin were administered for 14 days. However, plasma concentrations of Rosuvastatin

increased to a clinically significant extent (about 3-fold) in patients with severe renal

54


impairment (CLCr < 30 mL/min/1.73 m2) compared with healthy subjects (CLCr >80

mL/min/l.73 m2).

Hemodialysis: Steady-state plasma concentrations of Rosuvastatin in patients on chronic

hemodialysis were approximately 50% greater compared with healthy volunteer subjects with

normal renal function.

Hepatic Insufficiency: In patients with chronic alcohol liver disease, plasma concentrations

of Rosuvastatin were modestly increased. In patients with Child-Pugh A disease, Cmax and

AUC were increased by 60% and 5%, respectively, as compared with patients with normal

liver function. In patients with Child-Pugh B disease, Cmax and AUC were increased 100%

and 21%, respectively, compared with patients with normal liver function.

Pregnancy and Lactation

Atherosclerosis is a chronic process and discontinuation of lipid-lowering drugs during

pregnancy should have little impact on the outcome of long-term therapy of primary

hypercholesterolemia. Cholesterol and other products of cholesterol biosynthesis are essential

components for fetal development (including synthesis of steroids and cell membranes). Since

HMG-CoA reductase inhibitors decrease cholesterol synthesis and possibly the synthesis of

other biologically active substances derived from cholesterol, they may cause fetal harm when

administered to pregnant women. Therefore, HMG-CoA reductase inhibitors are

contraindicated during pregnancy and in nursing mothers. “Rosuvastatin should be

administered to women of childbearing age only when such patients are highly unlikely to

conceive and have been informed of the potential hazards”. If the patient becomes pregnant

while taking this drug, therapy should be discontinued immediately and the patient apprised

of the potential hazard to the fetus.

4.12.5 Therapeutic Indication and Usage:

As an adjunct to diet to reduce elevated total-C, LDL-C, ApoB, nonHDL-C, and TG

levels and to increase HDL-C in patients with primary hypercholesterolemia

(heterozygous familial and nonfamilial) and mixed Dyslipidaemia (Fredrickson Type IIa

and IIb).

As an adjunct to diet for the treatment of patients with elevated serum TG levels

(Fredrickson Type IV).

55


To reduce LDL-C, total-C, and ApoB in patients with homozygous familial

hypercholesterolemia as an adjunct to other lipid-lowering treatments (e.g., LDL

apheresis) or if such treatments are unavailable.

4.12.6 Contraindications

Rosuvastatin is contraindicated in patients with a known hypersensitivity to any

component of this product.

Rosuvastatin is contraindicated in patients with active liver disease or with

unexplained persistent elevations of serum transaminase

4.12.7 Drug-Drug Interactions

Cytochrome P450 3A4: In vitro and in vivo data indicate that Rosuvastatin clearance is not

dependent on metabolism by cytochrome P450 3A4 to a clinically significant extent. This has

been confirmed in studies with known cytochrome P450 3A4 inhibitors (Ketoconazole,

erythromycin, itraconazole).

Ketoconazole: Coadministration of Ketoconazole (200 mg twice daily for 7 days) with

Rosuvastatin (80 mg) resulted in no change in plasma concentrations of Rosuvastatin.

Erythromycin: Coadministration of erythromycin (500 mg four times daily for 7 days) with

Rosuvastatin (80 mg) decreased AUC and Cmax of Rosuvastatin by 20% and 31%,

respectively. These reductions are not considered clinically significant.

Itraconazole: Itraconazole (200 mg once daily for 5 days) resulted in a 39% and 28%

increase in AUC of rosuvastatin after 10 mg and 80 mg dosing, respectively. These increases

are not considered clinically significant.

Fluconazole: Coadministration of fluconazole (200 mg once daily for 11 days) with

Rosuvastatin (80 mg) resulted in a 14% increase in AUC of Rosuvastatin. This increase is not

considered clinically significant.

Cyclosporine: Coadministration of cyclosporine with Rosuvastatin resulted in no significant

changes in cyclosporine plasma concentrations. However, Cmax and AUC of Rosuvastatin

increased 11- and 7-fold, respectively, compared with historical data in healthy subjects.

These increases are considered to be clinically significant

Warfarin: Coadministration of Warfarin (25 mg) with Rosuvastatin (40 mg) did not change

Warfarin plasma concentrations but increased the International Normalized Ratio (1NR).

56


Digoxin: Coadministration of digoxin (0.5 mg) with Rosuvastatin (40 mg) resulted in no

change to digoxin plasma concentrations.

Fenofibrate: Coadministration of fenofibrate (67 mg three times daily) with Rosuvastatin (10

mg) resulted in no significant changes in plasma concentrations of Rosuvastatin or

fenofibrate.

Gemfibrozil: Coadministration of gemfibrozil (600 mg twice daily for 7 days) with

Rosuvastatin (80 mg) resulted in a 90% and 120% increase for AUC and Cmax of

Rosuvastatin, respectively. This increase is considered to be clinically significant.

Ezetimibe: Coadministration of ezetimibe (10 mg) with Rosuvastatin (40 mg) resulted in no

significant changes in plasma concentrations of Rosuvastatin or ezetimibe.

Antacid: Coadministration of an antacid (aluminum and magnesium hydroxide combination)

with Rosuvastatin (40 mg) resulted in a decrease in plasma concentrations of Rosuvastatin by

54%. However, when the antacid was given 2 hours after Rosuvastatin, there were no

clinically significant changes in plasma concentrations of Rosuvastatin.

Oral contraceptives: Coadministration of oral contraceptives (ethinyl estradiol and

norgestrel) with Rosuvastatin resulted in an increase in plasma concentrations of ethinyl

estradiol and norgestrel by 26% and 34%, respectively.

Lopinavir/Ritonavir: Coadministration of Rosuvastatin and a combination product of two

protease inhibitors (400 mg lopinavir / 100 mg ritonavir) in healthy volunteers was associated

with an approximately 2-fold and 5-fold increase in Rosuvastatin steady-state AUC (0-24) and

Cmax respectively. Interactions between Rosuvastatin and other protease inhibitors have not

been examined.

4.12.8 Adverse Reactions

Rosuvastatin is generally well tolerated. Adverse reactions have usually been mild and

transient. In clinical studies of 10,275 patients, 3.7% were discontinued due to adverse

experiences attributable to Rosuvastatin. The most frequent adverse events thought to be

related to Rosuvastatin were myalgia, constipation, asthenia, abdominal pain, and nausea.

Clinical Adverse Experiences

Adverse experiences, regardless of causality assessment, reported in ≥2% of patients in

placebo-controlled clinical studies of Rosuvastatin are shown in Table 11; discontinuations

57


due to adverse events in these studies of up to 12 weeks duration occurred in 3% of patients

on Rosuvastatin and 5% on placebo.

Table 11 Adverse Events in Placebo-Controlled Studies

Rosuvastatin Placebo

Adverse Event N=744 N=382

Pharyngitis 9.0 7.6

Headache 5.5 5.0

Diarrhea 3.4 2.9

Dyspepsia 3.4 3.1

Nausea 3.4 3.1

Myalgia 2.8 1.3

Asthenia 23 26

Back Pain 2.6 2.4

Flu syndrome 2.3 1.8

Urinary tract infection 2.3 1.6

Rhinitis 2.2 2.1

Sinusitis 2.0 1.8

In addition, the following adverse events were reported, regardless of causality assessment, in

≥1% of 10,275 patients treated with Rosuvastatin in clinical studies. The events in italics

occurred in ≥2% of these patients.

Body as a Whole: Abdominal pain, accidental injury, chest pain, infection, pain, pelvic pain,

and neck pain.

Cardiovascular System: Hypertension, angina pectoris, vasodilatation, and palpitation.

Digestive System: Constipation, gastroenteritis, vomiting, flatulence, periodontal abscess,

and gastritis.

Endocrine: Diabetes mellitus.

58


Hemic and Lymphatic System: Anaemia and ecchymosis.

Metabolic and Nutritional Disorders: Peripheral edema.

Musculoskeletal System: Arthritis, arthralgia, and pathological fracture.

Nervous System: Dizziness, insomnia, hypertonia, paresthesia, depression, anxiety, vertigo

and neuralgia.

Respiratory System: Bronchitis, cough increased dyspnea, pneumonia, and asthma.

Skin and Appendages: Rash and pruritus.

Laboratory Abnormalities: In the Rosuvastatin clinical trial program, dipstick-positive

proteinuria and microscopic hematuria were observed among Rosuvastatin-treated patients,

predominantly in patients dosed above the recommended dose range (i.e., 80 mg). However,

this finding was more frequent in patients taking Rosuvastatin 40 mg, when compared to

lower doses of Rosuvastatin or comparator statins, though it was generally transient and was

not associated with worsening renal function.

Other abnormal laboratory values reported were elevated creatine phosphokinase,

transaminases, hyperglycemia, glutamyl transpeptidase, alkaline phosphatase, bilirubin, and

thyroid function abnormalities.

Other adverse events reported less frequently than 1% in the Rosuvastatin clinical study

program, regardless of causality assessment, included arrhythmia, hepatitis, hypersensitivity

reactions (i.e., face edema, thrombocytopenia, leukopenia, vesiculobullous rash, urticaria, and

angioedema), kidney failure, syncope, myasthenia, myositis, pancreatitis, photosensitivity

reaction, myopathy, and rhabdomyolysis.

4.12.9 Precautions

General

Before instituting therapy with Rosuvastatin, an attempt should be made to control

hypercholesterolemia with appropriate diet and exercise, weight reduction in obese patients,

and treatment of underlying medical problems. Administration of Rosuvastatin 20 mg to

patients with severe renal impairment (CLcr <30 mL/min/1.73 m2) resulted in a 3-fold

increase in plasma concentrations of Rosuvastatin compared with healthy volunteers. The

result of a large pharmacokinetic study conducted in the US demonstrated an approximate 2-

fold elevation in median exposure in Asian subjects (having either Filipino, Chinese,

Japanese, Korean, Vietnamese or Asian-Indian origin) compared with a Caucasian control

59


group. This increase should be considered when making Rosuvastatin dosing decisions for

Asian patients.

4.12.10 Overdosage

There is no specific treatment in the event of overdose. In the event of overdose, the patient

should be treated symptomatically and supportive measures instituted as required.

Hemodialysis does not significantly enhance clearance of Rosuvastatin.

4.12.11 Dosage and Administration

The patient should be placed on a standard cholesterol-lowering diet before receiving

Rosuvastatin and should continue on this diet during treatment. Rosuvastatin can be

administered as a single dose at any time of day, with or without food.

Hypercholesterolemia (Heterozygous Familial and Nonfamilial) and Mixed

Dyslipidemia (Fredrickson Type Ila and lIb)

The dose range for Rosuvastatin is 5 to 40 mg once daily. Therapy with Rosuvastatin should

be individualized according to goal of therapy and response. The usual recommended starting

dose of Rosuvastatin is 10 mg once daily. However, initiation of therapy with 5 mg once daily

should be considered for patients requiring less aggressive LDL-C reductions, who have

predisposing factors for myopathy, and as noted below for special populations such as

patients taking cyclosporine, Asian patients, and patients with severe renal insufficiency. For

patients with marked hypercholesterolemia (LDL-C >190 rng/dL) and aggressive lipid

targets, a 20-mg starting dose may be considered. After initiation and/or upon titration of

Rosuvastatin, lipid levels should be analyzed within 2 to 4 weeks and dosage adjusted

accordingly. The 40-mg dose of Rosuvastatin is reserved only for those patients who have not

achieved their LDL-C goal utilizing the 20 mg of Rosuvastatin once daily. When initiating

statin therapy or switching from another statin therapy, the appropriate Rosuvastatin starting

dose should first be utilized, and only then titrated according to the patient’s individualized

goal of therapy.

