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
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 concentrationtime 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 (92.90116.99), 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.
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
Bioequivalence study of Rosuvastatin calcium 40mg Tablet under Fed Condition 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 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 nonorally 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
Bioequivalence study of Rosuvastatin calcium 40mg Tablet under Fed Condition 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 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. 4
Bioequivalence study of Rosuvastatin calcium 40mg Tablet under Fed Condition 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 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 5
Bioequivalence study of Rosuvastatin calcium 40mg Tablet under Fed Condition (housed) during the study to ensure compliance with regard to dosing schedule as well as other protocol requirements.9
1.3 Dyslipidemia
Dyslipidemia 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:
RISK FACTORS OF DYSLIPIDEMIA
Emerging 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 6 (ABI) Tests for myocardial ischemia
Non Modifiable Risk Factors
A) Life Style Risk Factors Obesity Physical Inactivity Atherogenic Diet B) Hypertension C) Cigarette Smoking D) Thrombogenic/ haemostatic State E) Diabetes
Age Male Sex Family History of Premature CHD
Epidemiologic, angiographic and postmortem studies have documented a causal relationship between elevated serum cholesterol levels and the genesis of coronary heart disease.10,11 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 7
Bioequivalence study of Rosuvastatin calcium 40mg Tablet under Fed Condition 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 Table:1 Adult Treatment Panel (ATP III) Classification of LDL, Total and HDL Cholesterol and Triglycerides (mg/dL)14 Total Cholesterol <200 200-239 240 LDL Cholesterol <100 100-129 130-159 160-189 190 HDL Cholesterol <40 60 Triglyceride <150 150-199 200-499 500 Normal Borderline high High Very high Low High Optimal Near or above optimal Borderline High High Very high Desirable Borderline High 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
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
Bioequivalence study of Rosuvastatin calcium 40mg Tablet under Fed Condition administration of single tablet of either test or reference formulation under fed condition in each period. Secondary objective was to evaluate the safety of Rosuvastatin calcium 40 mg in healthy human volunteers.
10
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 11
Bioequivalence study of Rosuvastatin calcium 40mg Tablet under Fed Condition (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 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 2060%, 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 forcetitration 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 12
Bioequivalence study of Rosuvastatin calcium 40mg Tablet under Fed Condition 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 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 200400 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 Hypercholesterolemia Hypertriglyceridaemia Mixed Hyperlipidaemia Low HDL cholesterol First choice Statin Fibrate Statin Statin If refractory Add cholesterol absorption inhibitor, Bile acid sequestrant, or nicotinic acid Add nicotinic acid or 3 fatty acids Substitute or add fibrate (not gemfibrizil+ 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 1040 mg, although there was no significant difference between Rosuvastatin 40 mg and atorvastatin 80 mg in this respect
13
Fig 1-Comparative LDL lowering efficacy of Rosuvastatin, atorvastatin, simvastatin, and pravastatin .12
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.
14
Bioequivalence study of Rosuvastatin calcium 40mg Tablet under Fed Condition 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 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 (IC 50) 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 benefitrisk profile across this dose range.20
15
Bioequivalence study of Rosuvastatin calcium 40mg Tablet under Fed Condition Table: 3 Outcomes of LDL-Cholesterol Lowering Therapy in CHD21 Intervention No. of Trials 1 3 6 12 No. Treated 421 1,992 1,200 17,405 Mean Cholesterol reduction(%) 22 9 11 20 CHD incidence (%) change -43 -21 -24 -30 CHD Mortality (%)change -30 -32 -21 -29
Surgery Sequestrants Diet Statins
16
17
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 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 Solubility
The 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 17.5. 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 Permeability
18
Bioequivalence study of Rosuvastatin calcium 40mg Tablet under Fed Condition The 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 Studies

The drug substance for which a waiver is being requested should be highly soluble and highly permeable. 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. 19
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).
20
Rosuvastatin Calcium 40 mg
Fig 3: A typical plasma drug concentration time curve obtained after a single oral dose
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. 21
Bioequivalence study of Rosuvastatin calcium 40mg Tablet under Fed Condition 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/mLhours. 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
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.
22
Bioequivalence study of Rosuvastatin calcium 40mg Tablet under Fed Condition 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 drugs 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 F= Bioavailable Dose Administered Dose
4.2.1 Absolute bioavailability25

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. 23
Bioequivalence study of Rosuvastatin calcium 40mg Tablet under Fed Condition 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. [AUC]A [AUC]B [D]B [D]A
Relative Bioavailability =
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. 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 24
Bioequivalence study of Rosuvastatin calcium 40mg Tablet under Fed Condition 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. 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:
25
Bioequivalence study of Rosuvastatin calcium 40mg Tablet under Fed Condition 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

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 halflives 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 26
Bioequivalence study of Rosuvastatin calcium 40mg Tablet under Fed Condition 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 [D]i.v [AUC]i.v [D]oral
F=
[AUC]test [D]std [AUC]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

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 27
Bioequivalence study of Rosuvastatin calcium 40mg Tablet under Fed Condition 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]oral F= [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: 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 28
Bioequivalence study of Rosuvastatin calcium 40mg Tablet under Fed Condition 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 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.
29
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 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.
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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 (z) 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 (z) 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 subjects 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 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 31
Bioequivalence study of Rosuvastatin calcium 40mg Tablet under Fed Condition AUC0-t, AUC0-inf., Cmax, Tmax, z, 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 Design26 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 32
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. 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. 33
Bioequivalence study of Rosuvastatin calcium 40mg Tablet under Fed Condition 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 Sequence 1 2 T R 2 R T 3 T R 4 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 Sequence 1 2 T R 2 R T 3 T R
III. Food Effect Study Design:5,37

34
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:

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. 35
Bioequivalence study of Rosuvastatin calcium 40mg Tablet under Fed Condition 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 mixedeffects 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. 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
36
Bioequivalence study of Rosuvastatin calcium 40mg Tablet under Fed Condition 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 logtransformed 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 nonreplicated crossover designs.
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 37
Bioequivalence study of Rosuvastatin calcium 40mg Tablet under Fed Condition 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 IIa IIb III IV V
Chylomicrons LDL LDL and VLDL IDL VLDL VLDL& chylomicrons
Normal to
Normal
None seen
Normal to
Normal to
+++ +++ +++ + +
38

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 Lipoprotein Clinical Findings Genetic Lipoprotein Lipase Deficiency Familial Hypercholesterolemia Familial Hepatic Lipase Deficiency Familial Dysbetalipoproteinemia LDL receptor Hepatic Lipase (LIPC) ApoE VLDL remnants Chylomicron & VLDL remnants Familial apolipoprotein C-II deficiency Sitosterolemia ABCG5 or ABCG8 LDL ApoC-II Chylomicron LDL Defects LPL Elevated Chylomicron Eruptive xanthomas, Hepatospleenomegaly Pancreatitis Tendon Xanthomas,CHD Premature Atherosclerosis Palmar & tuberoeruptive Xanthomas,CHD,PVD Eruptive xanthomas, Hepatospleenomegaly Pancreatitis Tendon Xanthomas,CHD AR AD AR AR AR AD Transmission 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
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Table:6 Selected Causes of Secondary Dyslipidemia11 Increased LDL Cholesterol Level Diabetes mellitus Hypothyroidism Nephrotic syndrome Obstructive liver disease Drugs: Anabolic steroids Progestins Beta-adrenergic blockers (without intrinsic Sympathomimetic action) Thiazides Increased Triglyceride Level Renal insufficiency Alcoholism Diabetes mellitus Hypothyroidism Obesity Drugs: Beta-adrenergic blockers (without intrinsic sympathomimetic action) Bile acid binding resins Estrogens Ticlopidine Decreased HDL Cholesterol Level 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
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 40
Bioequivalence study of Rosuvastatin calcium 40mg Tablet under Fed Condition 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.
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Table:7 Molecular Properties of Lipoproteins Lipoprotein class Chylomicrons and remnants Density of Flotation, g/ml <<1.006 Major lipid constituent Dietary triglycerides and cholesterol Tg: chol ratio 10:1 Site of Synthesis Intestine Mechanism(s) of catabolism Triglyceride hydrolysis by LPL, ApoE-mediated remnant uptake by liver Triglyceride hydrolysis by LPL 50% converted to LDL mediated by HL, 50% apoEmediated uptake by Liver. ApoB-100mediated uptake by LDL receptor (~75% in liver) Transfer of cholesteryl ester to VLDL and LDL, Uptake of HDL cholesterol by Hepatocytes Lp(a) 1.05-1.09 Cholesteryl esters NS Liver Unknown
VLDL IDL
<1.006 1.006-1.019
"Endogenous" or hepatic triglycerides Cholesteryl esters and "endogenous" triglycerides Cholesteryl esters Phospholipid, cholesteryl esters
5:1 1:1
Liver Product of VLDL catabolism
LDL
1.019-1.063
NS
Product of VLDL catabolism Intestine, liver, plasma
HDL
1.063-1.21
NS
Abbreviations: apo, apolipoprotein; CHOL, cholesterol; HDL, high-density lipoproteins; IDL, intermediatedensity 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 42
Bioequivalence study of Rosuvastatin calcium 40mg Tablet under Fed Condition 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)
Fig 4-Lipid Synthesis, Metabolism and Transport14
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Table:8 Types of Apoproteins Apolipoprotein ApoA-I ApoA-II ApoA-IV ApoA-V ApoB-48 Primary Source Intestine, liver liver Intestine liver Intestine Lipoprotein Association HDL, chylomicrons HDL, chylomicrons HDL, chylomicrons VLDL chylomicrons Function Structural protein for HDL; activates LCAT Structural protein for HDL Unknown Unknown 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 Unknown Cofactor for LPL Inhibits lipoprotein binding to receptors
ApoC-I ApoC-II
Liver Liver Liver
Chylomicrons, VLDL, HDL Chylomicrons, VLDL, HDL Chylomicrons, VLDL, HDL HDL
ApoC-III
ApoD
Spleen, brain, testes, adrenals
Unknown
ApoE
Liver
Chylomicron remnants, IDL, HDL Chylomicrons, VLDL, LDL, HDL 44
Ligand for binding to LDL receptor B2 glycoprotein I
ApoH
Liver

