PHARMACOKINETICS OF IBUPROFEN UNDER ... - University of … of Ibupr… · PHARMACOKINETICS OF IBUPROFEN UNDER HEAD-DOWN-BED-REST TECHNIQUE (HDBR) IN HEALTHY HUMANS By Mahmoud Musa

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PHARMACOKINETICS OF IBUPROFEN UNDER

HEAD-DOWN-BED-REST TECHNIQUE (HDBR)

IN HEALTHY HUMANS

By

Mahmoud Musa Eid Abu Dhair

A thesis Submitted inPartial Fulfillment of the

Master of ScienceIn Pharmaceutical Science

At

Petra UniversityFaculty of Pharmacy

Amman-JordanMay 2011

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Abstract

PHARMACOKINETICS OF IBUPROFEN UNDER HEAD-DOWN-BED-REST

TECHNIQUE (HDBR) IN HEALTHY HUMANS

By

Mahmoud Musa Eid Abu Dhair

Petra University 2011

Under the supervision of Prof. Nasir Idkaidek and Prof. Tawfiq Arafat

Space medicine is a new field that is growing fast as the international

space station becomes a reality. Medications had been taken since the first flight

to space and, since then, had been used for several indications. A large number of

studies had been performed in the past 30 years in the attempt to understand how

the human body responds to the lack of gravity.

Microgravity can be simulated on earth by using head-down-bed-rest

technique (HDBR). This involves sleeping while the body is down by 6° from

head side. The aim of this study is to compare the pharmacokinetic parameters of

ibuprofen under head-down-bed-rest technique & normal position in plasma and

saliva in six healthy human volunteers.

The study has been conducted according to open, completely randomized,

single dose, two periods, one treatment sequential design, under fasting conditions

in healthy volunteers. A total number of 6 healthy subjects had completed the

trial. A wash-out period of 7 days separated the two periods.

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Dedicated to ... My parents

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ACKNOWLEDGMENTS

First and foremost, I would like to express my deep and sincere gratitude

to my supervisory Professor Nasir Idkaidek, Faculty of Pharmacy, Petra

University, for his guidance, continuous support, encouragement, and for giving

me the opportunity to work on such an exciting project. His wide knowledge and

his logical way of thinking have been of great value for me.

I am deeply grateful to my co-supervisor Professor Tawfiq Arafat, Dean of

Faculty of Pharmacy, Petra University, for his high support and guidance, which

came always in the right moment. Prof. Arafat opened the doors for this research

at “Jordan Center for Pharmaceutical Research” where I carried out my research.

I also want to thank my examination committee members (Prof.

Mohammed Shubair and Dr. Mutasim Al-Gazawi) for their support and guidance,

which came always in the right moment.

I warmly thank Mr. Hamza Alhorob, Mr. Mahmoud Jamal, Mr. Ahmad

Abu Awad and Eng. Monther Melhim from Jordan Center for Pharmaceutical

Research for their valuable advice and friendly help.

The overall support of Petra University is gratefully acknowledged.

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Table of Contents

Contents Page

Title Page 1

Examination Committee 2

Abstract 3

Dedication 5

Acknowledgment 6

Table of Contents 7

List of Figures 11

List of Tables 12

List of Abbreviations 13

Chapter One: Introduction

No. Contents Page

1. Introduction 16

1.1 Physiological Changes in Space 16

1.2 Pharmacological Changes During Space Flight 18

1.3 Current Practice for Use of Medications in Space 19

1.4 Pharmacokinetics in Space 20

1.4.1 Absorption and Bioavailability 20

1.4.2 Distribution 22

1.4.3 Elimination and Clearance 22

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1.4.4 Metabolism 23

1.5 Pharmacodynamics in Space 23

1.6 Head-Down-Bed-Rest (HDBR) 24

1.7 Ibuprofen 26

1.8 Objective 27

Chapter Two: Experimental

2. Experimental 29

2.1 Design 29

2.2 Volunteers 30

2.2.1 Selection of Subjects 30

2.2.1.1 Inclusion Criteria 31

2.2.1.2 Exclusion Criteria 31

2.3 Medication 33

2.3.1 Reference Drug 33

2.3.2 Blinding 33

2.4 Dosage and Duration of Treatment 33

2.4.1 Randomization List 34

2.5 Benefit – Risk Evaluation 34

2.6 Study Procedure 35

2.6.1 General Procedure 35

2.6.2 Special Procedures 36

2.6.3 Daily Activities in Each Period of the Trial 36

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2.6.3.1 Entry Examination 36