Table:12 DEVELOPMENT STATUS 35

60


Indication Country Status Date

Hypercholesterolemia UK Launched 20-MAY-2003

Hypercholesterolemia US Launched 16-SEP-2003

Hypercholesterolemia Canada Launched 19-FEB-2003

Hyperlipidemia Canada Launched 19-FEB-2003

Hyperlipidemia Netherlands Launched 03-MAR-2003

Hyperlipidemia UK Launched 20-MAY-2003

Hyperlipidemia US Launched 16-SEP-2003

Hypercholesterolemia Western Europe Launched 06-OCT-2004

Hyperlipidemia Western Europe Launched 06-OCT-2004

Hyperlipidemia Japan Launched 30-APR-2005

Hyperlipidemia South Korea Launched 31-DEC-2005

Atherosclerosis US Launched 07-DEC-2007

Atherosclerosis Western Europe Launched 07-DEC-2007

Hypercholesterolemia Japan Launched 27-APR-2007

61


5. STUDY FLOW CHARTS

5.1 Pre-study:

62


5.2 During Study:

63


5.3 Post Study:

64


65


6.0 METHODOLOGY

66


6.1 Study Title

An Open Label, Randomised, 2-Period, 2-Treatment, 2-Sequence, Crossover, Single-Dose

Bioequivalence Study of Rosuvastatin calcium 40mg Tablet [Test formulation; Torrent

Pharmaceuticals Ltd., India] Versus Rosuvastatin Calcium® 40mg Tablet [Reference

formulation; AstraZeneca LP, USA] in Healthy Human Volunteers Under Fed Condition.

6.2 Study Site

This research work was done at Bio Evaluation Centre, Torrent Pharmaceuticals Ltd., Village

Bhat, Gandhinagar-382428, Gujarat, India.

6.3 Ethics (IEC) Approval

The Protocol and corresponding Informed Consent Forms (English and Gujarati language),

Case Report Forms (Period I and II) were reviewed and discussed in the IEC meeting held on

October 28, 2009. Subjects were not enrolled into the study until the IEC approved the

protocol and the ICF.

6.4 Ethical Conduct of the Study

The study was conducted according to current version of the Declaration of Helsinki (Tokyo,

2008) and in compliance to the current ICH-GCP Guidelines. All the procedures in the study

were carried out as per local regulatory requirements, US-FDA’s guidance for industry:

Bioavailability and Bioequivalence studies, 2003 and in-house Standard Operating Procedures

(SOP’s).

6.5 Informed Consent Procedure

Screening Procedure: General screening for participation in the clinical study was done

within 28 days prior to first dosing.

After successful completion of initial screening, volunteers were called for the study specific

screening test (i.e. Serum Creatine kinase), and ICF for study specific screening was signed

by the volunteers. A total of 50 volunteers participated in the informed consent presentation.

The Informed consent form was issued to all the volunteers in vernacular (Gujarati) language.

The volunteers read the ICF which summarized the discussion prior to check-in. Sufficient

time was given to the volunteers to read, understand and clarify the doubts on the contents of

the ICF. Hence, a total of 48 volunteer had given their consent and enrolled in the study.

67


6.6 Selection of Study PopulationScreening of the volunteers was done and those found to be healthy and met all inclusion and

exclusion criteria were included in the study. The screening procedure of clinical

examination, reading of 12 lead electrocardiogram, radiological investigation (chest X-ray,

and laboratory investigation testing of hematology, biochemistry (including creatinine

kinase), serology and urine analysis conducted not more than 28 days prior to first dosing.

Total 48 healthy volunteers, aged 19 to 42 years with BMI between 18.57-26.62 kg/m 2 were

enrolled in the study on the basis of the following inclusion and exclusion criteria.

6.6.1 Inclusion CriteriaVolunteers meeting following criteria were enrolled: Sex: male, Age: 18 - 45 years, Volunteer

with BMI of 18-27 (inclusive both) kg/m2 with minimum of 50kg weight, Healthy and willing

to participate in the study, Volunteer willing to adhere to the protocol requirements and to

provide written informed consent, Non-smokers or smoker who smokes less than 10

cigarettes per day.

6.6.2 Exclusion CriteriaThe volunteers were excluded from the study based on the following criteria: Clinically

relevant abnormalities in the results of the laboratory screening evaluation (including

creatinine kinase), Clinically significant abnormal ECG or Chest X-ray, Systolic blood

pressure less than 100 mm Hg or more than 140 mm Hg and diastolic blood pressure less than

60 mm Hg or more than 90 mm Hg, Pulse rate less than 50/minute or more than 100/minute,

Oral temperature less than 95°F or more than 98.6°F, Respiratory rate less than 12/minute or

more than 20/minute, History of allergy to the test drug or any drug chemically similar to the

drug under investigation, History of alcohol or drug abuse, Positive breath alcohol test,

Recent history of kidney or liver dysfunction, History of consumption of prescribed

medication since last 14 days or OTC medication since last 07 days before beginning of the

study, Volunteers suffering from any chronic illness such as arthritis, asthma etc., HIV, HCV,

HBsAg positive volunteers, Opiate, tetra hydrocannabinol, amphetamine, barbiturates,

benzodiazepines, Cocaine positive volunteers based on urine test, Volunteers suffering from

any psychiatric (acute or chronic) illness requiring medications, Administration of any study

drug in the period 0 to 3 months before entry to the study, History of significant blood loss

68


due to any reason, including blood donation in the past 3 months, History of pre-existing

bleeding disorder, Existence of any surgical or medical condition, which, in the judgement of

the chief investigator and/or clinical investigator/physician, might interfere with the

absorption, distribution, metabolism or excretion of the drug or likely to compromise the

safety of volunteers, Inability to communicate or co-operate due to language problem, poor

mental development or impaired cerebral function.

Volunteers meeting Inclusion criteria and not fulfilling Exclusion criteria were enrolled in the

study.

6.7 Sample Size

The sample size calculation was based on an expected intra subject variability for

Rosuvastatin Cmax around 27%, minimum 46 subjects were required to achieve a sufficient

power. As there was no subject replacement during the study, 2 more subjects were enrolled

along with the above considering potential dropouts. Hence, a total of 48 subjects were

enrolled for study.

6.7 Duration of studyTotal duration of study was of 13 days from the day of check-in of first period till the end of

second period. Upon entering into study, subjects were confined in the clinical facility of Bio

Evaluation Centre to ensure 10 hours overnight fasting before high fat high calories breakfast

until 24 hours post-dose sample collection in each of the two periods.

Clinical Phase: November 27, 2009 to December 09, 2009.

6.8 Identity of investigational products

The identity of Test Formulation includes Generic name (Rosuvastatin Calcium), Company

(Torrent Pharmaceuticals Ltd., India), Mode of administration and dose (Single Tablet of

40mg was given orally in each period with approximately 240 ml of water under fed

condition)

The identity of Reference Formulation includes Generic name (Rosuvastatin Calcium), Brand

name (Crestor®), Company (AstraZeneca LP, USA), Mode of administration and dose (Single

Tablet of 40mg was given orally in each period with approximately 240 ml of water under fed

condition)

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6.9 Storage, Dispensing and Accountability Procedures for Investigational

Products

All drug supplies were stored at or below 25°C in accordance with the manufacturer’s

instructions; separately from normal practice stocks, locked and only accessible for authorized

personnel. The temperature and the humidity in the storage room were continuously

monitored. The storage conditions were checked by the study personnel.

The label on the dispensing container contained “FOR CLINICAL RESEARCH PURPOSE

ONLY”, Study Code, Batch No., Generic Name, Brand Name, Storage Condition, Expiry

date/Use by date/Retest date, No. of units received, and prepared by with sign. and date.

The dispensed drugs were delivered to study area approximately 30 minutes before dosing by

the Pharmacist, till that time dispensed study drugs were kept under controlled access and

specified condition in pharmacy. One extra units of test and reference drug were dispensed in

each period of each batch.

Each label of container contained study code, date of dosing, enrolment no., period,

randomization code, drug name and signature of pharmacist. The label were prepared as

duplicate as given below.

Study code:

Period: Enrolment No:

Randomization code:

Drug name:

Date of dosing :

Sign:

FOR CLINICAL RESEARCH PURPOSE

ONLY

Study code:

Period: Enrolment No:

Randomization code:

Drug name:

Date of dosing:

Sign:

FOR CLINICAL RESEARCH PURPOSE

ONLY

After completion of dosing activity, the dispensed but unused study drugs were sent back to

the pharmacy. The extra dispensed, not dosed or un-dispensed study drugs were disposed.

Randomisation schedule for all 48 volunteers was generated before the start of study.

Volunteers were administered each treatment (A or B) during the two period of the study

according to the randomization schedule.

The randomization was balanced and the code was kept under controlled access.

70


The drug accountability was maintained by pharmacist through out study under supervision of

chief investigator. All the study drugs (i.e. dispensed but un-dosed) returned from bio study

was sent back to pharmacy and recorded.

No concomitant drug therapy was allowed during the study except one(s) used due to an

adverse event.

6.10 Treatments Administered

After supervised overnight fast of at least 10 hours, a standard high fat high calorie breakfast

was served 30 minute prior to dosing. After finishing high fat high calorie breakfast,

volunteers were administered a single oral dose of either reference or test product based on

randomization along with 240ml of drinking water in each period.

The drug was administered on following dates and times:

Period-I: 08:00 hours to 08:22 hours on November 28, 2009Period-II: 08:00 hours to 08:22 hours on December 05, 2009

6.11 Removal of Volunteers from Therapy Volunteers were informed that they were free to withdraw from the study at any time without

giving any reason for doing so.

Volunteers may be discontinued from the study for any of the following reasons:

1. Volunteers not willing to continue with the study, irrespective of the reason.

2. Volunteer experiences adverse event, when withdrawal would be in the best interest of the

volunteer’s safety.

3. Volunteer suffers from significant illness or undergoes surgery during the course of the

study.

4. Violation of the protocol by the volunteer.

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A total of 48 healthy, adult male volunteers were enrolled in the study and out of these

enrolled volunteers, Enrolment No. 18, 27 in period I and Enrolment No. 45 in period II had

been medically withdrawn due to vomiting. Enrolment No. 43 did not report in period II.

Enrolment No. 25 voluntarily withdrawn on the day of enrolment of period II. Hence, total 43

volunteers had completed the clinical phase according to the study protocol.

6.12 Study Volunteers

6.12.1 Disposition of Volunteers

Flow chart summarizes the volunteer’s disposition

Batch 2

Batch 1

6.13 Washout period

The administration of each product was followed by a sufficiently long period of time to

ensure complete elimination of the drug (washout period) before the next administration. The

washout period was a minimum of 10 half-lives of the administered drug because at more

than 7-8 half lives a pharmacokinetic carry-over effect can be excluded. The half-life of

Rosuvastatin calcium is approximately 19 hours, so minimum wash-out period of at least 07

days was chosen.

72

No. of volunteers attended ICF

Presentation: 50

No. of volunteers enrolled and Randomized = 48

Not Checked InTwo volunteers did not give consent to participate in the study

Period I

Test drug A = 24Reference drug B = 24

Period II

Test drug A = 22Reference drug B = 22 Dropout: 01 Withdrawn: 03


6.14 Drug Concentration Measurements

6.14.1 Collection of Blood Samples for Pharmacokinetic Measurements

(A)Sampling schedule Twenty six (26) blood samples of 6 ml each were collected from each volunteer in each

period. In the morning of dosing day after vitals measurement, a pre-dose blood (0.0) sample

was taken. And other venous blood samples were withdrawn at 0.33, 0.67, 1.0, 1.5, 2.0, 2.25,

2.5, 2.75, 3.0, 3.25, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 7.0, 8.0, 10.0, 12.0, 16.0, 24.0 and ambulatory

sample at 48.0, 72.0 and 96.0 hours post dose in each period except for few volunteers who

did not report for ambulatory samples and volunteers who were withdrawn and dropout from

the study in P-II.