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.
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 proteinsthe 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 6070 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 2030 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 1015 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

45
Diet: Dietary modification is an important component in the management of hyperlipidemia.

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

Table: 9 Summary of the Major Drugs Used for the Treatment of Hyperlipidemia Drug HMG-CoA reductase inhibitors: Lovastatin Pravastatin Simvastatin Fluvastatin Atorvastatin Rosuvastatin Major Indications Starting Dose Maximal Dose Mechanism Common Side Effects
Elevated LDL
20 mg daily 40 mg qhs 20 mg qhs 20 mg qhs 10 mg qhs 10 mg qhs
Cholesterol 80 mg daily synthesis, 80 mg qhs hepatic LDL 80 mg qhs receptors 80 mg qhs VLDL 80 mg qhs production 40 mg qhs
Myalgias, arthralgias, elevated transaminases, dyspepsia
Bile acid sequestrants: Cholestyramine Colestipol Elevated LDL Colesevelam
4 g daily 5 g daily 3750 mg daily
32 g daily 40 g daily 4375 mg daily
Bile acid excretion And LDL receptors
Bloating, constipation, elevated triglycerides
Nicotinic acid: IR SR ER
Elevated LDL 100 mg tid and TG, 250 mg bid Low HDL, 500 mgqhs
2 g tid 1.5 g bid 2 g q hs
VLDL hepatic synthesis
Cutaneous flushing; GI upset; elevated glucose, uric acid, and liver function tests Dyspepsia, myalgia, gallstones, elevated transaminases
Fibric acid Derivatives: Gemfibrozil Fenofibrate
Elevated TG, elevated remnants
600 mg bid 160 mg qd
LPL, VLDL 600 mg bid synthesis 160 mg qd
Fish oils Cholesterol
Severely elevated TG Elevated LDL
3 g daily 10 mg daily 47
Chylomicron Dyspepsia, and diarrhea, fishy 12 g daily VLDL odor to breath production 10 mg daily Intestinal Elevated
Bioequivalence study of Rosuvastatin calcium 40mg Tablet under Fed Condition absorption inhibitors: Ezetimibe cholesterol absorption transaminases
HMG-CoA reductase inhibitors (statins): By inhibiting cholesterol biosynthesis, HMGCoA 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 HMGCoA 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 acidbinding 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 lipidlowering agent. Fibric Acid Derivatives (Fibrates): Fibric acid derivatives, or fibrates, are agonists of PPAR , 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. 48
Bioequivalence study of Rosuvastatin calcium 40mg Tablet under Fed Condition Fibrates are the most effective drugs available for reducing triglyceride levels, and they also raise HDL-C levels. 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
49
Bioequivalence study of Rosuvastatin calcium 40mg Tablet under Fed Condition 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 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 LDLC 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 LowDensity-Lipoprotein Cholesterol (LDL-C) from Baseline.13 20 -25 26 -30 31 -35 36 -40 41 -50 51 -55 (%) (%) (%) (%) (%) (%) Atorvastatin Fluvastatin Lovastatin Pravastatin Rosuvastatin Simvastatin 20 10 10 40 20 20 10 10 80 40 40 20 20 80 5 40 10 80 20, 40 40 80
50
4.11 Extra cardiac Effects of Rosuvastatin:

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 51
Bioequivalence study of Rosuvastatin calcium 40mg Tablet under Fed Condition 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 (2,3dimethoxy-5-methyl-6-decaprenyl-1,4-benzoquinone), is a major participant in electron 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
18, 4.12 DRUG PROFILE18,34
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, 20-25C (68-77F), Protect from moisture.
4.12.1 Clinical Pharmacology 4.12.2 Mechanism of Action:
52
Bioequivalence study of Rosuvastatin calcium 40mg Tablet under Fed Condition 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 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 53
Bioequivalence study of Rosuvastatin calcium 40mg Tablet under Fed Condition 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 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 -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 54
Bioequivalence study of Rosuvastatin calcium 40mg Tablet under Fed Condition 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 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. 55
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). 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.
56
Bioequivalence study of Rosuvastatin calcium 40mg Tablet under Fed Condition 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). 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 been examined. 57
(0-24)
and
Cmax respectively. Interactions between Rosuvastatin and other protease inhibitors have not
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 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 Adverse Event Pharyngitis Headache Diarrhea Dyspepsia Nausea Myalgia Asthenia Back Pain Flu syndrome Urinary tract infection Rhinitis Sinusitis N=744 9.0 5.5 3.4 3.4 3.4 2.8 23 2.6 2.3 2.3 2.2 2.0 Placebo N=382 7.6 5.0 2.9 3.1 3.1 1.3 26 2.4 1.8 1.6 2.1 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.
58
Bioequivalence study of Rosuvastatin calcium 40mg Tablet under Fed Condition Cardiovascular System: Hypertension, angina pectoris, vasodilatation, and palpitation. Digestive System: Constipation, gastroenteritis, vomiting, flatulence, periodontal abscess, and gastritis. Endocrine: Diabetes mellitus. 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, 59
Bioequivalence study of Rosuvastatin calcium 40mg Tablet under Fed Condition 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 2fold elevation in median exposure in Asian subjects (having either Filipino, Chinese, Japanese, Korean, Vietnamese or Asian-Indian origin) compared with a Caucasian control 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 patients individualized goal of therapy. 60
Table:12 DEVELOPMENT STATUS 35

Indication Hypercholesterolemia Hypercholesterolemia Hypercholesterolemia Hyperlipidemia Hyperlipidemia Hyperlipidemia Country Status Date
UK US Canada Canada Netherlands UK US
Launched Launched Launched Launched Launched Launched Launched
20-MAY-2003 16-SEP-2003 19-FEB-2003 19-FEB-2003 03-MAR-2003 20-MAY-2003 16-SEP-2003
Hyperlipidemia
Hypercholesterolemia Hyperlipidemia Hyperlipidemia Hyperlipidemia Atherosclerosis Atherosclerosis Hypercholesterolemia
Western Europe Western Europe Japan South Korea US Western Europe Japan
Launched Launched Launched Launched Launched Launched Launched
06-OCT-2004 06-OCT-2004 30-APR-2005 31-DEC-2005 07-DEC-2007 07-DEC-2007 27-APR-2007
61
5. STUDY FLOW CHARTS

62
5.1 Pre-study:
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-FDAs guidance for industry: Bioavailability and Bioequivalence studies, 2003 and in-house Standard Operating Procedures (SOPs).
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 Population