2.6.3.2 Period I of the Trial 37

2.6.3.3 Wash Out 38

2.6.3.4 Period II of the Trial 38

2.6.3.5 Final Examination 39

2.6.4 Restrictions 39

2.6.5 Drug Administration 40

2.7 Assay Method of Ibuprofen 40

2.7.1 Instrumentation 40

2.7.2 Reagents 41

2.7.3 Standards and Stock Solutions 41

2.7.3.1 Preparation of Glibenclamide, (Internal Standard) 41

2.7.3.2 Preparation of Ibuprofen in Plasma 41

2.7.3.3 Preparation of Quality Control Samples in Plasma 42

2.7.4 Description of Method 43

2.7.4.1 Sample Preparation 43

2.7.4.2 Chromatographic Conditions 43

2.7.5 Method Validation 44

2.8 Data Analysis 44

Chapter Three: Results and Discussion

3. Results and Discussion 47

3.1 Plasma Concentrations for All the Volunteers in Normal

and Head-Down-Bed-Rest positions

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3.2 Data for Maximum Concentrations (Cmax) 57

3.3 Data for K elimination 58

3.4 Data for Area Under the Curve ( AUC) 59

3.5 Data for Volume of Distribution ( V/F ) and

Clearance (CL)

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3.6 Data and Statistical Comparisons for (AUMClast ) and

(AUMCinf_obs)

62

3.7 Data for ( MRT ) 63

3.8 Pharmacokinetic Parameter Means (CV%) of

Ibuprofen Under Normal and Head-Down-Bed-Rest

Positions

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Chapter Four: Conclusions

4. Conclusions 68

References 69

Appendices

1 Institutional Review Board (IRB) Approval 72

2 Jordan Food and Drug Administration (JFDA)

Approval

74

3 Informed Constant Form 76

4 Winnonlin Output 89

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List of Figures

No. Contents Page

Fig. 1 Fluid Shift from the Lower to the Upper Part of the BodyInduced by Real and Simulated Weightlessness.

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Fig 2 Ibuprofen Plasma Concentrations of Volunteer 1 inNormal and Head-Down-Bed-Rest Positions.

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Fig 8 Ibuprofen Plasma Concentrations of 6 Volunteers Averagein Normal and Head-Down-Bed-Rest Positions.

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List of Tables

No. Contents Page

Table 1 Randomization List. 34

Table 2 Ibuprofen Plasma Concentrations (µg/ml) after 600 mgOral Dose in 6 Volunteers in Normal Position.

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Table 3 Ibuprofen Plasma Concentrations (µg/ml) after 600 mgOral Dose in 6 Volunteers in Head-Down-Bed-RestPosition.

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Table 4: Cmax and Tmax in Both Phases for All the

Volunteers.

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Table 5: Kel in Both Phases for All the Volunteers. 58

Table 6.a AUClast, AUC0-4, AUCinf_obs and AUC_%Extrap inHead-Down-Bed-Rest Phase for All the Volunteers.

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Table 6.b AUClast, AUC0-4, AUCinf_obs and AUC_%Extrap inNormal Phase for All the Volunteers.

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Table 7 V/F and Cl/F in Both Phases for All the Volunteers. 61

Table 8 AUMClast and AUMCinf_obs in Both Phases for All theVolunteers.

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Table 9 MRT in Both Phases for All the Volunteers. 63

Table 10 Pharmacokinetic Parameter Means (CV%) of IbuprofenUnder Normal and Head-Down-Bed-Rest Positions.