About 3.0 ml of blood was collected for post study safety analysis from each of the volunteers

dosed at least once in the study.

Blood sampling up to ± 2 minutes of the planned time of in-house sampling and up to ±1 hr in

ambulatory sample was considered as an acceptable deviation. Beyond that, time deviation

was taken in to consideration for further pharmacokinetic parameters, except for pre dose

samples, which is always reported as zero hour sample (0).

(B) Sample collection

All blood samples were collected in pre-labeled tubes containing 5 IU diluted heparin in

normal saline for each ml of blood. Samples were collected through an indwelling cannula

placed in a forearm vein. The cannula was kept in situ as long as possible by injecting, about

0.5 ml of 5 IU/ml of heparin in normal saline solution to maintain the cannula patent. While

sampling through the cannula, blood samples were collected after discarding first 0.5 ml of

heparinised blood from the tubing of the cannula. Tubes were shaken gently to ensure the

proper mixing of blood with anticoagulant. The cannula was removed from the volunteers in

each period before checkout or it was removed upon volunteer’s request during study.

Samples were collected in tubes containing 5IU diluted heparin for each ml of blood pre-

labeled as shown below:

73


The total volume of blood drawn from each volunteer (except for volunteer no. 03, 23 & 25

who did not report for ambulatory samples) completing this study approximates 338 ml. This

amount includes all blood samples (26 x 6.0 ml) in each period, 0.5ml heparinised blood at in

house time point, 3.0ml blood for post study safety. Additional blood sample was taken for

post study follow-up laboratory investigation for Enrolment no. 02, 15, 18, 19, 23, 26, 27, 28,

30 and 38 to repeat the post study investigations.

(C) Sample Processing

After collection of blood samples from all the volunteers at each time point, tubes containing

blood samples were kept in box containing coolant bags and transferred for centrifugation.

The centrifugation was carried out at 3500 RPM for 15 minutes at 20oC. The plasma samples

then separated in tubes, were subsequently stored at ≤- 40°C until withdrawn for analysis.

All the plasma samples containing vials during the study were stored in biochemical

laboratory and at the end of the study the samples were transferred to bio-analytical

department for analysis in insulated box containing coolant bags. The samples received at the

analytical facility were frozen and in good condition

6.14.2 Method of MeasurementThe Rosuvastatin concentration levels in plasma were determined by a validated LC-MS/MS

method for samples from 48 volunteers in the Bioanalytical department of Torrent

Pharmaceuticals Ltd., India. The analyst did not have access to the randomization schedule

during the course of the analysis.

6.14.3 Randomization and Blinding

The order of receiving the test and reference products for each subject during the periods of

the study will be determined according to randomization schedule (Generated using SAS®

Version 9.1.3). The randomization will be balanced and the code will be kept under

controlled access.

This study was comprised of a randomized open label. However analysts were blinded to the

sequence of administration of Test and Reference formulation to minimize bias.

74


6.15 Discussion of the Study Design

A monocentric, randomized, crossover design is typically employed in bioequivalence

studies, and the same was considered to be the most appropriate for this study.

The study was open label in nature, because blood concentration levels can not be influenced

by the knowledge of the identity of the treatment. Only analyst was blinded to the

randomization schedule.

Sampling was done up to 96.0 hours post dose such that plasma concentration could be

measured for more than four longest registered half-lives of Rosuvastatin.

The washout period of 07 days was maintained to allow the complete elimination of the drug

before subsequent dosing and to avoid carry over effects.

The objective of this study was, however, not the investigation of the pharmacokinetics of

different drugs, but the comparison of the pharmacokinetic profiles of the drug entities itself.

The group of volunteers under investigation could be as homogeneous as possible that’s why

only male volunteers were recruited to maintain homogeneity in study populations. This study

was conducted under fed condition. The volunteers were administered a single oral dose of

Rosuvastatin Calcium 40mg tablet of either test (A) or the reference (B) product in each

period in one of the two sequences AB or BA as per the randomization.

The following precautions were incorporated into the study to minimize bias: Volunteers were

sequentially assigned to randomly ordered treatment, Volunteers enrolment was dependent on

satisfactory fulfillment of the given inclusion criteria and exclusion criteria, The

circumstances when individual volunteers were withdrawn prior to completion of the study

were specified, The analyst was blinded to the randomization schedule.

6.16 Safety AssessmentSafety assessments were done based on clinical observations, laboratory data at the beginning

and at the end of the study, and evaluation of adverse events observed during the course of the

study.

Screening assessment comprised of personal details, detailed medical history followed by

general physical examination and laboratory investigations (hematology, biochemistry

75


including creatinine kinase, urine analysis and serology), ECG and X-ray (which were done

prior to including the volunteer into the study).

After successful completion of initial screening, volunteers were called for the study specific

screening test (i.e. Creatinine kinase).

Volunteers were instructed to report any side effect (nature, severity, onset and

disappearance) whenever it appears. Vitals (blood pressure and pulse rate) were monitored at

enrolment, pre-dose, 2.0, 4.0 and 6.0 hours post-dose, discharge and whenever necessary.

Vital signs like sitting blood pressure, radial pulse, respiratory rate and oral temperature were

measured and recorded at the time of volunteer check-in, pre-dose (in the morning of the day

of dosing) and at checkout in each period. All volunteers had their vitals within clinically

acceptable range.

At the beginning of second period volunteers were questioned concerning unusual symptoms,

which may have occurred after the previous administration of the test or reference drug.

Clinical examination of all the volunteers was done at the time of check-in and at checkout.

For the safety of the volunteers, hematology and biochemistry (including creatinine kinase)

investigations were repeated at the end of the study. The abnormal post study laboratory

investigations were considered as adverse events and were followed up until resolution

Analytical Phase: December 10, 2009 to December 21, 2009

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6.17 Pharmacokinetic assessmentThe following pharmacokinetic parameters were determined for test and reference drugs in

each subject using WinNonlin® 5.2

a) Cmax Maximum concentration of drug observed in plasma.b) Tmax Time required reaching maximum concentration of drug in

plasma.c) AUC(0-t) Area under the plasma concentration vs time curve from time

zero to the last measurable concentration time t. It was calculated using linear trapezoidal method. (AUCt)

d) AUC(0-inf) Area under the plasma concentration vs time curve from time zero to time infinity. (AUCinf)

e) AUC%Extrap Extrapolated AUC percentage of total AUC.f) Kel Elimination Rate constantg) Thalf Time taken by plasma concentration to reduce to 50% during

the elimination phase

6.17.1 Primary Pharmacokinetic Variable(s)

The Cmax, AUC(0-t) and AUC(0-inf) for Rosuvastatin were the primary pharmacokinetic

variables calculated in the study. The bioequivalence criteria were based on the 90%

Confidence Intervals of the above parameters for Rosuvastatin

6.18 Statistical Methods and Determination of Sample Size

6.18.1 Statistical and Analytical PlansPlasma concentration-time data were to be presented for both test and reference product of

Rosuvastatin. The pharmacokinetic parameters [Tmax, Cmax, AUC(0-t), AUC(0-inf), AUC_

%Extrap, Kel and Thalf] were to be calculated for Rosuvastatin.

Summary StatisticsDescriptive statistics were to be calculated for concentration-time data and all

pharmacokinetic parameters of Rosuvastatin.

77


Ratio AnalysisDifference of test and reference formulation and ratio analysis of test over reference

formulation were to be calculated for log-transformed and un-transformed primary

pharmacokinetic parameters Cmax, AUC(0-t) and AUC(0-inf) of Rosuvastatin respectively.

Assessment of ANOVAAnalysis of Variance was to be performed on the log-transformed data of Cmax, AUC(0-t)

and AUC(0-inf) for Rosuvastatin. The sequence, subject within sequence, period and

formulation were to be considered as sources of variation. The sequence effect was to be

tested using the subjects within sequence effect as an error term. The formulation and period

effects were to be tested against the residual mean square error. Probability (p) values were to

be derived from Type III sums of squares.

Subject Variability Inter-subject and Intra-subject percentage coefficient of variance (%CV) were to be calculated

for Cmax, AUC(0-t) and AUC(0-inf) of Rosuvastatin.

Standards of Bioequivalence The bioequivalence acceptance interval was set to range of 80%-125% for Cmax, AUC(0-t)

and AUC(0-inf) of Rosuvastatin.

To justify the bioequivalence claim; the 90% confidence interval of the intra-individual mean

ratio (Test/Reference) were computed for the log-transformed primary pharmacokinetic

parameters [Cmax, AUC(0-t) and AUC(0-inf)] of Rosuvastatin.

Evaluation of TmaxNon-parametric Wilcoxon Signed Rank test was to be performed on actual value of test and

reference formulation of secondary pharmacokinetic parameter Tmax for Rosuvastatin.

Level of SignificanceFor all analyses, effects or differences were to be considered statistically significant if the

probability found less than 0.05

78


Software detailsPharmacokinetic and bioequivalence analyses were to be performed using the WinNonlin®

software version 5.2 and further statistical analysis was to be performed using the SAS®

software version 9.1.3

6.19 Protocol DeviationsFollowing protocol deviations were recorded during clinical phase conduct of the study:

• Time point deviation

• Missing sample deviation

6.20 Data Sets Analyzed

Total 48 volunteers were enrolled and amongst that 43 volunteers had completed the study

according to the protocol. Total 05 volunteers (Vol. No. 27, 18, 43, 25 and 45) had not

completed the study as per the protocol.

As per the protocol, samples from all volunteers were analyzed in the Bioanalytical laboratory

to determine the plasma concentrations of Rosuvastatin. However data of withdrawn (Vol.

No. 18, 27, 25 & 45) and dropout (Vol. No. 43) volunteers were excluded from the

pharmacokinetic and statistical evaluation. Hence, total 43 volunteers were evaluated for

statistical and bioequivalence analysis of Rosuvastatin.

6.21 Pharmacokinetic Results and Tabulations of Individual vol. data

6.21.1 Pharmacokinetic Analysis

Non-compartmental method (WinNonlin® 5.2) was used to estimate pharmacokinetic

parameters of Rosuvastatin: [Tmax, Cmax, AUC(0-t), AUC(0-inf), AUC_%Extrap, Kel and Thalf].

Linear Trapezoidal (Linear Interpolation) method was used for AUC computation. The BLQ

(Below Limit of Quantification) values at the initial absorption or at the terminal elimination

phase were considered as zero. Missing values such as SNS (Sample Not Submitted) were

ignored from the pharmacokinetic evaluations.

The result summary for primary and secondary pharmacokinetic parameters of product Test

(A) and Reference (B) are presented in below table for Rosuvastatin.

79


7.0 RESULTS

80


7.1 Study Subjects

7.1.1 Measurements of Treatment Compliance

Compliance for dosing was assessed by monitoring the subject till he swallowed tablet and

then a thorough check of the oral cavity was done by the study personnel using a torch. The

duplicate label of dispensed container was then pasted on the ‘Dosing’ section of individual

Case Report Form (CRF). It was further confirmed by the measurement of plasma levels of

the drug taken in the study.