Screening 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 Criteria

Volunteers 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 Criteria

The 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 95F or more than 98.6F, 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
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Bioequivalence study of Rosuvastatin calcium 40mg Tablet under Fed Condition 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 study

Total 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 25C in accordance with the manufacturers 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: Drug name: Date of dosing : Sign: FOR CLINICAL RESEARCH PURPOSE ONLY Enrolment No: Randomization code: Study code: Period: Drug name: Date of dosing: Sign: FOR CLINICAL RESEARCH PURPOSE ONLY Enrolment No: Randomization code:
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
Bioequivalence study of Rosuvastatin calcium 40mg Tablet under Fed Condition 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, 2009 Period-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 volunteers 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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Bioequivalence study of Rosuvastatin calcium 40mg Tablet under Fed Condition 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 6.12.1
Study Volunteers Disposition of Volunteers

Flow chart summarizes the volunteers disposition No. of volunteers attended ICF Presentation: 50
No. of volunteers enrolled and Randomized = 48
Not Checked In Two volunteers did not give consent to participate in the study
Period I Test drug A = 24 Reference drug B = 24
Period II Batch 2 Test drug A = 22 Reference drug B = 22 Dropout: 01 Batch 1 Withdrawn: 03
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.
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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 volunteers request during study. Samples were collected in tubes containing 5IU diluted heparin for each ml of blood prelabeled as shown below:
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Bioequivalence study of Rosuvastatin calcium 40mg Tablet under Fed Condition 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 - 40C 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 Measurement

The 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). 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. The randomization will be balanced and the code will be kept under
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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 thats 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 Assessment

Safety 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
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Bioequivalence study of Rosuvastatin calcium 40mg Tablet under Fed Condition 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 assessment

The following pharmacokinetic parameters were determined for test and reference drugs in each subject using WinNonlin 5.2 a) b) c) d) e) f) g) Cmax Tmax AUC(0-t) AUC(0-inf) AUC%Extrap Kel Thalf Maximum concentration of drug observed in plasma. Time required reaching maximum concentration of drug in plasma. 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) Area under the plasma concentration vs time curve from time zero to time infinity. (AUCinf) Extrapolated AUC percentage of total AUC. Elimination Rate constant 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 Plans
Plasma 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 Statistics
Descriptive statistics were to be calculated for concentration-time data and all pharmacokinetic parameters of Rosuvastatin.
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Ratio Analysis
Difference 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 ANOVA
Analysis 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 Tmax
Non-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 Significance
For all analyses, effects or differences were to be considered statistically significant if the probability found less than 0.05 78
Software details
Pharmacokinetic 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 Deviations