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List of Abbreviations

µG MicrogravityABR Anti-orthostatic Bed RestADME Absorption, Distribution, Metabolism, ExcretionADR Adverse Drug ReactionAE Adverse EventALC EthanolART Adverse Reaction TerminologyAUC Area Under Plasma Concentration-Time CurveAUMC Area Under Moment CurveBA BioavailabilityBE BioequivalenceBILI BilirubinBMI Body Mass IndexBP Blood PressureCa CalciumCLR Renal ClearanceCmax Maximal Plasma Drug ConcentrationCNS Central Nervous SystemCp Concentration PointCREA CreatinineCRF Case Report FormGCP Good Clinical PracticeGIT Gastrointestinal TractGMP Good Manufacturing Practicehr HourHR Heart RateHB HemoglobinHBS-AG Hepatitis B AntigenHcvg Hepatitis C AntigenHDBR Head-Down-Bed-Rest

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HIV Human Immunodeficiency VirusHPLC High Performance Liquid ChromatographyIEC Independent Ethics CommitteeIRB Institutional Review Boardi.v IntravenousJCPR Jordan Center for Pharmaceutical ResearchJFDA Jordan Food and Drug AdministrationK PotassiumKel Terminal Rate ConstantLLOQ Lower Limit Of Quantificationmin. MinuteMRT Mean Residence TimeNa SodiumNASA National Aeronautics and Space AdministrationPBC PlateletspH Hydrogen Ion ConcentrationPK Pharmacokinetic ParameterRBC Red Blood CountSAE Serious Adverse EventSMS Space Motion SicknessSOP Standard Operating ProcedureT Timet0.5 Terminal Elimination Half –LifeTmax Time of Maximum Plasma Drug ConcentrationVd Volume of DistributionWBC White Blood CountWHO World Health Organization

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Chapter 1:

Introduction

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1. Introduction:

Space medicine is a new field that is growing fast as the international

space station becomes a reality. Medications had been taken since the first flight

to space and, since then, had been used for several indications such as space

motion sickness, sleeplessness, headache, nausea, vomiting, back pain, and

congestion. As the duration of space missions get longer, it is even more likely

that astronauts will encounter some of the acute illnesses that are frequently seen

on Earth. Microgravity environment induces several physiological changes in the

human body.

National Aeronautics and Space Administration (NASA) has ongoing

studies on how the astronauts' health is affected by space travel, & how the

gravity affects the pharmacokinetics of the medications

The gravity may define as the force that attracts a body toward the center

of the earth, while microgravity is a condition, especially in space orbit, where

the force of gravity is so weak that weightlessness results.

1.1 Physiological Changes in Space:

Exposure to the microgravity (µG) environment of space flight causes

several physiological changes to the body. These changes are believed to occur in

two phases : The initial response phase takes place during the first 3 to 6 weeks of

flight, when gradual readjustments in all body systems take place. This phase is

characterized by changes in neurovestibular receptors, sympathetic and

parasympathetic function, fluid volume, metabolism, hemodynamics, and

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endocrine regulation. One of the most noticeable changes early in flight is the

fluid shifts. Fluids are redistributed from the extremities to the upper body and

neck region, causing two common syndromes called “bird legs” and “puffy face”

(Nicogossian et al 1994).

This fluid shift causes heart to enlarge in order to handle the increased

blood flow. This "flooding" in the upper part of the body causes the body -

through negative feedback - to correct this situation by getting rid of some of the

excess fluids. The human becomes less thirsty than normal, and the kidneys

increase the output of the urine. Both these actions decrease the over all quantity

of fluids and electrolytes. Once the fluid level decreases, the heart shrinks back to

its normal shape and size.

This decrease in fluid volume leads to electrolyte imbalance and decrease

the plasma volume (Blood volume may decrease by 10 %) (Edgar et al 2004).

The adaptation phase begins after several months in space. It is during this

period that changes such as regional bone and muscle mass loss, changes in

neurotransmitter and receptor functions, and perhaps decreased immune responses

and central nervous system (CNS) function take place(Edgar et al 2004).

A large number of studies had been performed in the past 30 years in

attempt to understand how the human body responds to the lack of gravity. As of

today, most of the physiological changes experienced in space have been fairly

well characterized and understood. Countermeasures to the most significant ones,

such as cardiovascular deconditioning, loss of plasma volume, and muscle and

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bone degeneration are being developed and tested. It is not the scope of this work

to review the details of all the physiological changes observed in space.

Several general and specific reviews (Graebe et al 2004, Zhang LF 2001,

Wang et al 2001, Watenpaugh DE 2001, Harm et al 2001 and Fitts et al 2000) had

been published recently. These publications should be consulted for a more

detailed explanation of all the physiological alterations that occur as a

consequence of adaptation to space µG.