7.2 Pharmacokinetic Data

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Table 13: Mean Plasma Concentration for Rosuvastatin

Time(hr)

RosuvastatinTest (A)

RosuvastatinReference (B)

N Mean ± SD CV% N Mean ± SD CV%0.00 43 0.000 0.00 - 43 0.013 0.08 655.70.33 43 0.956 1.14 119.7 43 1.222 1.72 141.10.67 43 2.740 2.40 87.4 43 3.025 3.52 116.31.00 43 5.923 4.71 79.5 43 6.218 5.15 82.81.50 43 12.527 8.47 67.6 43 13.625 10.46 76.72.00 43 18.665 9.89 53.0 43 19.180 12.04 62.82.25 43 21.707 10.51 48.4 43 21.355 11.86 55.52.50 43 24.626 12.13 49.2 43 23.004 12.50 54.42.75 43 27.238 13.33 48.9 43 25.359 12.84 50.63.00 43 27.794 12.08 43.4 43 26.420 13.24 50.13.25 43 27.864 12.04 43.2 43 27.972 14.91 53.33.50 43 29.525 12.92 43.7 43 30.085 18.49 61.54.00 43 29.490 14.85 50.4 43 29.023 16.37 56.44.50 43 28.596 12.80 44.8 43 30.562 18.41 60.25.00 43 26.681 12.03 45.1 43 27.047 13.82 51.15.50 43 31.583 13.13 41.6 43 32.082 16.08 50.16.00 43 25.316 11.56 45.7 43 25.210 13.14 52.17.00 43 16.562 7.40 44.7 43 16.448 8.07 49.18.00 43 12.973 5.54 42.7 43 12.754 6.34 49.710.00 43 9.632 4.30 44.6 43 9.082 4.50 49.512.00 43 6.832 2.94 43.1 43 6.705 3.32 49.616.00 43 4.500 1.69 37.5 43 4.468 2.25 50.424.00 43 2.839 1.05 37.0 43 2.858 1.24 43.548.00 43 0.915 0.48 52.4 43 0.937 0.60 64.072.00 a42 0.205 0.25 121.8 43 0.207 0.29 140.896.00 b41 0.029 0.13 463.6 43 0.054 0.18 337.9

a 72.00hr= Subject-3 (Period-I) / SNSb 96.00hr= Subject-3 & 23 (Period-I) / SNS

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Table 14: Pharmacokinetic Parameters of the Test Formulation for Rosuvastatin

Test Formulation: Rosuvastatin

Subject Sequence Tmax(hr)

Cmax(ng/mL)

AUC(0-t)(hr.ng/mL)

AUC(0-inf)(hr.ng/mL)

AUC_%Extrap(%)

Kel(1/hr)

Thalf(hr)

1 BA 2.25 28.654 292.959 302.976 3.306 0.039 17.902 AB 5.50 31.509 270.476 287.542 5.935 0.036 19.173 AB 5.50 28.394 236.481 260.709 9.293 0.039 17.714 BA 2.75 41.204 273.966 281.839 2.793 0.059 11.765 BA 5.50 24.659 203.581 214.312 5.007 0.052 13.386 AB 5.50 26.533 180.250 186.161 3.175 0.060 11.487 AB 6.00 51.257 358.426 368.538 2.744 0.044 15.648 BA 2.75 17.565 176.652 194.821 9.326 0.044 15.829 AB 5.50 24.964 212.599 222.324 4.374 0.060 11.5010 BA 4.00 81.865 369.000 377.693 2.302 0.083 8.3611 AB 3.25 59.797 452.617 463.436 2.335 0.044 15.6912 BA 5.50 57.658 535.985 549.676 2.491 0.046 14.9413 BA 2.75 62.574 334.981 343.841 2.577 0.040 17.2014 AB 5.50 16.968 196.890 203.734 3.359 0.045 15.5515 AB 5.50 43.881 375.583 401.673 6.495 0.029 24.1116 BA 3.50 32.350 300.919 313.014 3.864 0.060 11.4817 BA 3.50 50.461 408.055 420.667 2.998 0.040 17.1819 AB 2.75 25.725 246.580 262.635 6.113 0.049 14.0520 AB 2.50 16.835 96.820 107.588 10.009 0.134 5.1821 AB 5.50 48.997 374.618 383.788 2.389 0.071 9.7922 BA 5.50 42.104 358.988 368.078 2.469 0.046 15.0023 AB 5.00 59.831 511.811 520.347 1.640 0.048 14.4024 BA 3.50 36.238 356.346 361.377 1.392 0.061 11.3626 AB 5.50 37.669 318.911 325.833 2.124 0.046 15.0928 BA 3.25 31.602 254.920 279.203 8.697 0.038 18.2829 AB 6.00 43.299 338.120 355.796 4.968 0.052 13.3030 BA 2.00 42.306 333.712 345.033 3.281 0.056 12.3031 BA 2.50 26.403 196.985 202.070 2.516 0.063 10.9532 AB 5.50 59.206 478.966 483.943 1.028 0.062 11.1333 BA 4.00 66.219 558.623 573.960 2.672 0.043 16.1134 BA 3.00 21.572 222.171 233.683 4.926 0.035 19.7035 AB 2.00 45.066 461.641 469.052 1.580 0.050 13.8136 AB 5.50 51.000 526.991 537.969 2.041 0.046 15.0737 BA 3.50 22.158 194.612 202.655 3.969 0.066 10.4838 BA 2.75 50.193 298.338 320.549 6.929 0.046 15.2039 AB 2.00 23.156 166.060 173.171 4.106 0.059 11.7940 AB 5.50 20.901 236.098 246.789 4.332 0.041 17.1141 AB 2.75 30.986 232.577 244.616 4.921 0.048 14.5642 BA 4.50 35.938 346.110 374.044 7.468 0.044 15.7544 BA 3.50 28.292 253.916 268.057 5.275 0.053 13.1046 AB 3.50 13.588 139.655 163.054 14.350 0.033 21.1747 AB 3.25 28.044 251.095 258.084 2.708 0.064 10.77

83


Test Formulation: Rosuvastatin


Cmax(ng/mL)

AUC(0-t)(hr.ng/mL)


AUC_%Extrap(%)

Kel(1/hr)

Thalf(hr)

48 BA 5.50 24.663 226.143 233.082 2.977 0.062 11.19N 43 43 43 43 43 43 43

Mean 4.08 37.495 306.052 318.312 4.355 0.052 14.31SD 1.3 15.78 113.59 113.51 2.76 0.02 3.5Min 2.00 13.588 96.820 107.588 1.028 0.029 5.18

Median 3.50 32.350 292.959 302.976 3.306 0.048 14.56Max 6.00 81.865 558.623 573.960 14.350 0.134 24.11CV% 32.9 42.1 37.1 35.7 63.3 32.6 24.7

Geometric Mean 3.85 34.381 285.497 298.621 3.688 0.050 13.85

84


Table 15: Pharmacokinetic Parameters of the Reference Formulation for Rosuvastatin

Reference Formulation: Rosuvastatin


Cmax(ng/mL)

AUC(0-t)(hr.ng/mL)


AUC_%Extrap(%)

Kel(1/hr)

Thalf(hr)

1 BA 5.50 39.919 361.034 372.843 3.167 0.038 18.312 AB 5.50 4.157 34.728 45.204 23.176 0.085 8.163 AB 5.00 41.984 383.319 406.785 5.769 0.028 25.184 BA 3.25 36.470 249.986 258.516 3.299 0.063 10.975 BA 5.50 24.182 205.877 219.095 6.033 0.050 13.766 AB 5.50 28.528 195.317 201.708 3.169 0.061 11.367 AB 6.00 71.427 485.422 495.825 2.098 0.047 14.818 BA 2.00 22.501 156.036 167.149 6.649 0.049 14.249 AB 5.50 23.002 235.694 253.415 6.993 0.051 13.7210 BA 4.50 89.275 379.498 387.279 2.009 0.043 16.2911 AB 3.25 73.185 473.630 488.629 3.070 0.059 11.8112 BA 5.50 51.591 473.554 483.108 1.978 0.050 13.7413 BA 4.00 19.627 191.715 199.602 3.951 0.038 18.1014 AB 3.50 23.136 248.539 256.931 3.266 0.044 15.8115 AB 4.50 38.540 432.677 444.278 2.611 0.034 20.2016 BA 5.50 22.198 214.561 223.922 4.181 0.057 12.0617 BA 5.50 50.026 380.924 387.655 1.736 0.052 13.4419 AB 3.50 37.126 305.794 326.542 6.354 0.045 15.5120 AB 2.25 16.023 97.179 103.837 6.411 0.143 4.8621 AB 5.50 33.455 324.462 450.279 27.942 0.017 41.1822 BA 5.50 41.031 357.366 363.469 1.679 0.052 13.2623 AB 3.50 77.729 610.900 632.938 3.482 0.041 17.1124 BA 2.50 21.558 227.772 243.851 6.594 0.050 13.9126 AB 2.50 24.473 189.050 213.119 11.294 0.066 10.4528 BA 5.50 42.652 291.504 306.355 4.848 0.052 13.3329 AB 5.50 45.470 393.974 406.221 3.015 0.063 10.9430 BA 3.25 28.561 273.225 278.989 2.066 0.073 9.5131 BA 5.50 31.642 215.929 220.185 1.933 0.074 9.4232 AB 3.25 54.890 515.457 520.841 1.034 0.056 12.4033 BA 3.50 78.055 560.106 570.366 1.799 0.050 13.8634 BA 2.25 27.932 203.466 224.065 9.194 0.037 18.6635 AB 5.50 51.365 506.748 516.818 1.948 0.048 14.3636 AB 3.50 86.206 670.463 691.231 3.005 0.039 17.9537 BA 5.50 14.343 142.065 149.777 5.149 0.056 12.3438 BA 2.75 43.924 290.953 306.393 5.039 0.053 13.0839 AB 5.50 18.611 154.331 167.627 7.932 0.043 16.1740 AB 6.00 20.690 246.141 256.873 4.178 0.036 19.5241 AB 2.00 49.842 309.675 317.399 2.434 0.060 11.5442 BA 2.75 40.994 381.077 403.788 5.624 0.039 17.8144 BA 2.25 28.556 195.247 199.697 2.228 0.071 9.7346 AB 3.50 20.532 177.018 189.332 6.504 0.049 14.2347 AB 5.50 24.835 226.840 235.192 3.551 0.060 11.55

85


Reference Formulation: Rosuvastatin


Cmax(ng/mL)

AUC(0-t)(hr.ng/mL)


AUC_%Extrap(%)

Kel(1/hr)

Thalf(hr)

48 BA 2.00 21.059 191.989 202.153 5.028 0.053 13.10N 43 43 43 43 43 43 43

Mean 4.20 38.170 306.075 320.681 5.196 0.053 14.60SD 1.4 20.44 142.40 145.13 5.10 0.02 5.5Min 2.00 4.157 34.728 45.204 1.034 0.017 4.86

Median 4.50 33.455 273.225 278.989 3.551 0.050 13.74Max 6.00 89.275 670.463 691.231 27.942 0.143 41.18CV% 32.5 53.6 46.5 45.3 98.1 36.0 37.7

Geometric Mean 3.96 33.011 271.124 286.441 3.989 0.050 13.83

7.2 MEAN PLASMA CONCENTRATION GRAPHS

Figure 6-Linear Mean plasma concentration vs. time curve of Rosuvastatin

Figure 7-Semilog Mean plasma concentration vs. time curve for Rosuvastatin

86


87


Table 16: Ratio analysis for Pharmacokinetic parameters for Rosuvastatin

Rosuvastatin: Cmax

Subject Sequence A (Test) B (Ref) %Ratio[Test/Ref]

Ln[A(Test)]

Ln[B(Ref)]

Diff[Ln(Test-Ref)]