Following 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.
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7.0 RESULTS
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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) 0.00 0.33 0.67 1.00 1.50 2.00 2.25 2.50 2.75 3.00 3.25 3.50 4.00 4.50 5.00 5.50 6.00 7.00 8.00 10.00 12.00 16.00 24.00 48.00 72.00 96.00
a b
N 43 43 43 43 43 43 43 43 43 43 43 43 43 43 43 43 43 43 43 43 43 43 43 43 a 42 b 41
Rosuvastatin Test (A) Mean SD 0.000 0.00 0.956 1.14 2.740 2.40 5.923 4.71 12.527 8.47 18.665 9.89 21.707 10.51 24.626 12.13 27.238 13.33 27.794 12.08 27.864 12.04 29.525 12.92 29.490 14.85 28.596 12.80 26.681 12.03 31.583 13.13 25.316 11.56 16.562 7.40 12.973 5.54 9.632 4.30 6.832 2.94 4.500 1.69 2.839 1.05 0.915 0.48 0.205 0.25 0.029 0.13
CV% 119.7 87.4 79.5 67.6 53.0 48.4 49.2 48.9 43.4 43.2 43.7 50.4 44.8 45.1 41.6 45.7 44.7 42.7 44.6 43.1 37.5 37.0 52.4 121.8 463.6
N 43 43 43 43 43 43 43 43 43 43 43 43 43 43 43 43 43 43 43 43 43 43 43 43 43 43
Rosuvastatin Reference (B) Mean SD 0.013 0.08 1.222 1.72 3.025 3.52 6.218 5.15 13.625 10.46 19.180 12.04 21.355 11.86 23.004 12.50 25.359 12.84 26.420 13.24 27.972 14.91 30.085 18.49 29.023 16.37 30.562 18.41 27.047 13.82 32.082 16.08 25.210 13.14 16.448 8.07 12.754 6.34 9.082 4.50 6.705 3.32 4.468 2.25 2.858 1.24 0.937 0.60 0.207 0.29 0.054 0.18
CV% 655.7 141.1 116.3 82.8 76.7 62.8 55.5 54.4 50.6 50.1 53.3 61.5 56.4 60.2 51.1 50.1 52.1 49.1 49.7 49.5 49.6 50.4 43.5 64.0 140.8 337.9
72.00hr= Subject-3 (Period-I) / SNS 96.00hr= Subject-3 & 23 (Period-I) / SNS
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Bioequivalence study of Rosuvastatin calcium 40mg Tablet under Fed Condition Table 14: Pharmacokinetic Parameters of the Test Formulation for Rosuvastatin Test Formulation: Rosuvastatin Tmax Cmax AUC(0-t) AUC(0-inf) AUC_% Kel Thalf Subject Sequence (hr) (ng/mL) (hr.ng/mL) (hr.ng/mL) Extrap(%) (1/hr) (hr) 1 BA 2.25 28.654 292.959 302.976 3.306 0.039 17.90 2 AB 5.50 31.509 270.476 287.542 5.935 0.036 19.17 3 AB 5.50 28.394 236.481 260.709 9.293 0.039 17.71 4 BA 2.75 41.204 273.966 281.839 2.793 0.059 11.76 5 BA 5.50 24.659 203.581 214.312 5.007 0.052 13.38 6 AB 5.50 26.533 180.250 186.161 3.175 0.060 11.48 7 AB 6.00 51.257 358.426 368.538 2.744 0.044 15.64 8 BA 2.75 17.565 176.652 194.821 9.326 0.044 15.82 9 AB 5.50 24.964 212.599 222.324 4.374 0.060 11.50 10 BA 4.00 81.865 369.000 377.693 2.302 0.083 8.36 11 AB 3.25 59.797 452.617 463.436 2.335 0.044 15.69 12 BA 5.50 57.658 535.985 549.676 2.491 0.046 14.94 13 BA 2.75 62.574 334.981 343.841 2.577 0.040 17.20 14 AB 5.50 16.968 196.890 203.734 3.359 0.045 15.55 15 AB 5.50 43.881 375.583 401.673 6.495 0.029 24.11 16 BA 3.50 32.350 300.919 313.014 3.864 0.060 11.48 17 BA 3.50 50.461 408.055 420.667 2.998 0.040 17.18 19 AB 2.75 25.725 246.580 262.635 6.113 0.049 14.05 20 AB 2.50 16.835 96.820 107.588 10.009 0.134 5.18 21 AB 5.50 48.997 374.618 383.788 2.389 0.071 9.79 22 BA 5.50 42.104 358.988 368.078 2.469 0.046 15.00 23 AB 5.00 59.831 511.811 520.347 1.640 0.048 14.40 24 BA 3.50 36.238 356.346 361.377 1.392 0.061 11.36 26 AB 5.50 37.669 318.911 325.833 2.124 0.046 15.09 28 BA 3.25 31.602 254.920 279.203 8.697 0.038 18.28 29 AB 6.00 43.299 338.120 355.796 4.968 0.052 13.30 30 BA 2.00 42.306 333.712 345.033 3.281 0.056 12.30 31 BA 2.50 26.403 196.985 202.070 2.516 0.063 10.95 32 AB 5.50 59.206 478.966 483.943 1.028 0.062 11.13 33 BA 4.00 66.219 558.623 573.960 2.672 0.043 16.11 34 BA 3.00 21.572 222.171 233.683 4.926 0.035 19.70 35 AB 2.00 45.066 461.641 469.052 1.580 0.050 13.81 36 AB 5.50 51.000 526.991 537.969 2.041 0.046 15.07 37 BA 3.50 22.158 194.612 202.655 3.969 0.066 10.48 38 BA 2.75 50.193 298.338 320.549 6.929 0.046 15.20 39 AB 2.00 23.156 166.060 173.171 4.106 0.059 11.79 40 AB 5.50 20.901 236.098 246.789 4.332 0.041 17.11 41 AB 2.75 30.986 232.577 244.616 4.921 0.048 14.56 42 BA 4.50 35.938 346.110 374.044 7.468 0.044 15.75 44 BA 3.50 28.292 253.916 268.057 5.275 0.053 13.10 46 AB 3.50 13.588 139.655 163.054 14.350 0.033 21.17 47 AB 3.25 28.044 251.095 258.084 2.708 0.064 10.77 83
Bioequivalence study of Rosuvastatin calcium 40mg Tablet under Fed Condition Test Formulation: Rosuvastatin Tmax Cmax AUC(0-t) AUC(0-inf) AUC_% Kel Thalf Subject Sequence (hr) (ng/mL) (hr.ng/mL) (hr.ng/mL) Extrap(%) (1/hr) (hr) 48 BA 5.50 24.663 226.143 233.082 2.977 0.062 11.19 N 43 43 43 43 43 43 43 Mean 4.08 37.495 306.052 318.312 4.355 0.052 14.31 SD 1.3 15.78 113.59 113.51 2.76 0.02 3.5 Min 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 14.56 Max 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 24.7 Geometric Mean 3.85 34.381 285.497 298.621 3.688 0.050 13.85
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Bioequivalence study of Rosuvastatin calcium 40mg Tablet under Fed Condition Table 15: Pharmacokinetic Parameters of the Reference Formulation for Rosuvastatin Reference Formulation: Rosuvastatin Tmax Cmax AUC(0-t) AUC(0-inf) AUC_% Kel Thalf Subject Sequence (hr) (ng/mL) (hr.ng/mL) (hr.ng/mL) Extrap(%) (1/hr) (hr) 1 BA 5.50 39.919 361.034 372.843 3.167 0.038 18.31 2 AB 5.50 4.157 34.728 45.204 23.176 0.085 8.16 3 AB 5.00 41.984 383.319 406.785 5.769 0.028 25.18 4 BA 3.25 36.470 249.986 258.516 3.299 0.063 10.97 5 BA 5.50 24.182 205.877 219.095 6.033 0.050 13.76 6 AB 5.50 28.528 195.317 201.708 3.169 0.061 11.36 7 AB 6.00 71.427 485.422 495.825 2.098 0.047 14.81 8 BA 2.00 22.501 156.036 167.149 6.649 0.049 14.24 9 AB 5.50 23.002 235.694 253.415 6.993 0.051 13.72 10 BA 4.50 89.275 379.498 387.279 2.009 0.043 16.29 11 AB 3.25 73.185 473.630 488.629 3.070 0.059 11.81 12 BA 5.50 51.591 473.554 483.108 1.978 0.050 13.74 13 BA 4.00 19.627 191.715 199.602 3.951 0.038 18.10 14 AB 3.50 23.136 248.539 256.931 3.266 0.044 15.81 15 AB 4.50 38.540 432.677 444.278 2.611 0.034 20.20 16 BA 5.50 22.198 214.561 223.922 4.181 0.057 12.06 17 BA 5.50 50.026 380.924 387.655 1.736 0.052 13.44 19 AB 3.50 37.126 305.794 326.542 6.354 0.045 15.51 20 AB 2.25 16.023 97.179 103.837 6.411 0.143 4.86 21 AB 5.50 33.455 324.462 450.279 27.942 0.017 41.18 22 BA 5.50 41.031 357.366 363.469 1.679 0.052 13.26 23 AB 3.50 77.729 610.900 632.938 3.482 0.041 17.11 24 BA 2.50 21.558 227.772 243.851 6.594 0.050 13.91 26 AB 2.50 24.473 189.050 213.119 11.294 0.066 10.45 28 BA 5.50 42.652 291.504 306.355 4.848 0.052 13.33 29 AB 5.50 45.470 393.974 406.221 3.015 0.063 10.94 30 BA 3.25 28.561 273.225 278.989 2.066 0.073 9.51 31 BA 5.50 31.642 215.929 220.185 1.933 0.074 9.42 32 AB 3.25 54.890 515.457 520.841 1.034 0.056 12.40 33 BA 3.50 78.055 560.106 570.366 1.799 0.050 13.86 34 BA 2.25 27.932 203.466 224.065 9.194 0.037 18.66 35 AB 5.50 51.365 506.748 516.818 1.948 0.048 14.36 36 AB 3.50 86.206 670.463 691.231 3.005 0.039 17.95 37 BA 5.50 14.343 142.065 149.777 5.149 0.056 12.34 38 BA 2.75 43.924 290.953 306.393 5.039 0.053 13.08 39 AB 5.50 18.611 154.331 167.627 7.932 0.043 16.17 40 AB 6.00 20.690 246.141 256.873 4.178 0.036 19.52 41 AB 2.00 49.842 309.675 317.399 2.434 0.060 11.54 42 BA 2.75 40.994 381.077 403.788 5.624 0.039 17.81 44 BA 2.25 28.556 195.247 199.697 2.228 0.071 9.73 46 AB 3.50 20.532 177.018 189.332 6.504 0.049 14.23 47 AB 5.50 24.835 226.840 235.192 3.551 0.060 11.55 85
Bioequivalence study of Rosuvastatin calcium 40mg Tablet under Fed Condition Reference Formulation: Rosuvastatin Tmax Cmax AUC(0-t) AUC(0-inf) AUC_% Kel Thalf Subject Sequence (hr) (ng/mL) (hr.ng/mL) (hr.ng/mL) Extrap(%) (1/hr) (hr) 48 BA 2.00 21.059 191.989 202.153 5.028 0.053 13.10 N 43 43 43 43 43 43 43 Mean 4.20 38.170 306.075 320.681 5.196 0.053 14.60 SD 1.4 20.44 142.40 145.13 5.10 0.02 5.5 Min 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 13.74 Max 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 37.7 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
Bioequivalence study of Rosuvastatin calcium 40mg Tablet under Fed Condition Table 16: Ratio analysis for Pharmacokinetic parameters for Rosuvastatin Rosuvastatin: Cmax Subject Sequence A (Test) 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 19 20 21 22 23 24 26 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 44 46 BA AB AB BA BA AB AB BA AB BA AB BA BA AB AB BA BA AB AB AB BA AB BA AB BA AB BA BA AB BA BA AB AB BA BA AB AB AB BA BA AB 28.654 31.509 28.394 41.204 24.659 26.533 51.257 17.565 24.964 81.865 59.797 57.658 62.574 16.968 43.881 32.350 50.461 25.725 16.835 48.997 42.104 59.831 36.238 37.669 31.602 43.299 42.306 26.403 59.206 66.219 21.572 45.066 51.000 22.158 50.193 23.156 20.901 30.986 35.938 28.292 13.588 B (Ref) 39.919 4.157 41.984 36.470 24.182 28.528 71.427 22.501 23.002 89.275 73.185 51.591 19.627 23.136 38.540 22.198 50.026 37.126 16.023 33.455 41.031 77.729 21.558 24.473 42.652 45.470 28.561 31.642 54.890 78.055 27.932 51.365 86.206 14.343 43.924 18.611 20.690 49.842 40.994 28.556 20.532 %Ratio Ln Ln [Test/Ref] [A(Test)] [B(Ref)] 71.780 3.355 3.687 757.975 3.450 1.425 67.631 3.346 3.737 112.981 3.719 3.596 101.973 3.205 3.186 93.007 3.278 3.351 71.761 3.937 4.269 78.063 2.866 3.114 108.530 3.217 3.136 91.700 4.405 4.492 81.707 4.091 4.293 111.760 4.055 3.943 318.816 4.136 2.977 73.340 2.831 3.141 113.858 3.781 3.652 145.734 3.477 3.100 100.870 3.921 3.913 69.291 3.247 3.614 105.068 2.823 2.774 146.456 3.892 3.510 102.615 3.740 3.714 76.974 4.092 4.353 168.095 3.590 3.071 153.921 3.629 3.198 74.093 3.453 3.753 95.225 3.768 3.817 148.125 3.745 3.352 83.443 3.273 3.454 107.863 4.081 4.005 84.836 4.193 4.357 77.230 3.071 3.330 87.737 3.808 3.939 59.161 3.932 4.457 154.487 3.098 2.663 114.272 3.916 3.782 124.421 3.142 2.924 101.020 3.040 3.030 62.168 3.434 3.909 87.666 3.582 3.713 99.076 3.343 3.352 66.180 2.609 3.022 Diff [Ln(Test-Ref)] -0.332 2.025 -0.391 0.122 0.020 -0.072 -0.332 -0.248 0.082 -0.087 -0.202 0.111 1.159 -0.310 0.130 0.377 0.009 -0.367 0.049 0.382 0.026 -0.262 0.519 0.431 -0.300 -0.049 0.393 -0.181 0.076 -0.164 -0.258 -0.131 -0.525 0.435 0.133 0.219 0.010 -0.475 -0.132 -0.009 -0.413