1.2 Pharmacological Changes During Space Flight:

Pharmacological effects can be viewed as the result of pharmacokinetic

(PK) and pharmacodynamic (PD) contributions. Pharmacokinetics deals with

what the body does to the drug. It describes the time course of drug and

metabolite concentrations in the body. Pharmacodynamics describes the

relationships between pharmacological effects and drug/metabolite

concentrations. The combination of the two, PK-PD modeling, leads to the

identification of the optimum dose and dosage regimen to achieve the desired

therapeutic effect. For many drugs, the relationship between PK and PD has been

studied in normal gravity. However, the established normal gravity dosing

recommendations may not be applicable in space because of possible changes in

PK or PD or both.

As previously indicated, space travel induces several physiological

changes in the human body. The first signs of exposure to a zero-gravity

atmosphere include symptoms such as headache, nausea, vomiting, congestion,

back-pain, and sleeplessness. Space motion sickness (SMS), as is commonly

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known, is experienced by 50% or more of astronauts during the first few days of

exposure to the µG environment of space. It is no surprise that since the first

mercury flight in 1967, medications have been a common and necessary part of

space missions.

1.3 Current Practice for Use of Medications in Space:

All pharmacological agents that are used on the shuttle are carefully

reviewed for potential benefits as well as potential negative effects on the

individual or the mission. Astronauts test all frequently used medications before

mission assignment, to identify potential side-effects, performance-related

problems, and individual therapeutic effect.

This procedure helps to evaluate the effect of these medications in space,

although precise predictions are still not possible. Critical issues such as “What

are the pharmacodynamics and pharmacokinetics of drug therapy in

weightlessness?” and “What drug forms, administration routes, and treatment

regimen must be used to optimize therapeutic intervention in weightlessness?” are

still largely unknown.

For example, the crewmembers’ responses to their preferred sleeping

medication are evaluated preflight to determine any side effects or idiosyncratic

reactions. Different doses are tested in each individual to adjust for the decreased

bioavailability of drugs as a result of alterations in gastrointestinal function in µG

(Santy et al 1988).

Most of the medications taken during space flight were taken for space

motion sickness (SMS), sleep disturbances, headache, backache, and sinus

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congestion. Commonly used sleep medications were temazepam, zolpidem,

triazolam, and flurazepam. The medications taken for pain were acetaminophen,

aspirin and ibuprofen. Promethazine was the predominant medication used for

SMS, either by itself, or in combination with dextroamphetamine (Putcha et al

1999).

1.4 Pharmacokinetics in Space:

1.4.1 Absorption and Bioavailability:

Drug absorption from gastrointestinal tract (GIT) depends on many factors

such as gastric emptying rate, intestinal motility, blood perfusion and mean

residence time. Gastric emptying rate depends on volume of the meal,

temperature of food, body position and also earth gravity. Similarly, intestinal

motility, blood perfusion and mean residence time will also change at

microgravity.

The oral route is generally preferred for administering drugs. However,

experience with the administration of medications in the µG environment has

stimulated a reevaluation of the relative advantages and disadvantages of the

various dosage forms formulations. Few data exist on bioavailability in space, but

(Pool SL and Nicogossian A 1983) showed that a few orally administered

medications taken during flight were less effective than expected.

Preflight and in-flight salivary levels of acetaminophen where shown to

differ, probably due to changes in gastrointestinal transit time. In-flight salivary

concentration-time curves of scopolamine /dextroamphetamine, given as

conventional oral tablets, also were shown to be erratic and exhibited higher intra-

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and inter-individual variability compared to those of preflight data (Cintron et al

1987).

Of all factors that can influence the absorption of orally administered

drugs, the rate of absorption may be the most variable parameter noted in space

(Benet LZ. 1988). Gastric emptying is highly influenced by several factors:

volume, calories, exercise, size and density of particles, temperature, viscosity,

and osmolality, and factors associated with physiologic responses (such as

splanchnic blood flow, body position, and electrolyte balance). Gastric emptying

in µG can also be altered by changes in particle size discrimination by the

stomach, which is strongly dependent on the force of gravity.

Also, particles are no longer restricted to the lower pyloric region of the stomach

by gravity, but move throughout all regions of the stomach. This array of factors

can lead to variability in drug plasma levels. Another important consideration

concerning gastric emptying is the effect of motion sickness (Amidon et al 1991).