1 BA 28.654 39.919 71.780 3.355 3.687 -0.3322 AB 31.509 4.157 757.975 3.450 1.425 2.0253 AB 28.394 41.984 67.631 3.346 3.737 -0.3914 BA 41.204 36.470 112.981 3.719 3.596 0.1225 BA 24.659 24.182 101.973 3.205 3.186 0.0206 AB 26.533 28.528 93.007 3.278 3.351 -0.0727 AB 51.257 71.427 71.761 3.937 4.269 -0.3328 BA 17.565 22.501 78.063 2.866 3.114 -0.2489 AB 24.964 23.002 108.530 3.217 3.136 0.08210 BA 81.865 89.275 91.700 4.405 4.492 -0.08711 AB 59.797 73.185 81.707 4.091 4.293 -0.20212 BA 57.658 51.591 111.760 4.055 3.943 0.11113 BA 62.574 19.627 318.816 4.136 2.977 1.15914 AB 16.968 23.136 73.340 2.831 3.141 -0.31015 AB 43.881 38.540 113.858 3.781 3.652 0.13016 BA 32.350 22.198 145.734 3.477 3.100 0.37717 BA 50.461 50.026 100.870 3.921 3.913 0.00919 AB 25.725 37.126 69.291 3.247 3.614 -0.36720 AB 16.835 16.023 105.068 2.823 2.774 0.04921 AB 48.997 33.455 146.456 3.892 3.510 0.38222 BA 42.104 41.031 102.615 3.740 3.714 0.02623 AB 59.831 77.729 76.974 4.092 4.353 -0.26224 BA 36.238 21.558 168.095 3.590 3.071 0.51926 AB 37.669 24.473 153.921 3.629 3.198 0.43128 BA 31.602 42.652 74.093 3.453 3.753 -0.30029 AB 43.299 45.470 95.225 3.768 3.817 -0.04930 BA 42.306 28.561 148.125 3.745 3.352 0.39331 BA 26.403 31.642 83.443 3.273 3.454 -0.18132 AB 59.206 54.890 107.863 4.081 4.005 0.07633 BA 66.219 78.055 84.836 4.193 4.357 -0.16434 BA 21.572 27.932 77.230 3.071 3.330 -0.25835 AB 45.066 51.365 87.737 3.808 3.939 -0.13136 AB 51.000 86.206 59.161 3.932 4.457 -0.52537 BA 22.158 14.343 154.487 3.098 2.663 0.43538 BA 50.193 43.924 114.272 3.916 3.782 0.13339 AB 23.156 18.611 124.421 3.142 2.924 0.21940 AB 20.901 20.690 101.020 3.040 3.030 0.01041 AB 30.986 49.842 62.168 3.434 3.909 -0.47542 BA 35.938 40.994 87.666 3.582 3.713 -0.13244 BA 28.292 28.556 99.076 3.343 3.352 -0.00946 AB 13.588 20.532 66.180 2.609 3.022 -0.413

88


Rosuvastatin: Cmax


Ln[A(Test)]

Ln[B(Ref)]

Diff[Ln(Test-Ref)]

47 AB 28.044 24.835 112.921 3.334 3.212 0.12248 BA 24.663 21.059 117.114 3.205 3.047 0.158

N 43 43 43 43 43 43Mean 37.495 38.170 120.487 3.537 3.497 0.041

SD 15.78 20.44 108.43 0.43 0.58 0.45Min 13.588 4.157 59.161 2.609 1.425 -0.525

Median 32.350 33.455 100.870 3.477 3.510 0.009Max 81.865 89.275 757.975 4.405 4.492 2.025CV% 42.1 53.6 90.0 12.0 16.5 1096.4

Geometric Mean 34.381 33.011 104.148 3.512 3.441 -

Rosuvastatin: AUC(0-t)


Ln[A(Test)]

Ln[B(Ref)]

Diff[Ln(Test-Ref)]

1 BA 292.959 361.034 81.144 5.680 5.889 -0.2092 AB 270.476 34.728 778.847 5.600 3.548 2.0533 AB 236.481 383.319 61.693 5.466 5.949 -0.4834 BA 273.966 249.986 109.592 5.613 5.521 0.0925 BA 203.581 205.877 98.885 5.316 5.327 -0.0116 AB 180.250 195.317 92.286 5.194 5.275 -0.0807 AB 358.426 485.422 73.838 5.882 6.185 -0.3038 BA 176.652 156.036 113.213 5.174 5.050 0.1249 AB 212.599 235.694 90.201 5.359 5.463 -0.10310 BA 369.000 379.498 97.234 5.911 5.939 -0.02811 AB 452.617 473.630 95.563 6.115 6.160 -0.04512 BA 535.985 473.554 113.184 6.284 6.160 0.12413 BA 334.981 191.715 174.728 5.814 5.256 0.55814 AB 196.890 248.539 79.219 5.283 5.516 -0.23315 AB 375.583 432.677 86.805 5.928 6.070 -0.14216 BA 300.919 214.561 140.249 5.707 5.369 0.33817 BA 408.055 380.924 107.122 6.011 5.943 0.06919 AB 246.580 305.794 80.636 5.508 5.723 -0.21520 AB 96.820 97.179 99.630 4.573 4.577 -0.00421 AB 374.618 324.462 115.458 5.926 5.782 0.14422 BA 358.988 357.366 100.454 5.883 5.879 0.00523 AB 511.811 610.900 83.780 6.238 6.415 -0.17724 BA 356.346 227.772 156.448 5.876 5.428 0.44826 AB 318.911 189.050 168.692 5.765 5.242 0.52328 BA 254.920 291.504 87.450 5.541 5.675 -0.13429 AB 338.120 393.974 85.823 5.823 5.976 -0.15330 BA 333.712 273.225 122.138 5.810 5.610 0.200

89


Rosuvastatin: AUC(0-t)


Ln[A(Test)]

Ln[B(Ref)]

Diff[Ln(Test-Ref)]

31 BA 196.985 215.929 91.227 5.283 5.375 -0.09232 AB 478.966 515.457 92.921 6.172 6.245 -0.07333 BA 558.623 560.106 99.735 6.325 6.328 -0.00334 BA 222.171 203.466 109.193 5.403 5.315 0.08835 AB 461.641 506.748 91.099 6.135 6.228 -0.09336 AB 526.991 670.463 78.601 6.267 6.508 -0.24137 BA 194.612 142.065 136.988 5.271 4.956 0.31538 BA 298.338 290.953 102.538 5.698 5.673 0.02539 AB 166.060 154.331 107.600 5.112 5.039 0.07340 AB 236.098 246.141 95.920 5.464 5.506 -0.04241 AB 232.577 309.675 75.104 5.449 5.736 -0.28642 BA 346.110 381.077 90.824 5.847 5.943 -0.09644 BA 253.916 195.247 130.049 5.537 5.274 0.26346 AB 139.655 177.018 78.893 4.939 5.176 -0.23747 AB 251.095 226.840 110.693 5.526 5.424 0.10248 BA 226.143 191.989 117.790 5.421 5.257 0.164

N 43 43 43 43 43 43Mean 306.052 306.075 118.686 5.654 5.603 0.052

SD 113.59 142.40 105.91 0.39 0.54 0.38Min 96.820 34.728 61.693 4.573 3.548 -0.483

Median 292.959 273.225 98.885 5.680 5.610 -0.011Max 558.623 670.463 778.847 6.325 6.508 2.053CV% 37.1 46.5 89.2 6.8 9.7 739.8

Geometric Mean 285.497 271.124 105.301 5.641 5.574 -

Rosuvastatin: AUC(0-inf)


Ln[A(Test)]

Ln[B(Ref)]

Diff[Ln(Test-Ref)]

1 BA 302.976 372.843 81.261 5.714 5.921 -0.2082 AB 287.542 45.204 636.098 5.661 3.811 1.8503 AB 260.709 406.785 64.090 5.563 6.008 -0.4454 BA 281.839 258.516 109.022 5.641 5.555 0.0865 BA 214.312 219.095 97.817 5.367 5.390 -0.0226 AB 186.161 201.708 92.292 5.227 5.307 -0.0807 AB 368.538 495.825 74.328 5.910 6.206 -0.2978 BA 194.821 167.149 116.555 5.272 5.119 0.1539 AB 222.324 253.415 87.731 5.404 5.535 -0.13110 BA 377.693 387.279 97.525 5.934 5.959 -0.02511 AB 463.436 488.629 94.844 6.139 6.192 -0.05312 BA 549.676 483.108 113.779 6.309 6.180 0.129

90


Rosuvastatin: AUC(0-inf)


Ln[A(Test)]

Ln[B(Ref)]

Diff[Ln(Test-Ref)]

13 BA 343.841 199.602 172.263 5.840 5.296 0.54414 AB 203.734 256.931 79.295 5.317 5.549 -0.23215 AB 401.673 444.278 90.410 5.996 6.096 -0.10116 BA 313.014 223.922 139.787 5.746 5.411 0.33517 BA 420.667 387.655 108.516 6.042 5.960 0.08219 AB 262.635 326.542 80.429 5.571 5.789 -0.21820 AB 107.588 103.837 103.613 4.678 4.643 0.03521 AB 383.788 450.279 85.233 5.950 6.110 -0.16022 BA 368.078 363.469 101.268 5.908 5.896 0.01323 AB 520.347 632.938 82.211 6.254 6.450 -0.19624 BA 361.377 243.851 148.196 5.890 5.497 0.39326 AB 325.833 213.119 152.888 5.786 5.362 0.42528 BA 279.203 306.355 91.137 5.632 5.725 -0.09329 AB 355.796 406.221 87.587 5.874 6.007 -0.13330 BA 345.033 278.989 123.673 5.844 5.631 0.21231 BA 202.070 220.185 91.773 5.309 5.394 -0.08632 AB 483.943 520.841 92.916 6.182 6.255 -0.07333 BA 573.960 570.366 100.630 6.353 6.346 0.00634 BA 233.683 224.065 104.293 5.454 5.412 0.04235 AB 469.052 516.818 90.758 6.151 6.248 -0.09736 AB 537.969 691.231 77.828 6.288 6.538 -0.25137 BA 202.655 149.777 135.305 5.312 5.009 0.30238 BA 320.549 306.393 104.620 5.770 5.725 0.04539 AB 173.171 167.627 103.307 5.154 5.122 0.03340 AB 246.789 256.873 96.074 5.509 5.549 -0.04041 AB 244.616 317.399 77.069 5.500 5.760 -0.26042 BA 374.044 403.788 92.634 5.924 6.001 -0.07744 BA 268.057 199.697 134.232 5.591 5.297 0.29446 AB 163.054 189.332 86.120 5.094 5.244 -0.14947 AB 258.084 235.192 109.734 5.553 5.460 0.09348 BA 233.082 202.153 115.300 5.451 5.309 0.142

N 43 43 43 43 43 43Mean 318.312 320.681 114.522 5.699 5.658 0.042

SD 113.51 145.13 84.51 0.37 0.51 0.35Min 107.588 45.204 64.090 4.678 3.811 -0.445

Median 302.976 278.989 97.525 5.714 5.631 -0.025Max 573.960 691.231 636.098 6.353 6.538 1.850CV% 35.7 45.3 73.8 6.5 9.1 840.5

Geometric Mean 298.621 286.441 104.252 5.687 5.633 -

91


Table 17: Summary Table of Pharmacokinetic Variables for Rosuvastatin

Test : Rosuvastatin

Statistics: Tmax(hr)

Cmax(ng/mL)

AUC(0-t)(hr.ng/

mL)

AUC(0-inf)(hr.ng/

mL)

AUC_Extrap

(%)

Kel(1/hr)

Thalf(hr)

N 43 43 43 43 43 43 43Mean 4.08 37.495 306.052 318.312 4.355 0.052 14.31SD 1.3 15.78 113.59 113.51 2.76 0.02 3.5Min 2.00 13.588 96.820 107.588 1.028 0.029 5.18

Median 3.50 32.350 292.959 302.976 3.306 0.048 14.56Max 6.00 81.865 558.623 573.960 14.350 0.134 24.11%CV 32.9 42.1 37.1 35.7 63.3 32.6 24.7GM 3.85 34.381 285.497 298.621 3.688 0.050 13.85

Reference: Rosuvastatin

Statistics: Tmax

(hr)Cmax

(ng/mL)

AUC(0-t)

(hr.ng/mL)

AUC(0-inf)

(hr.ng/mL)

AUC_Extrap

(%)

Kel(1/hr)

Thalf

(hr)

N 43 43 43 43 43 43 43Mean 4.20 38.170 306.075 320.681 5.196 0.053 14.60SD 1.4 20.44 142.40 145.13 5.10 0.02 5.5Min 2.00 4.157 34.728 45.204 1.034 0.017 4.86

Median 4.50 33.455 273.225 278.989 3.551 0.050 13.74Max 6.00 89.275 670.463 691.231 27.942 0.143 41.18%CV 32.5 53.6 46.5 45.3 98.1 36.0 37.7GM 3.96 33.011 271.124 286.441 3.989 0.050 13.83

The log-transformed pharmacokinetic parameters Cmax, AUC(0-t) and AUC(0-inf) of

Rosuvastatin were subjected to analysis of variance (ANOVA) with the main effects of

sequence, formulation and period at 5% level of significance. The ANOVA (p-values) are

presented in below table with percentage intra-subject variability of Rosuvastatin.