88
Bioequivalence study of Rosuvastatin calcium 40mg Tablet under Fed Condition Rosuvastatin: Cmax Subject Sequence A (Test) 47 48 AB BA 28.044 24.663 43 37.495 15.78 13.588 32.350 81.865 42.1 34.381 B (Ref) 24.835 21.059 43 38.170 20.44 4.157 33.455 89.275 53.6 33.011 %Ratio Ln Ln [Test/Ref] [A(Test)] [B(Ref)] 112.921 3.334 3.212 117.114 3.205 3.047 43 43 43 120.487 3.537 3.497 108.43 0.43 0.58 59.161 2.609 1.425 100.870 3.477 3.510 757.975 4.405 4.492 90.0 12.0 16.5 104.148 3.512 3.441 Diff [Ln(Test-Ref)] 0.122 0.158 43 0.041 0.45 -0.525 0.009 2.025 1096.4 -
N Mean SD Min Median Max CV% Geometric Mean
Rosuvastatin: AUC(0-t) Subject Sequence A (Test) 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 19 20 21 22 23 24 26 28 29 30 BA AB AB BA BA AB AB BA AB BA AB BA BA AB AB BA BA AB AB AB BA AB BA AB BA AB BA 292.959 270.476 236.481 273.966 203.581 180.250 358.426 176.652 212.599 369.000 452.617 535.985 334.981 196.890 375.583 300.919 408.055 246.580 96.820 374.618 358.988 511.811 356.346 318.911 254.920 338.120 333.712 B (Ref) 361.034 34.728 383.319 249.986 205.877 195.317 485.422 156.036 235.694 379.498 473.630 473.554 191.715 248.539 432.677 214.561 380.924 305.794 97.179 324.462 357.366 610.900 227.772 189.050 291.504 393.974 273.225 %Ratio Ln Ln [Test/Ref] [A(Test)] [B(Ref)] 81.144 5.680 5.889 778.847 5.600 3.548 61.693 5.466 5.949 109.592 5.613 5.521 98.885 5.316 5.327 92.286 5.194 5.275 73.838 5.882 6.185 113.213 5.174 5.050 90.201 5.359 5.463 97.234 5.911 5.939 95.563 6.115 6.160 113.184 6.284 6.160 174.728 5.814 5.256 79.219 5.283 5.516 86.805 5.928 6.070 140.249 5.707 5.369 107.122 6.011 5.943 80.636 5.508 5.723 99.630 4.573 4.577 115.458 5.926 5.782 100.454 5.883 5.879 83.780 6.238 6.415 156.448 5.876 5.428 168.692 5.765 5.242 87.450 5.541 5.675 85.823 5.823 5.976 122.138 5.810 5.610 Diff [Ln(Test-Ref)] -0.209 2.053 -0.483 0.092 -0.011 -0.080 -0.303 0.124 -0.103 -0.028 -0.045 0.124 0.558 -0.233 -0.142 0.338 0.069 -0.215 -0.004 0.144 0.005 -0.177 0.448 0.523 -0.134 -0.153 0.200
89
Rosuvastatin: AUC(0-t) Subject Sequence A (Test) 31 32 33 34 35 36 37 38 39 40 41 42 44 46 47 48 BA AB BA BA AB AB BA BA AB AB AB BA BA AB AB BA 196.985 478.966 558.623 222.171 461.641 526.991 194.612 298.338 166.060 236.098 232.577 346.110 253.916 139.655 251.095 226.143 43 306.052 113.59 96.820 292.959 558.623 37.1 285.497 B (Ref) 215.929 515.457 560.106 203.466 506.748 670.463 142.065 290.953 154.331 246.141 309.675 381.077 195.247 177.018 226.840 191.989 43 306.075 142.40 34.728 273.225 670.463 46.5 271.124 %Ratio Ln Ln [Test/Ref] [A(Test)] [B(Ref)] 91.227 5.283 5.375 92.921 6.172 6.245 99.735 6.325 6.328 109.193 5.403 5.315 91.099 6.135 6.228 78.601 6.267 6.508 136.988 5.271 4.956 102.538 5.698 5.673 107.600 5.112 5.039 95.920 5.464 5.506 75.104 5.449 5.736 90.824 5.847 5.943 130.049 5.537 5.274 78.893 4.939 5.176 110.693 5.526 5.424 117.790 5.421 5.257 43 43 43 118.686 5.654 5.603 105.91 0.39 0.54 61.693 4.573 3.548 98.885 5.680 5.610 778.847 6.325 6.508 89.2 6.8 9.7 105.301 5.641 5.574 Diff [Ln(Test-Ref)] -0.092 -0.073 -0.003 0.088 -0.093 -0.241 0.315 0.025 0.073 -0.042 -0.286 -0.096 0.263 -0.237 0.102 0.164 43 0.052 0.38 -0.483 -0.011 2.053 739.8 -
Rosuvastatin: AUC(0-inf) Subject Sequence A (Test) 1 2 3 4 5 6 7 8 9 10 11 12 13 BA AB AB BA BA AB AB BA AB BA AB BA BA 302.976 287.542 260.709 281.839 214.312 186.161 368.538 194.821 222.324 377.693 463.436 549.676 343.841 B (Ref) 372.843 45.204 406.785 258.516 219.095 201.708 495.825 167.149 253.415 387.279 488.629 483.108 199.602 %Ratio Ln Ln [Test/Ref] [A(Test)] [B(Ref)] 81.261 5.714 5.921 636.098 5.661 3.811 64.090 5.563 6.008 109.022 5.641 5.555 97.817 5.367 5.390 92.292 5.227 5.307 74.328 5.910 6.206 116.555 5.272 5.119 87.731 5.404 5.535 97.525 5.934 5.959 94.844 6.139 6.192 113.779 6.309 6.180 172.263 5.840 5.296 90 Diff [Ln(Test-Ref)] -0.208 1.850 -0.445 0.086 -0.022 -0.080 -0.297 0.153 -0.131 -0.025 -0.053 0.129 0.544
Rosuvastatin: AUC(0-inf) Subject Sequence A (Test) 14 15 16 17 19 20 21 22 23 24 26 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 44 46 47 48 AB AB BA BA AB AB AB BA AB BA AB BA AB BA BA AB BA BA AB AB BA BA AB AB AB BA BA AB AB BA 203.734 401.673 313.014 420.667 262.635 107.588 383.788 368.078 520.347 361.377 325.833 279.203 355.796 345.033 202.070 483.943 573.960 233.683 469.052 537.969 202.655 320.549 173.171 246.789 244.616 374.044 268.057 163.054 258.084 233.082 43 318.312 113.51 107.588 302.976 573.960 35.7 298.621 B (Ref) 256.931 444.278 223.922 387.655 326.542 103.837 450.279 363.469 632.938 243.851 213.119 306.355 406.221 278.989 220.185 520.841 570.366 224.065 516.818 691.231 149.777 306.393 167.627 256.873 317.399 403.788 199.697 189.332 235.192 202.153 43 320.681 145.13 45.204 278.989 691.231 45.3 286.441 %Ratio Ln Ln [Test/Ref] [A(Test)] [B(Ref)] 79.295 5.317 5.549 90.410 5.996 6.096 139.787 5.746 5.411 108.516 6.042 5.960 80.429 5.571 5.789 103.613 4.678 4.643 85.233 5.950 6.110 101.268 5.908 5.896 82.211 6.254 6.450 148.196 5.890 5.497 152.888 5.786 5.362 91.137 5.632 5.725 87.587 5.874 6.007 123.673 5.844 5.631 91.773 5.309 5.394 92.916 6.182 6.255 100.630 6.353 6.346 104.293 5.454 5.412 90.758 6.151 6.248 77.828 6.288 6.538 135.305 5.312 5.009 104.620 5.770 5.725 103.307 5.154 5.122 96.074 5.509 5.549 77.069 5.500 5.760 92.634 5.924 6.001 134.232 5.591 5.297 86.120 5.094 5.244 109.734 5.553 5.460 115.300 5.451 5.309 43 43 43 114.522 5.699 5.658 84.51 0.37 0.51 64.090 4.678 3.811 97.525 5.714 5.631 636.098 6.353 6.538 73.8 6.5 9.1 104.252 5.687 5.633 Diff [Ln(Test-Ref)] -0.232 -0.101 0.335 0.082 -0.218 0.035 -0.160 0.013 -0.196 0.393 0.425 -0.093 -0.133 0.212 -0.086 -0.073 0.006 0.042 -0.097 -0.251 0.302 0.045 0.033 -0.040 -0.260 -0.077 0.294 -0.149 0.093 0.142 43 0.042 0.35 -0.445 -0.025 1.850 840.5 -
91
Bioequivalence study of Rosuvastatin calcium 40mg Tablet under Fed Condition Table 17: Summary Table of Pharmacokinetic Variables for Rosuvastatin Test : Rosuvastatin Tmax Cmax AUC(0-t) Statistics: (hr) (ng/mL) (hr.ng/mL) N Mean SD Min Median Max %CV GM 43 4.08 1.3 2.00 3.50 6.00 32.9 3.85 43 37.495 15.78 13.588 32.350 81.865 42.1 34.381 43 306.052 113.59 96.820 292.959 558.623 37.1 285.497 AUC AUC_ (0-inf) Extrap (hr.ng/mL) (%) 43 43 318.312 4.355 113.51 2.76 107.588 1.028 302.976 3.306 573.960 14.350 35.7 63.3 298.621 3.688 Kel (1/hr) 43 0.052 0.02 0.029 0.048 0.134 32.6 0.050 Thalf (hr) 43 14.31 3.5 5.18 14.56 24.11 24.7 13.85
Reference: Rosuvastatin Statistics: N Mean SD Min Median Max %CV GM Tmax (hr) 43 4.20 1.4 2.00 4.50 6.00 32.5 3.96 AUC_ Cmax AUC(0-t) AUC(0-inf) Extrap (ng/mL) (hr.ng/mL) (hr.ng/mL) (%) 43 43 43 43 38.170 306.075 320.681 5.196 20.44 142.40 145.13 5.10 4.157 34.728 45.204 1.034 33.455 273.225 278.989 3.551 89.275 670.463 691.231 27.942 53.6 46.5 45.3 98.1 33.011 271.124 286.441 3.989 Kel (1/hr) 43 0.053 0.02 0.017 0.050 0.143 36.0 0.050 Thalf (hr) 43 14.60 5.5 4.86 13.74 41.18 37.7 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.
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Bioequivalence study of Rosuvastatin calcium 40mg Tablet under Fed Condition Table 18: Summary Tables for ANOVA Results PK Parameters [N=43] Ln(Cmax) Ln(AUC(0-t)) Ln(AUC(0-inf)) ANOVA Results Probability (p)-value Sequence Formulation Period 0.69 0.55 0.55 0.95 0.37 0.36 0.96 0.42 0.23 Intra-subject CV (%) 32.57 27.58 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] Ln(Cmax) Ln(AUC(0-t)) Ln(AUC(0-inf))
90% Confidence Interval (Lower limit-Upper limit) 92.90-116.99 95.56-116.32 95.49-114.17
Geometric LSM Ratio (%) (Test/Reference) 104.25 105.43 104.41
Intra Subject CV% 32.57 27.58 24.98
Table 20: Extract of Analysis of Variance and Construction of Confidence Interval for Rosuvastatin
93
Bioequivalence study of Rosuvastatin calcium 40mg Tablet under Fed Condition Statistics [N=43]
Ln(Cmax)
Ln(AUC(0-t))
Ln(AUC(0-inf))
Type III Tests of Analysis Variance [Probability (p)values] Sequence Formulation Period Error Term Mean Square Error* Mean Square-Subject(Seq)** 0.10 0.42 0.07 0.38 0.06 0.35 0.69 0.55 0.55 0.95 0.37 0.36 0.96 0.42 0.23
Intra-subject and Inter-subject Variability: Intra-subject CV (%) Inter-subject CV (%) %Geometric Least Square Mean Test (A) 34.420 33.017 104.25 285.704 270.982 105.43 298.822 286.201 104.41 Reference (B) %Geometric LSM Ratio (Test/Ref) 90% Confidence Interval Lower CI (%) Upper CI (%) Power 92.90 116.99 0.94 95.56 116.32 0.98 95.49 114.17 0.99 32.57 41.80 27.58 40.49 24.98 39.29
*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
Bioequivalence study of Rosuvastatin calcium 40mg Tablet under Fed Condition 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 physicians 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
En No 25 27 18 45 AE Date & time of onset 28/11/09, 08:50 28/11/09, 08:18 28/11/09, 16:20 05/12/09, 08:54 Date & Time of resolution 28/11/09, 10:50 NA NA NA Severity Serious Study drug -ness related Not serious Not serious Not serious Not serious Possible Unlikely Unlikely Unlikely Treatment required None None Cap. Domstal-0 None Current Status Recovered Recovered Recovered Recovered Volunteer status Continued withdrawn Withdrawn Withdrawn
Giddiness Vomiting Vomiting Vomiting
Mild Mild Mild Mild
Table 22 Summary of post study adverse event