Stewart (Stewart et al 1994) showed that motion sickness can be considered the

major source of gastric stasis in space. By using nuclear medicine techniques,

Stewart et al observed that the additive effect of scopolamine and promethazine

on gastric emptying in Malaise-III (M-III) subjects was little, confirming the

strong inhibitory effect of motion sickness on gastric motility.

Intestinal transit rate in a gravity environment is highly dependent on the

motility state of the GI tract either fasted or fed partly because of the higher

viscosity of chyme in the fed state. In space, the absence of gravity may tend to

increase the transit rate along the small intestine by decreasing the dimensionless

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ratio of gravitational forces to viscous forces. In zero gravity, therefore, these

alterations in GI emptying and intestinal transit rate could lead to inefficient

absorption and erratic plasma levels.

1.4.2 Distribution:

Physiological changes like the decrease in total body water and plasma

volume, tissue perfusion, and muscle mass loss may alter the volume of

distribution of drugs. This may have an impact on the plasma and tissue

concentrations achieved after administering a drug in space, and depending on the

magnitude of the change, may require a completely new dosing scheme to be

designed to avoid subtherapeutic or toxic concentrations.

Drug binding in blood occurs to different structures as plasma proteins,

lipids and erythrocytes. As only the unbound fraction of drug is active, changes in

total plasma proteins will affect pharmacokinetics. During space flight no

significant changes in plasma protein concentrations had been observed

(Nicogossian et al 1994), but a reduction in red blood cell mass (hemoglobin and

number of erythrocytes) is reported. Altered tissue binding is observed as a result

of protein loss, muscle atrophy and decrease in lean body mass (Edgar et al 2004).

1.4.3 Elimination and Clearance:

Clearance is defined as the volume of blood cleared per time unit, and

hence, can be expressed as the product of blood flow across an eliminating organ

and the respective extraction ratio. Because plasma volume is decreased during

space flight there might be a possibility of changes in organ perfusion. Studies in

anti-orthostatic bed rest did not show a significant change in hepatic blood flow or

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effective renal plasma flow. However, this may be because of differences in the

underlying mechanisms that cause the fluid shifts in ground-based models and in

space flight. Therefore, these studies need to be confirmed in space before any

conclusions can be made (Edgar et al 2004).

1.4.4 Metabolism:

Amounts of cytochrome P-450 isoforms and other enzymes were shown to

decrease during space flight and simulated µG, suggesting that metabolism may

also be altered by space flight (Merrill et al 1990).

Although generalized predictions of the effect of µG on drug metabolism

cannot be made based on these observations, these alterations could result in

higher plasma levels of some drugs, especially low- extraction drugs, and

increased incidence of adverse effects.

1.5 Pharmacodynamics in Space:

Little is known about the effect of µG on pharmacodynamics.

Pharmacodynamic changes due to microgravity are hard to evaluate since the

dose-response relation is a hybrid: it depends on pharmacokinetic characteristics,

pharmacodynamic characteristics or both.

The pharmacological changes that may be observed during space flights are more

likely to be resulting from the physiological perturbations that arise from

exposure to µG. Intra and inter-individual variability in pharmacological response

to medications can also be caused by factors such as stress and lack of sleep.

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1.6 Head-Down-Bed-Rest (HDBR):

Microgravity can be simulated on earth by using head-down-bed-rest technique.

This involves sleeping while the body is down by 6° from head side. Head-Down-

Bed-Rest (HDBR) has proved its usefulness as a reliable simulation model for the

most physiological effects of spaceflight. As well as continuing to search for

better understanding of the physiological changes induced, upward fluid shift,

unloading the body’s upright weight, absence of work against gravity, reduced

energy requirements and reduction in overall sensory stimulation.

The upward fluid shift by acting on central volume receptors induces a

10–15% reduction in plasma volume, Calcium excretion is increased from the

beginning of bed rest leading to a sustained negative calcium balance. Calcium

absorption is reduced. Body weight, muscle mass, muscle strength is reduced.

Bone density, stiffness of bones of the lower limbs and spinal cord and bone

architecture are altered (Pavy-Le et al 2007).