92


Table 18: Summary Tables for ANOVA Results

PK Parameters[N=43]

ANOVA ResultsProbability (p)-value Intra-subject

CV (%)Sequence Formulation PeriodLn(Cmax) 0.69 0.55 0.55 32.57

Ln(AUC(0-t)) 0.95 0.37 0.36 27.58Ln(AUC(0-inf)) 0.96 0.42 0.23 24.98

Ratio analysis of untransformed and difference of log transformed primary pharmacokinetic

parameters [Cmax, AUC(0-t) and AUC(0-inf)] for test & reference formulation were

calculated for Rosuvastatin. The percentage geometric least square mean (LSM) ratio of test

and reference values was expressed as point estimates of relative bioavailability.

Average bioequivalence was evaluated based on the 90% CI for the intra-individual mean

ratio of log-transformed Cmax, AUC(0-t) and AUC(0-inf) of the test to the reference

formulation for Rosuvastatin were found within the accepted bioequivalence range of

80.00%-125.00%.

The Geometric LSM ratio and 90% confidence interval of Rosuvastatin are presented in

below table.

Table 19: 90% Confidence Intervals, %Geometric LSM Ratio and %Intra-subject CV

PK Parameters[N=43]

90% Confidence Interval(Lower limit-Upper limit)

Geometric LSM Ratio (%)

(Test/Reference)

Intra SubjectCV%

Ln(Cmax) 92.90-116.99 104.25 32.57

Ln(AUC(0-t)) 95.56-116.32 105.43 27.58

Ln(AUC(0-inf)) 95.49-114.17 104.41 24.98

Table 20: Extract of Analysis of Variance and Construction of Confidence Interval for Rosuvastatin

93


Statistics [N=43] Ln(Cmax) Ln(AUC(0-t)) Ln(AUC(0-inf))

Type III Tests of Analysis Variance [Probability (p)–values]

Sequence 0.69 0.95 0.96

Formulation 0.55 0.37 0.42

Period 0.55 0.36 0.23

Error Term

Mean Square Error* 0.10 0.07 0.06

Mean Square-Subject(Seq)** 0.42 0.38 0.35

Intra-subject and Inter-subject Variability:

Intra-subject CV (%) 32.57 27.58 24.98

Inter-subject CV (%) 41.80 40.49 39.29

%Geometric Least Square Mean

Test (A) 34.420 285.704 298.822

Reference (B) 33.017 270.982 286.201%Geometric LSM Ratio

(Test/Ref) 104.25 105.43 104.41

90% Confidence Interval

Lower CI (%) 92.90 95.56 95.49

Upper CI (%) 116.99 116.32 114.17

Power 0.94 0.98 0.99

*Mean Square Error =An Error term of Formulation and Period effect

** Mean Square - Subject(Seq) Error =An Error term of Sequence effect

7.4 Safety Evaluation

7.4.1 Brief Summary of Adverse Events

94


There was no serious AE reported in the study. Total four incidences of adverse events

reported during clinical phase where three volunteers reported vomiting (i.e. enrolment no. 27

and 45 were reported vomiting following the administration of test drug while enrolment no

18 was reported vomiting following the administration of reference drug) and enrolment no.

25 was reported giddiness in period I following the administration of reference drug. Total

elevan adverse events were reported during post study safety evaluation. These AEs were

mild in nature and resolved completely without sequelae (except enrolment no. 18 who was

given the treatment of Capsule of Domstal-O).

Total 11 volunteers’ laboratory parameters were considered clinically significant as per

physician’s judgment and were advised for follow up to the facility. All post study AEs were

mild in nature and resolved completely without sequelae (except for enrolment no. 19 & 30

who were lost to follow up).

Table 21 and 22 presents the details of the AE which were reported during study and post

study laboratory investigations.

Table 21 – Summary of adverse event during the clinical phase

EnNo AE

Date & time of onset

Date & Time of

resolutionSeverity Serious

-ness

Study drug

related

Treatment required

Current Status

Volunteer status

25 Giddiness

28/11/09, 08:50

28/11/09, 10:50 Mild Not

serious Possible None Recovered Continued

27 Vomiting 28/11/09, 08:18 NA Mild Not

serious Unlikely None Recovered withdrawn


serious Unlikely Cap. Domstal-0 Recovered Withdrawn


serious Unlikely None Recovered Withdrawn

Table 22 – Summary of post study adverse event

En.No.

AbnormalLaboratory parameter

with Values

Date &Time of last

Dose(hh:mm)

Date ofSample

collection Severity Seriousness

Date of Repeatsample( if any)

Out ComeStudy drug

related

Treatmentrequired

95


02 Triglycerides – 332.52 mg/dl

05/12/0908:02 09/12/09 Mild Not serious 14/12/09 Resolved Unlikely None

27 S. Creatinine – 1.75 mg/dl

28/11/0908:04 28/11/09 Mild Not Serious 08/12/09 Resolved Unlikely None

18

S. Creatinine – 2.42 mg/dl

28/11/0908:10 28/11/09 Mild Not serious 30/11/09 Resolved Unlikely None

Creatinine Kinase – 282.18 U/L

28/11/0908:10 28/11/09 Mild Not serious

30/11/09( 1110.44)10/12/09(229.77)

Resolved Possible None

15 Creatinine Kinase – 1108.07 U/L

05/12/0908:04 09/12/09 Mild Not serious

14/12/09(1182.62)18/12/09(175.36)

Resolved Possible None

19 S. Potassium – 5.7 mEq/L

05/12/0908:12 09/12/09 Mild Not serious Lost to

follow-upLost to

follow-up Unlikely None


05/12/0908:20 09/12/09 Mild Not Serious 14/12/09 Resolved Possible None



28 RBS – 207.82 mg/dl



05/12/0908:10 09/12/09 Mild Not Serious Lost to

follow-upLost to

follow-up Possible None



7.4.3 Clinical Laboratory EvaluationScreening was carried out before the start of the study and post study safety assessment was

done at the end of the clinical part for each volunteer. In general the laboratory parameters

were within the normal range of the laboratory or clinically not significant values except few

volunteers (enrolment No. 02, 15, 18, 19, 23, 26, 27, 28, 30 and 38). Each laboratory

parameter outside of normal range was assessed by the physician/investigator for clinical

96


relevance. The majority of the out-of-range laboratory values were only marginally outside of

the respective reference ranges.

97


8.0 DISCUSSION

The quantification of Rosuvastatin in plasma samples was performed in accordance with GLP

requirements. The analytical methods LC MS-MS allowed specific and sensitive

98


determination of Rosuvastatin in plasma. The calibration ranges validated for the analysis of

plasma samples showed linearity between 0.300 ng/ml to 60.000 ng/ml for Rosuvastatin and

the validation parameters of the method fulfilled international requirements for method

validation.

The bioequivalence evaluation was based on the intra-individual ratios of the primary target

parameters Cmax, AUC(0-t) and AUC(0-inf) of Rosuvastatin.

Mean peak concentrations (Cmax) found to be 37.495 ng/ml for Test (A) formulation and

38.170 ng/ml for Reference (B) formulation. No statistical significant difference was

observed for sequence, formulation and period effect at 5% level of significance. The

percentage intra-subject CV was found to be 32.57%. The 90% confidence interval for the

log-transformed Cmax for the Test and Reference formulation was 92.90 to 116.99.

Mean area under the curve AUC(0-t) found to be 306.052 hr.ng/ml for Test (A) formulation and

306.075 hr.ng/ml for Reference (B) formulation. No statistical significant difference was

observed for sequence, formulation and period effect at 5% level of significance. The

percentage intra-subject CV was found to be 27.58%. The 90% confidence interval for the

log-transformed AUC(0-t) for the Test and Reference formulation was 95.56 to 116.32.

Mean AUC(0-inf) found to be 318.312 hr.ng/ml for Test (A) formulation and 320.681 hr.ng/ml

for Reference (B) formulation. No statistical significant difference was observed for

sequence, formulation and period effect at 5% level of significance. The percentage intra-

subject CV was found to be 24.98%. The 90% confidence interval for the log-transformed

AUC(0-inf) for the Test and Reference formulation was 95.49 to 114.17.

Ratio analysis of untransformed and difference of log transformed primary pharmacokinetic

parameters [Cmax, AUC(0-t) and AUC(0-inf)] for test & reference formulation were calculated

for Rosuvastatin. The percentage geometric LSM ratio was expressed as point estimates of

relative bioavailability and it was found to be 104.25, 105.43 and 104.41 for Cmax, AUC(0-t)

and AUC(0-inf) for Rosuvastatin.

Actual values of secondary pharmacokinetic parameter Tmax were compared for test and

reference using Non-parametric Wilcoxon Signed Rank test for Rosuvastatin. No statistical

99


significant difference found between formulations (p value= 0.71). The median time to reach

peak plasma concentrations (Tmax) was found to be 3.50 hr with range 2.00 hr - 6.00 hr for

test (A) formulation. For reference (B) formulation median time found to be 4.50 hr with

range 2.00 hr-6.00 hr.

The 90% confidence interval for an intra individual means ratio for log-transformed Cmax,

AUC(0-t) and AUC(0-inf) of the test to the reference formulation were within the range 80.00%-

125.00% for Rosuvastatin. Hence, the test (A) formulation was bioequivalent to reference (B)

formulation.

These observations confirms that the test formulation Rosuvastatin 40 mg tablet (Test,

Torrent Pharmaceuticals Ltd., India) is bioequivalent with the Reference formulation i.e.

Crestor® 40 mg tablet (Reference, AstraZeneca Pharmaceuticals LP, USA) and is also well

tolerated on single dose administration in healthy, adult, male, human volunteers under fed

condition.

100


9.0 CONCLUSION:

Pharmacokinetic Conclusion:

The 90% confidence intervals of Cmax, AUC(0-t) and AUC(0-inf) are within the bioequivalence

acceptance limits of 80.00-125.00 % for Rosuvastatin Based on the results, it can be

concluded that the test product Rosuvastatin Calcium 40 mg tablet of Torrent Pharmaceuticals

101


Ltd., India is bioequivalent with the reference product Crestor® 40mg tablet of AstraZeneca

Pharmaceuticals LP, USA in healthy male human volunteers under fed conditions.

Safety Conclusion:No serious adverse event occurred during the course of the study. Upon conclusion of the

clinical portion of the study the results from the volunteers who completed post study

procedures, including laboratory tests and vital signs measurements confirmed the absence of

significant changes in the volunteers’ state of health.