En. No. Abnormal Laboratory parameter with Values Date & Date of Time of last Sample Dose collection (hh:mm) Seriousness Severity Date of Repeat sample ( if any) Out Come Study drug related Treatment required
95
02
Triglycerides 332.52 mg/dl
05/12/09 08:02
09/12/09
Mild
Not serious
14/12/09
Resolved
Unlikely
None
27
S. Creatinine 1.75 mg/dl
28/11/09 08:04
28/11/09
Mild
Not Serious
08/12/09
Resolved
Unlikely
None
S. Creatinine 2.42 mg/dl 18
28/11/09 08:10
28/11/09
Mild
Not serious
30/11/09 30/11/09 ( 1110.44) 10/12/09 (229.77) 14/12/09 (1182.62) 18/12/09 (175.36) Lost to follow-up
Resolved
Unlikely
None
Creatinine Kinase 28/11/09 282.18 U/L 08:10
28/11/09
Mild
Not serious
Resolved
Possible
None
15
09/12/09
Mild
Not serious
Resolved
Possible
None
19
S. Potassium 5.7 05/12/09 mEq/L 08:12
09/12/09
Mild
Not serious
Lost to follow-up
Unlikely
None
23
09/12/09
Mild
Not Serious
14/12/09
Resolved
Possible
None
26
S. Potassium 5.70 mEq/L
05/12/09 08:02
09/12/09
Mild
Not Serious
14/12/09
Resolved
Unlikely
None
28
RBS 207.82 mg/dl
05/12/09 08:06
09/12/09
Mild
Not Serious
14/12/09
Resolved
Unlikely
None
30
09/12/09
Mild
Not Serious
Lost to follow-up
Lost to follow-up
Possible
None
38
S. Potassium 5.6 05/12/09 mEq/L 08:02
09/12/09
Mild
Not Serious
14/12/09
Resolved
Unlikely
None
7.4.3 Clinical Laboratory Evaluation