Both formulations were well tolerated by healthy subjects, as a single dose administration and

no relevant differences in the safety profiles of the test and reference formulation were

observed.

102


10.0 REFERENCES1. EMEA, Committee For Proprietary Medicinal Products (CPMP), Note For Guidance on

The Investigation of Bioavailability and Bioequivalence,2001

2. Bioavailability and Bioequivalence Requirements, 21 CFR 320, Food and Drug

Administration (FDA), 2006

3. Guidance for Industry, Bioavailability and Bioequivalence Studies for Orally

Administered Drug Products -General Considerations, Food and Drug Administration

(FDA),Center for Drug Evaluation and Research (CDER), 2003

103


4. Guidance For Industry, Conduct and Analysis of Bioavailability and Bioequivalence

Studies - Part A: Oral Dosage Formulations Used for Systemic Effects, Health Canada,

1992.

5. Guidelines for Bioavailability Bioequivalence Studies, Central Drugs Standard Control

Organization (CDSCO), Directorate General of Health Services (DGHS) India,2005.

6. Schedule Y, Drugs And Cosmetics (IInd Amendment) Rules, 2005

7. Informed Consent Form (ICF) for BE Study of Rosuvastatin Calcium 40 mg tablet, Bio

Evaluation Centre, Torrent Pharmaceutical Ltd.,Gandhinagar.

8. EMEA, Committee for Medicinal Products For Human Use (CHMP), Guideline on the

Investigation of Bioequivalence, Draft, 2008.

9. Pharmaceutical Statistics Practical and Clinical Applications, 4th edition, Sanford

Bolton, Charles Bon. Vol 135 page 311-372

10. Endocrine and Metabolic Disorders, Merck Manual,2008

11. Ahmed SM, Clasen ME, and Donnelly JF, Management of Dyslipidaemia in Adults,

American family Physician, 1998

12. Thompson GR, Management of Dyslipidaemia, Heart,2004; (90):949–955

13. Goodman and Gillman’s drug therapy for hypercholesterolemia and dyslipidaemia, the

pharmacological basis of therapeutics,11th Ed. ,2006

14. Dyslipidemia, Essence series, A Cipla initiatives,2005

15. Bellosta S, Paoletti R, Corsini A, Safety of Statins Focus on Clinical Pharmacokinetics

and Drug Interactions, Circulation 2004,(109);50-57

16. CRESTORTM, Summary of the Product Characteristics, 2002

17. Smith SC, Clinical Treatment of Dyslipidaemia, Practice Patterns and Missed

Opportunities, Am J Cardiol (2000);86(suppl):62L–65L

18. Ebba Bergman, Patrik Forsell , Annica Tevell, Eva M. Perssona, Mikael Hedelandc, Ulf

Bondessonc, Lars Knutsonb, Hans Lennernas, Biliary secretion of rosuvastatin and bile

acids in humans during the absorption phase , european journal of pharmaceutical

sciences (2006);29;205–214

19. Fergus McTaggart, Comparative pharmacology of Rosuvastatin, Atherosclerosis

Supplements (2003); 4; 9-14

20. Evan A. Stein, John Amerena, Christie M. Ballantyne, Edmund Brice, Michel Farnier,

Robert M. Guthrie, Dror Harats, Long-Term Efficacy and Safety of Rosuvastatin 40 mg

in Patients With Severe Hypercholesterolemia, Am J Cardiol 2007(100):1387–1396

104


21. Third Report of the National Cholesterol Education Program (NCEP) Expert Panel on

Detection, Evaluation, and Treatment of High Blood Cholesterol in Adults (Adult

Treatment Panel III) Final Report, Circulation 2002;(106);3143- 3421

22. Kulkarni JS, Pawar AP, Shedbakar VP. Biopharmaceutics and Pharmacokinetics. CBS

Publishers and Distributors; 2006. p. 249-262.(01).

23. Brahmankar DM, Jaiswal SB. Biopharmaceutics and Pharmacokinetics A Treatise.

Vallabh Prakashan; 2005. p. 282-305(10).

24. Peter GW, Francis LS, Dighe SV. Pharmaceutical Bioequivalence. Vol.48. 347-348.

25. Shargel L, Yu AB. Applied biopharmaceutics & Pharmacokinetics. 4th ed. New York:

McGraw-Hill; 1999. p. 247-272

26. Guidance for Industry Statistical Approaches to Establishing Bioequivalence. U.S.

Department of Health and Human Services, Food and Drug Administration, Center for

Drug Evaluation and Research (CDER), 2001

27. Guidance for Industry: food-effect bioavailability and fed bioequivalence Studies. U.S.

Department of Health and Human Services, Food and Drug Administration, Center for

Drug Evaluation and Research (CDER), 2002.

28. Guidance for Industry for Method validation, U.S. Department of Health and Human

Services, Food and Drug Administration, Center for Drug Evaluation and Research

(CDER), May 2001.

29. Harrison’s Principles of Internal Medicine,16th Edition, Disorders Of Lipoprotein

Metabolism, 2286-2298.

30. Color Atlas of Pharmacology, 2nd edition, Drugs used in Hyperlipoproteinemias, 154-

157.

31. Ludovica Piconia, Maddalena Corgnalia, Roberto Da Rosa, Roberta Assalonia, Teodoro

Piliegob, Antonio Ceriello, The protective effect of rosuvastatin in human umbilical

endothelial cells exposed to constant or intermittent high glucose, Journal of Diabetes

and Its Complications,2008 (22) 38– 45.

32. Donald G. Vidt, Susan Harris, Fergus McTaggart, Marc Ditmarsch, Philip T. Sager, and

Jonathan M. Sorof, Effect of Short-Term Rosuvastatin Treatment on Estimated

Glomerular Filtration Rate, Am J Cardiol 2006;(97):1602–1606.

33. Emile G. Bliznakov, Coenzyme Q10, Lipid-Lowering Drugs (Statins) and Cholesterol,

The Journal of the American Nutraceutical Association, 2002, (5), 31-38.

34. CRESTOR® (Rosuvastatin calcium), Prescribing Information, US-FDA, 2007.

105


35. Rosuvastatin, Drug Report, Thomson Pharma®, 2010.

36. Guidance for Industry, Waiver of In Vivo Bioavailability and Bioequivalence Studies

for Immediate-Release Solid Oral Dosage Forms Based on a Biopharmaceutics

Classification System, US FDA, Aug 2000

37. Guidance for Industry: food-effect bioavailability and fasting bioequivalence Studies.

U.S. Department of Health and Human Services, Food and Drug Administration, Center

for Drug Evaluation and Research (CDER), 2002.

106


11.0 ANNEXURE

11.1 Demographic Data

Table 21: Demographic data of Individual volunteer

En. No. Sex Age

(Yrs)Height (cm)

Weight (Kg)

BMI* (Kg/m2) Race Smoking Date of

Screening1 Male 31 176.00 75.81 24.47 Asian None 23/11/09

2 Male 29 169.00 68.73 24.06 Asian None 07/11/09

3 Male 39 159.00 56.73 22.44 Asian None 19/11/09

4 Male 31 170.00 62.27 21.55 Asian None 23/11/09

5 Male 25 169.50 63.80 22.21 Asian None 24/11/09

6 Male 35 167.50 74.26 26.47 Asian None 19/11/09

7 Male 37 159.50 54.47 21.41 AsianCurrent Smoker-

Bidis- 1-0923/11/09

8 Male 23 174.00 58.32 19.26 Asian None 24/11/09

9 Male 19 171.00 54.31 18.57 Asian None 24/11/09

107


10 Male 24 168.50 65.90 23.21 Asian None 07/11/09

11 Male 25 168.00 65.85 23.33 Asian None 19/11/09

12 Male 41 165.00 60.82 22.34 Asian None 09/11/09

13 Male 38 167.00 61.48 22.04 Asian

Current Smoker-

Cigarettes- 1-09

23/11/09

14 Male 40 165.00 70.98 26.07 Asian None 23/11/09

15 Male 31 174.00 79.36 26.21 Asian None 16/11/09

16 Male 28 170.50 64.38 22.15 Asian None 16/11/09

17 Male 26 174.50 66.77 21.93 Asian None 24/11/09

18 Male 20 164.50 59.63 22.04 Asian None 09/11/09

19 Male 26 168.50 57.80 20.36 Asian None 19/11/09

20 Male 27 175.50 66.80 21.69 Asian None 19/11/09

21 Male 35 159.50 67.66 26.60 Asian None 11/11/09

22 Male 41 170.50 65.16 22.41 Asian

Ex-smoker- Discontinued > 3 months

ago

16/11/09

23 Male 28 154.00 53.00 22.35 Asian None 23/11/09

24 Male 35 159.50 65.71 25.83 Asian None 23/11/09

25 Male 37 171.00 56.21 19.22 Asian None 19/11/09

26 Male 40 178.00 71.50 22.57 Asian None 23/11/09

27 Male 25 162.00 55.32 21.08 AsianCurrent Smoker-

Bidis- 1-0907/11/09

28 Male 34 170.50 74.80 25.73 Asian None 13/11/09

29 Male 34 165.00 71.96 26.43 Asian None 23/11/09

30 Male 38 165.50 72.91 26.62 Asian None 24/11/09

31 Male 30 167.00 57.61 20.66 Asian

Ex-smoker [discontinued

>3 months ago]

23/11/09

32 Male 41 157.50 62.13 25.05 Asian None 13/11/09

33 Male 23 162.50 52.15 19.75 Asian None 11/11/09

34 Male 35 176.00 70.00 22.60 Asian None 23/11/09

35 Male 33 174.00 59.19 19.55 Asian None 11/11/09

36 Male 35 162.00 53.85 20.52 Asian

Ex-smoker [discontinued

>3 months ago]

11/11/09

108


37 Male 38 164.50 71.91 26.57 Asian None 04/11/09

38 Male 34 162.00 54.81 20.88 Asian None 23/11/09

39 Male 35 165.00 62.87 23.09 Asian None 16/11/09

40 Male 36 166.00 63.57 23.07 Asian None 06/11/09

41 Male 42 180.50 67.30 20.66 Asian None 19/11/09

42 Male 33 170.00 59.00 20.42 Asian None 06/11/09

43 Male 19 165.50 60.71 22.16 Asian None 16/11/09

44 Male 25 170.50 67.07 23.07 Asian None 19/11/09

45 Male 25 171.00 67.40 23.05 Asian None 23/11/09

46 Male 32 173.50 55.97 18.59 Asian None 24/11/09

47 Male 33 160.00 50.20 19.61 Asian None 23/11/09

48 Male 34 171.50 78.02 26.53 Asian None 19/11/09

31.77 167.73 63.68 22.646.3 5.8 7.3 2.442 180.50 79.36 26.6219 154.00 50.20 18.57