Screening 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
96
Bioequivalence study of Rosuvastatin calcium 40mg Tablet under Fed Condition parameter outside of normal range was assessed by the physician/investigator for clinical relevance. The majority of the out-of-range laboratory values were only marginally outside of the respective reference ranges.
97
8.0
DISCUSSION
98
Bioequivalence study of Rosuvastatin calcium 40mg Tablet under Fed Condition The quantification of Rosuvastatin in plasma samples was performed in accordance with GLP requirements. The analytical methods LC MS-MS allowed specific and sensitive 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 intrasubject 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.
99
Bioequivalence study of Rosuvastatin calcium 40mg Tablet under Fed Condition Actual values of secondary pharmacokinetic parameter Tmax were compared for test and reference using Non-parametric Wilcoxon Signed Rank test for Rosuvastatin. No statistical 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
Bioequivalence study of Rosuvastatin calcium 40mg Tablet under Fed Condition 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 REFERENCES
1. 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
Bioequivalence study of Rosuvastatin calcium 40mg Tablet under Fed Condition 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, 4 th 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):949955 13. Goodman and Gillmans 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):62L65L 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;205214 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):13871396 104
Bioequivalence study of Rosuvastatin calcium 40mg Tablet under Fed Condition 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. Harrisons Principles of Internal Medicine,16th Edition, Disorders Of Lipoprotein Metabolism, 2286-2298. 30. Color Atlas of Pharmacology, 2nd edition, Drugs used in Hyperlipoproteinemias, 154157. 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):16021606. 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
Bioequivalence study of Rosuvastatin calcium 40mg Tablet under Fed Condition 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. 1 2 3 4 5 6 7 8 9 Sex Male Male Male Male Male Male Male Male Male Age (Yrs) 31 29 39 31 25 35 37 23 19 Height (cm) 176.00 169.00 159.00 170.00 169.50 167.50 159.50 174.00 171.00 Weight (Kg) 75.81 68.73 56.73 62.27 63.80 74.26 54.47 58.32 54.31 BMI* (Kg/m2) 24.47 24.06 22.44 21.55 22.21 26.47 21.41 19.26 18.57 Race Asian Asian Asian Asian Asian Asian Asian Asian Asian Smoking None None None None None None Current SmokerBidis- 1-09 None None Date of Screening 23/11/09 07/11/09 19/11/09 23/11/09 24/11/09 19/11/09 23/11/09 24/11/09 24/11/09
107
10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36
Male Male Male Male Male Male Male Male Male Male Male Male Male Male Male Male Male Male Male Male Male Male Male Male Male Male Male
24 25 41 38 40 31 28 26 20 26 27 35 41 28 35 37 40 25 34 34 38 30 41 23 35 33 35
168.50 168.00 165.00 167.00 165.00 174.00 170.50 174.50 164.50 168.50 175.50 159.50 170.50 154.00 159.50 171.00 178.00 162.00 170.50 165.00 165.50 167.00 157.50 162.50 176.00 174.00 162.00
65.90 65.85 60.82 61.48 70.98 79.36 64.38 66.77 59.63 57.80 66.80 67.66 65.16 53.00 65.71 56.21 71.50 55.32 74.80 71.96 72.91 57.61 62.13 52.15 70.00 59.19 53.85
23.21 23.33 22.34 22.04 26.07 26.21 22.15 21.93 22.04 20.36 21.69 26.60 22.41 22.35 25.83 19.22 22.57 21.08 25.73 26.43 26.62 20.66 25.05 19.75 22.60 19.55 20.52
Asian Asian Asian Asian Asian Asian Asian Asian Asian Asian Asian Asian Asian Asian Asian Asian Asian Asian Asian Asian Asian Asian Asian Asian Asian Asian Asian
None None None Current SmokerCigarettes- 109 None None None None None None None None Ex-smokerDiscontinued > 3 months ago None None None None Current SmokerBidis- 1-09 None None None Ex-smoker [discontinued >3 months ago] None None None None Ex-smoker [discontinued >3 months ago]
07/11/09 19/11/09 09/11/09 23/11/09 23/11/09 16/11/09 16/11/09 24/11/09 09/11/09 19/11/09 19/11/09 11/11/09 16/11/09 23/11/09 23/11/09 19/11/09 23/11/09 07/11/09 13/11/09 23/11/09 24/11/09 23/11/09 13/11/09 11/11/09 23/11/09 11/11/09 11/11/09
108
37 38 39 40 41 42 43 44 45 46 47 48 31.77 6.3 42 19 20.4
Male Male Male Male Male Male Male Male Male Male Male Male 167.73 5.8 180.50 154.00 2.8
38 34 35 36 42 33 19 25 25 32 33 34
164.50 162.00 165.00 166.00 180.50 170.00 165.50 170.50 171.00 173.50 160.00 171.50 63.68 7.3 79.36 50.20 10.9 2.4
71.91 54.81 62.87 63.57 67.30 59.00 60.71 67.07 67.40 55.97 50.20 78.02 22.64 26.62 18.57 10.8
26.57 20.88 23.09 23.07 20.66 20.42 22.16 23.07 23.05 18.59 19.61 26.53
Asian Asian Asian Asian Asian Asian Asian Asian Asian Asian Asian Asian
None None None None None None None None None None None None
04/11/09 23/11/09 16/11/09 06/11/09 19/11/09 06/11/09 16/11/09 19/11/09 23/11/09 24/11/09 23/11/09 19/11/09
11.2 LABORATORY PARAMETERS WITH REFERENCE RANGE

Parameters Hematology WBC l Neutrophils % Lymphocytes % Monocytes % Eosinophils % Basophil % RBCx M/l Hb[g/dl] HCT% Platelets x K/l Biochemistry Urea (mg/dl) albumin (g/dl) Total Proteins (g/dl) Globulin (g/dl)* ALT [SGPT](U/l) Total Bilirubin (mg/dl) AST (SGOT] (U/L) Serum Alkaline Phosphatase (U/l) Normal Range Male Female 4.0-10.0 40.0-80.0 20.0-40.0 2.0-10.0 1.0-6.0 < 1-2 4.5-5.5 12.5-17.0 40.0-50.0 150-400 17-43 3.5-5.2 6.4-8.3 2.3-3.5 <45 0.3-1.2 <35 30-120 4.0-10.0 40.0-80.0 20.0-40.0 2.0-10.0 1.0-6.0 < 1-2 3.8-4.8 12.0-15.0 37.0-46.0 150-400 17-43 3.5-5.2 6.4-8.3 2.3-3.5 <34 <31 30-120 Observed Value
109

Random Blood Sugar (mg/dI) 45-130 Serum Creatinine (mg/dl) 0.84-1.25 Triglyceride (mg/dl) <150 Cholesterol (mg/dl) 150-250 Calcium (mg/dl) 8.8-10.6 Sodium (mEq/L) 136-145 Potassium (mEq/L) 3.5-5.1 Chloride (mEq/L) 98-106 GGT (U/L) <55 Uric Acid (mg/dl) 3.5-7.2 CK (U/l) 171 Urine analysis Glucose Negative Bilirubin Negative Ketone Negative Sp.Gr. 1.001-1.035 Blood Negative pH 5-9 Protein Negative U.bil gen 0.2-1.0 Nitrite Negative Leucocytes Negative Urine analysis: Physical and microscopic Quantity Color Appearance Clarity Epithelial cell 45-130 0.66-1.09 <150 150-250 8.8-10.6 136-145 3.5-5.1 98-106 <38 2.6-6.0 145 Negative Negative Negative 1.001-1.035 Negative 5-9 Negative 0.2-1.0 Negative Negative Pus cell RBC Cast Crystals Others
Abbriviations used:WBC-White blood cells, RBC-Red blood corpuscles, Hb-Hemoglobin,HCT-Hematocrit ALTAlanine Amino Trsnsferase,AST-Aspartste Amino Transferase. Normal ranges for hematology are given as per reference: Dade & Lewis Practical Hematology , 9 Edition, Page12 Globulin 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
110
Bioequivalence study of Rosuvastatin calcium 40mg Tablet under Fed Condition 11.3 SCHEDULE OF ASSESSMENT Type of Assessment Planned Study Day 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 screen Alchohol 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 Entry Examination (screening) -28 To 0 Minimum of 7 days Period I 0 1 2 3 4 5 Wash out Period II 0 1 2 3 4 5 Post study Safety Evaluation
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
111
Bioequivalence study of Rosuvastatin calcium 40mg Tablet under Fed Condition 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 01Urine only),drug screen, Clinical examination, Compliance Assessment, breath alcohol test, Check-in Dinner & Bed time DOSING DAY & DISCHARGE DAY ACTIVITY Scheduled Actual time (HH:MM) Activity time (Hrs) (Days) 05:30 (D1) Wake up call 06:00 to 07:30 (D1) Pre-dose Vital signs, Cannulation & Pre-dose blood sample collection 07.30 to 07.55 Breakfast 00.00 08:00 (D1) Study Drug Administration 00:33 08:20 (D1) Blood Draw 00:67 08:40 (D1) Blood Draw 01:00 09:00 (D1) Blood Draw 01:50 09:30 (D1) Blood Draw 02:00 10:00 (D1) Blood Draw followed by Vital Signs measurement 02:25 10:15 (D1) Blood Draw 02:50 10:30 (D1) Blood Draw 02:75 10:45 (D1) Blood Draw 03:00 11:00 (D1) Blood Draw 03:25 11:15 (D1) Blood Draw 03.50 11:30 (D1) Blood Draw 04:00 12:00 (D1) Blood Draw followed by Vital Signs measurement 04:50 12:30 (D1) Blood Draw 05:00 13:00 (D1) Blood Draw & lunch 05:50 13:30 (D1) Blood Draw 06:00 14:00 (D1) Blood Draw followed by Vital Signs measurement 07:00 15:00 (D1) Blood Draw 08:00 16:00 (D1) Blood Draw 09:00 17:00 (D1) Snacks 10:00 18:00 (D1) Blood Draw 12:00 20:00 (D1) Blood Draw 13:00 21:00 (D-1) Dinner 16:00 00:00 (D2) Blood Draw 08:00 (D2) Blood Draw followed by medical checkup, breakfast and 24:00 discharge SAMPLES AMBULATORY 08:00 (D-3) Blood draw (ambulatory sample) 48:00 08:00 (D-4) Blood draw (ambulatory sample) 72:00 08:00 (D-5) Blood draw (ambulatory sample) 96:00
Day-0; Entry day, Day-1; Drug administration day, Day-2; Discharge day, Day 3, 4 & 5: ambulatory sample
112
Bioequivalence study of Rosuvastatin calcium 40mg Tablet under Fed Condition 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.
113
I am Mr./Ms._____________________________________________________________, My father's name is _______________________________________________________, My mother's/spouses/guardians 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: Name of witness: Address of witness: Phone No.: Signature of the witness: Medical query resolved by: Written informed consent obtained by:
Date:
Date: Date: Date:
115
11.5 FOOD MENU (FED) CHECK-IN DAY Items Chapati (6 no.) Khichdi Kofta curry Rajmah Masala MixVegetable (Potato, Carrot Peas, Cauliflower) Roasted Papad (Black gram) (1 no.) DINNER Amount (gms or ml) 120 gm 150 gm 200 ml 100 gm 100 gm 07 gm STUDY DAY 1 Items Paneer Cutlet (3 no.) Chutney Milk + Sugar Veg Chana Salad Chapati (6 no.) Rice Butter milk Guj.Tuar Dal Paneer Tikka Masala Salad (Tomato, Cucumber, Carrot) Chhole Roasted Papad (Black gram) (1 no.) Dhokla Chutney (Green) Milk + Sugar BREAKFAST Amount (gms or ml) 120 gm 30 gm 200 ml+5 gm 35 gm LUNCH 120 gm 150 gm 200 ml 200 ml 100 gm 50 gm 100 gm 7 gm SNACKS 120 gm 30 gm 150 ml+ 3.75 gm DINNER 120 gm 150 gm 200 ml 100 gm Kcal 528.47 101.35 253.90 59.00 942.72 395.00 207.60 30.00 146.50 184.82 12.80 114.21 24.01 1114.94 240.06 27.54 190.43 458.03 395.00 207.6 140.00 163.50 Kcal 395.00 308.73 200.00 107.24 102.45 24.01 1137.43
Chapati (6 no.) Rice Sambar Sev Tomato
116
Bioequivalence study of Rosuvastatin calcium 40mg Tablet under Fed Condition Curd Moong (whole) Roasted Papad gram) (1 no.) 50 gm 100 gm (Black 7 gm CHECK-OUT DAY Items Aloo Paratha ( 1 no.) Chutney (Green) Milk + Sugar BREAKFAST Amount (gms or ml) 120 gm 30 gm 150 ml+ 3.75 gm Kcal 241.2 27.54 190.43 459.17 30.00 106.6 24.01 1066.71
Reference: Gopalan C. et al (2004). Nutritive value of Indian foods, NIN, Indian Council of Medical Research, Hyderabad.
BREAKFAST MENU
INGREDIENTS Paneer Cutlets Paneer Toast Bread Groundnut Potato Oil Onion Green Chilly Til AMOUNT 3 no. 50 gm 10 gm 20 gm 20 gm 20 ml 10 gm 04 gm 05 gm Paneer Cutlets Nutrients Amount Energy 528.47 K.cal Fat 40.64 gm Protein 15.88 gm Carbohydrate 24.37 gm
117
Chutney Nutrients Amount Energy 101.35 K.cal Fat 6.45gm Protein 4.78 gm Carbohydrate 6 gm Chutney Green chilly Cor.Leaves Groundnut
5 gm 15 gm 10 gm
Milk Milk Whole Milk Sugar 200 ml 5 gm Nutrients Energy Fat Protein Carbohydrate Amount 253.9 K.cal 13 gm 8.6 gm 14.97 gm
Veg Chana Salad Bengal Gram (Whole) Tomato Cabbage
15 gm 10 gm 10gm
Veg Chana Salad Nutrients Amount Energy 59 K.cal Fat 1.00 gm Protein 2.92 gm Carbohydrate 9.95 gm
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Bioequivalence study of Rosuvastatin calcium 40mg Tablet under Fed Condition

TITLE OF THE PROJECT

Bioequivalence study of Rosuvastatin calcium 40mg Tablet under Fed Condition

Bioequivalence study of Rosuvastatin calcium 40mg Tablet under Fed Condition

Bioequivalence study of Rosuvastatin calcium 40mg Tablet under Fed Condition

1. INTRODUCTION 1.1 Bioavailability and Bioequivalence

1.2 Importance of Bioequivalence Studies

Emerging Risk Factors

Modifiable Risk Factors

Non Modifiable Risk Factors

Age Male Sex Family History of Premature CHD

Bioequivalence study of Rosuvastatin calcium 40mg Tablet under Fed Condition

Bioequivalence study of Rosuvastatin calcium 40mg Tablet under Fed Condition

2.0 OBJECTIVES OF THE STUDY

Bioequivalence study of Rosuvastatin calcium 40mg Tablet under Fed Condition

3.0 RATIONALE OF DRUG Rosuvastatin:

3.1 Clinical Efficacy16,17

Bioequivalence study of Rosuvastatin calcium 40mg Tablet under Fed Condition

Surgery Sequestrants Diet Statins

Bioequivalence study of Rosuvastatin calcium 40mg Tablet under Fed Condition

Bioequivalence study of Rosuvastatin calcium 40mg Tablet under Fed Condition

4.1 Biopharmaceutics and Pharmacokinetics 4.1.1 Biopharmaceutics

4.1.1.3 A Waiver of In Vivo BA/BE Studies

Waiver is not applicable for narrow therapeutic index drug.36

4.1.2.1 Plasma drug concentration Time profile22

Bioequivalence study of Rosuvastatin calcium 40mg Tablet under Fed Condition

Fig 2-Pharmacokinetic profile for different routes of drug administration

Bioequivalence study of Rosuvastatin calcium 40mg Tablet under Fed Condition

4.1.2.2 Pharmacokinetic Parameters

1) Peak plasma concentration (Cmax)

2) Time of peak concentration (Tmax)

3) Area under the curve (AUC)

4.2.1 Absolute bioavailability25

4.2.2 Relative bioavailability25

4.2.3 Objective of Bioavailability studies23

4.2.4 Types of Bioavailability-Bioequivalence Studies22,23,24

4.2.5 Assessment of Bioequivalence5

4.2.6 Estimation of Bioavailability23

Plasma level- time studies

[AUC]test [D]std [AUC]std [D]test

Urinary excretion studies

Acute pharmacological response

Bioequivalence study of Rosuvastatin calcium 40mg Tablet under Fed Condition

4.4.1 Regulatory requirements

Bioequivalence study of Rosuvastatin calcium 40mg Tablet under Fed Condition

4.4.2 Sample collection and sampling times

4.4.3 Subjects with predose plasma concentrations

4.4.4 Data deletion due to vomiting

4.4.5 Rounding off of confidence interval values

4.4.6 Study Population

4.5 Types of Study Design26 I. Single-dose study Design

Bioequivalence study of Rosuvastatin calcium 40mg Tablet under Fed Condition

Multiple-dose study Design2,5

II. Replicated Crossover Designs

III. Food Effect Study Design:5,37

Bioequivalence study of Rosuvastatin calcium 40mg Tablet under Fed Condition

4.5.2 Sample Size and Dropouts:

4.6 Data Analysis26 4.6.1 Average Bioequivalence:

4.6.2 Population Bioequivalence:

4.6.3 Individual Bioequivalence:

I IIa IIb III IV V

Chylomicrons LDL LDL and VLDL IDL VLDL VLDL& chylomicrons

+++ +++ +++ + +

Bioequivalence study of Rosuvastatin calcium 40mg Tablet under Fed Condition