20.4 2.8 10.9 10.8

11.2 LABORATORY PARAMETERS WITH REFERENCE RANGE

ParametersNormal Range

Observed ValueMale Female

Hematology

WBC × µl 4.0-10.0 4.0-10.0

Neutrophils % 40.0-80.0 40.0-80.0

Lymphocytes % 20.0-40.0 20.0-40.0

Monocytes % 2.0-10.0 2.0-10.0

Eosinophils % 1.0-6.0 1.0-6.0

Basophil % < 1-2 < 1-2

RBCx M/µl 4.5-5.5 3.8-4.8

Hb[g/dl] 12.5-17.0 12.0-15.0

HCT% 40.0-50.0 37.0-46.0

Platelets x K/µl 150-400 150-400

Biochemistry

109


Urea (mg/dl) 17-43 17-43

albumin (g/dl) 3.5-5.2 3.5-5.2

Total Proteins (g/dl) 6.4-8.3 6.4-8.3

Globulin (g/dl)* 2.3-3.5 2.3-3.5

ALT [SGPT](U/l) <45 <34

Total Bilirubin (mg/dl) 0.3-1.2

AST (SGOT] (U/L) <35 <31

Serum Alkaline Phosphatase (U/l) 30-120 30-120

Random Blood Sugar (mg/dI) 45-130 45-130

Serum Creatinine (mg/dl) 0.84-1.25 0.66-1.09

Triglyceride (mg/dl) <150 <150

Cholesterol (mg/dl) 150-250 150-250

Calcium (mg/dl) 8.8-10.6 8.8-10.6

Sodium (mEq/L) 136-145 136-145

Potassium (mEq/L) 3.5-5.1 3.5-5.1

Chloride (mEq/L) 98-106 98-106

GGT (U/L) <55 <38

Uric Acid (mg/dl) 3.5-7.2 2.6-6.0

CK (U/l) ≤171 ≤ 145

Urine analysis

Glucose Negative Negative

Bilirubin Negative Negative

Ketone Negative Negative

Sp.Gr. 1.001-1.035 1.001-1.035

Blood Negative Negative

pH 5-9 5-9

Protein Negative Negative

U.bil gen 0.2-1.0 0.2-1.0

Nitrite Negative Negative

Leucocytes Negative Negative

Urine analysis: Physical and microscopic

Quantity Pus cell

Color RBC

Appearance Cast

110


Clarity Crystals

Epithelial cell Others

Abbriviations used:WBC-White blood cells, RBC-Red blood corpuscles, Hb-Hemoglobin,HCT-Hematocrit ALT-Alanine Amino Trsnsferase,AST-Aspartste Amino Transferase.Normal ranges for hematology are given as per reference: Dade & Lewis Practical Hematology , 9 Edition, Page- 12Globulin values are calculated from Total protein & Albumin.Normal Ranges for biochemistry are given as per kit literature•Btood glucose range (Random sampling) Clinical diagnosis and management by laboratory methods. Henry at al. Ch.9, p.199 .S. Electrolytes normal range as per Teitz text book of clinical chemistry* Normal ranges for hematology are given as per reference: Dacie & Lewis Practical Hematology , 9th Edition* Normal range for Cholesterol given as per Clinical Diagnosis and Managemenl by Laboratory Methods, Henry el at. 19th edition

111


11.3 SCHEDULE OF ASSESSMENT

Type of Assessment

Planned

EntryExaminatio

n(screening)

Period I Wash out Period II

Study Day -28 To 0 0 1 2 3 4 5

Minim

um of 7 days

0 1 2 3 4 5Post study

Safety Evaluation

Informed consent √ √

Demography √Medical and surgical history

√

Life style, habits √

Vital signs √ √ √ √ √ √ √12-lead ECG √Physical examination

√ √ √ √ √

Chest X – ray √

Laboratory examination Blood haematology Blood chemistry Serology Urine analysis

√√√√

√√

Drug abuse screenAlchohol breath test

√

√

√

√Inclusion criteria √ √Exclusion criteria √ √ √ √ √ √ √ √ √ √ √ √Decision on volunteer enrolment

√ √

Confinement √ √ √ √ √ √Check of restrictions

√ √ √ √ √ √ √ √ √ √ √ √

Fitness for dosing √ √Treatment administration as per randomization

√ √

Blood sampling for drug analysis

√ √ √ √ √ √ √ √ √ √

Adverse event questioning √ √ √ √ √ √ √ √ √ √ √ √ √

Day-0=Enrolment day, Day 1=Dosing day, Day 2=Discharge day, Note: Ambulatory sample will be collected at 48 hours (Day -3), 72 hours (Day -4) and 96 hours (Day-5) post dose post dos

112


SCHEDULE

Below mentioned schedule is the tentative time table of each period during study. These activities will be repeated after 07 days of wash-out period. Actual time of sampling shall be depend on dosing time.

ENROLLMENT DAY ACTIVITY Reporting, ICF Presentation and Obtaining Written Consent (period 01 only), Criteria Check

(period 01 only), Compliance Assessment, breath alcohol test, Urine drug screen, Clinical examination, Check-in

Dinner & Bed timeDOSING DAY & DISCHARGE DAY ACTIVITY

Scheduled time (Hrs)

Actual time (HH:MM) (Days) Activity

05:30 (D–1) Wake up call06:00 to 07:30 (D–1) Pre-dose Vital signs, Cannulation & Pre-dose blood sample

collection07.30 to 07.55 Breakfast00.00 08:00 (D–1) Study Drug Administration 00:33 08:20 (D–1) Blood Draw00:67 08:40 (D–1) Blood Draw01:00 09:00 (D–1) Blood Draw01:50 09:30 (D–1) Blood Draw02:00 10:00 (D–1) Blood Draw followed by Vital Signs measurement 02:25 10:15 (D–1) Blood Draw02:50 10:30 (D–1) Blood Draw02:75 10:45 (D–1) Blood Draw03:00 11:00 (D–1) Blood Draw03:25 11:15 (D–1) Blood Draw03.50 11:30 (D–1) Blood Draw04:00 12:00 (D–1) Blood Draw followed by Vital Signs measurement 04:50 12:30 (D–1) Blood Draw 05:00 13:00 (D–1) Blood Draw & lunch05:50 13:30 (D–1) Blood Draw06:00 14:00 (D–1) Blood Draw followed by Vital Signs measurement07:00 15:00 (D–1) Blood Draw08:00 16:00 (D–1) Blood Draw 09:00 17:00 (D–1) Snacks 10:00 18:00 (D–1) Blood Draw 12:00 20:00 (D–1) Blood Draw 13:00 21:00 (D-1) Dinner16:00 00:00 (D–2) Blood Draw24:00 08:00 (D–2) Blood Draw followed by medical checkup, breakfast and

discharge AMBULATORY SAMPLES 48:00 08:00 (D-3) Blood draw (ambulatory sample)72:00 08:00 (D-4) Blood draw (ambulatory sample)96:00 08:00 (D-5) Blood draw (ambulatory sample)

Day-0; Entry day, Day-1; Drug administration day, Day-2; Discharge day, Day 3, 4 & 5: ambulatory sample

113


11.4 VOLUNTEER DECLARATION AND SIGNATURE

• I am 18 years or older. I have given facts to the best of my knowledge to study personnel about my medical and family history.

• I have read this informed consent document, it is explained to my satisfaction, and I have understood it. Where I had doubts or questions, I had clarified them by study personnel.

• I understand that I am deemed medically fit enough to participate in this study and I will not gain any therapeutic benefit from participating in this study and its only for the purpose of research.

• I understand that I will not take up any financial encumbrance as a result of taking part in this study. All diagnostic costs and expenses related to any hospitalizations due to study drugs will be borne by Torrent Pharmaceuticals Ltd.

• I understand the risks to me of taking part in this study as explained in the adverse effects section of “Background information”. I understand that these risks include possible hospitalization.

• I understand that I have to be present at clinical facility of Torrent Pharmaceuticals Ltd. to comply with other instructions.

• I declare that I did not take part in a clinical study at any company in the past 3 months.

• I agree not to commit any misbehaviour or misconduct with any study personnel or with any staff member and disobeying that will make me liable for legal consequences.

• I agree not to cause any damage or loss of any property of Torrent Pharmaceuticals Ltd.• I am aware that my identity and personal details will be kept confidential and will not be

revealed to anyone except the IEC, the Regulatory Agency (ies) and the Sponsor's inspectors/auditors.

• I am aware that I can withdraw my consent from this study at any time during the course of the study even without disclosing the reason(s) thereof and that I shall not be deprived of any medical care that I should get for participation in this study and this will not take away my right for future participation in such studies.

• I am provided with the contact details of all the relevant persons whom I can contact for any study-related query or queries pertaining to my rights as a volunteer.

114


I am Mr./Ms._____________________________________________________________,

My father's name is _______________________________________________________,

My mother's/spouse’s/guardian’s name is ______________________________________,

My full residential address is ________________________________________________,

________________________________________________________________________

My phone number is: ______________________________________________________,

I hereby give my voluntary free consent (means without any coercion, misrepresentation and fraud) for including myself as a volunteer in the Single dose Bioequivalence study of Rosuvastatin Calcium 40 mg tablet (test formulation, Torrent Pharmaceuticals Ltd., India) Versus (Crestor®) 40 mg tablet (reference formulation, AstraZeneca Pharmaceuticals LP, USA) in healthy, adult, male, human Volunteers under fed conditions.

Signature of the volunteer: Date:Name of witness:Address of witness:

Phone No.:Signature of the witness: Date:Medical query resolved by: Date:Written informed consent obtained by: Date:

115


11.5 FOOD MENU (FED)

CHECK-IN DAY DINNER

Items Amount (gms or ml) KcalChapati (6 no.) 120 gm 395.00Khichdi 150 gm 308.73Kofta curry 200 ml 200.00Rajmah Masala 100 gm 107.24MixVegetable (Potato, Carrot Peas, Cauliflower) 100 gm 102.45

Roasted Papad (Black gram) (1 no.) 07 gm 24.01

1137.43STUDY DAY 1

BREAKFASTItems Amount (gms or ml) Kcal

Paneer Cutlet (3 no.) 120 gm 528.47Chutney 30 gm 101.35Milk + Sugar 200 ml+5 gm 253.90Veg Chana Salad 35 gm 59.00

942.72LUNCH

Chapati (6 no.) 120 gm 395.00Rice 150 gm 207.60Butter milk 200 ml 30.00Guj.Tuar Dal 200 ml 146.50Paneer Tikka Masala 100 gm 184.82Salad (Tomato, Cucumber, Carrot) 50 gm 12.80

Chhole 100 gm 114.21Roasted Papad (Black gram) (1 no.) 7 gm 24.01

1114.94SNACKS

Dhokla 120 gm 240.06Chutney (Green) 30 gm 27.54Milk + Sugar 150 ml+ 3.75 gm 190.43

458.03DINNER

Chapati (6 no.) 120 gm 395.00Rice 150 gm 207.6Sambar 200 ml 140.00Sev Tomato 100 gm 163.50Curd 50 gm 30.00Moong (whole) 100 gm 106.6

116


Roasted Papad (Black gram) (1 no.) 7 gm 24.01

1066.71CHECK-OUT DAY

BREAKFASTItems Amount (gms or ml) Kcal

Aloo Paratha ( 1 no.) 120 gm 241.2Chutney (Green) 30 gm 27.54Milk + Sugar 150 ml+ 3.75 gm 190.43

459.17

Reference: Gopalan C. et al (2004). Nutritive value of Indian foods, NIN, Indian Council of Medical Research, Hyderabad.

“BREAKFAST MENU”

117

INGREDIENTS AMOUNTPaneer Cutlets 3 no.Paneer 50 gm

Toast Bread 10 gm Groundnut 20 gm Potato 20 gm Oil 20 ml Onion 10 gm Green Chilly 04 gm Til 05 gm

Paneer CutletsNutrients Amount

Energy 528.47 K.calFat 40.64 gm

Protein 15.88 gmCarbohydrate 24.37 gm

ChutneyNutrients Amount

Energy 101.35 K.calFat 6.45gm

Protein 4.78 gmCarbohydrate 6 gm

Chutney Green chilly 5 gm

Cor.Leaves 15 gmGroundnut 10 gm

MilkWhole Milk 200 mlSugar 5 gm

MilkNutrients Amount

Energy 253.9 K.calFat 13 gm


Veg Chana SaladBengal Gram (Whole) 15 gmTomato 10 gmCabbage 10gm

Veg Chana SaladNutrients Amount

Energy 59 K.calFat 1.00 gm


final project

Documents

active moiety

active ingredient

active substance

site of action

range of bioequivalence

absolute bioavailability

relative bioavailability

fed condition title