Bed rest, ranging from several hours to several weeks or months, has been

the model most frequently used to simulate the physiological effects of

weightlessness, in particular on the cardiovascular system and body fluid

regulation. Most experiments since 1986 were performed using the –6 degree

HDBR model, in male volunteers. Bed rest induces a fluid shift from the lower to

the upper part of the body (Fig. 1a, b). This fluid shift results in a transient

increase of plasma volume, as more fluid moves into the vascular compartment

from the lower body than that is filtered out of capillaries into the upper body

(Pavy-Le et al 2007).

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Fig. 1 This shows the fluid shift from the lower to the upper part of the bodyinduced by real (a) and simulated weightlessness (b) (Pavy-Le et al 2007).

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1.7 Ibuprofen:

Ibuprofen is a non-steroidal anti-inflammatory agent used in painful and

inflammatory conditions. It is usually given 3 to 4 times daily in a dose of 200 -

800 mg. It is an inhibitor of cyclo-oxygenase enzyme. The usual dose by mouth is

1.2 to 1.8 gm daily in divided doses; if necessary the dose may be increased to 2.4

gm daily.

The main adverse effects are gastro-intestinal disturbances; reactions

range from abdominal discomfort, nausea and vomiting, and abdominal pain to

serious gastro-intestinal bleeding or activation of peptic ulcer. Central Nervous

System (CNS) related side effects include headache, dizziness, nervousness,

depression, drowsiness and insomnia. Hypersensitivity reactions may occur and

include fever and rashes. Ibuprofen or other non-steroidal anti-inflammatory

drugs should not be given to patients with active peptic ulcer.

Pharmacokinetics: Ibuprofen is rapidly absorbed after oral administration,

with peak serum or plasma levels generally appearing within 1.5 to 2 hours. Oral

absorption is estimated to be 80% of the dose. Both the rate of absorption and

peak plasma concentrations are reduced when the drug is taken with food, but,

bioavailability as measured by total area under the concentration-time curve is

minimally altered.

Ibuprofen has an elimination half-life of approximately 2 hours. It is

rapidly metabolized through oxidation and glucuronic acid conjugation with

urinary excretion of the inactive metabolites usually complete within 24 hours.

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Less than 10% is excreted unchanged in the urine. Clinical studies indicate

duration of clinical effect for up to 8 hours for fever and 6 hours for pain.

The therapeutic concentration of Ibuprofen is 20 to 30 mg/L

(Kleinbloesem et al 1995).

1.8 Objective:

The aim of this study is to compare the pharmacokinetic parameters of

ibuprofen under Head-Down-Bed-Rest technique & normal position in plasma

and saliva in six healthy human volunteers.

Ibuprofen will be used as model drug since no previous ground studies were

performed on it. Hence, our results can help understand more the effects of

microgravity conditions on pharmacokinetics and if needed to correct the dose

regimens for ibuprofen to be used in flight by crew members.

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Chapter2:

Experimental

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2. Experimental:

2.1 Design:

The study has been conducted according to open, completely randomized,

single dose, two periods, one treatment sequential design, under fasting conditions

in healthy volunteers.

A total number of 6 healthy subjects had completed the trial. A wash-out period

of 7 days was left between the two periods.

Under HDBR the volunteers were rested in a 6° head-down tilt position in

which subject has been able to eat and urinate. The selected subjects had been

randomized to receive a single 600 mg oral dose of Ibuprofen after a 10 hour

overnight fasting, in two different occasions: during a normal position and during

HDBR position.

The normal phase lasted 8 hours of sample and data collection and the

HDBR phase lasted 18 hours; 10 hours prior to drug administration and 8 hours of

data collection thereafter.

After volunteers received the dose by 4 hours, they had their first meal and

the second meal was after finishing blood sample collection. Subjects were

allowed to consume water all over the study.

Subjects were allowed to rest on an elbow for about 20 minutes during

meals, Physiological (BP, heart and respiratory rate) were monitored every 4

hours during wake hours, Labs (cell counts, differential, electrolytes, liver

enzymes, etc.) were measured once at admission and once immediately at the end

of the HDBR phase.

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Saliva samples (0.5-3 ml) & blood samples (5 ml) were collected at the

following times: pre-dose, and 0.25, 0.5, 0.75, 1, 1.33, 1.66, 2, 2.5, 3, 4, 5, 6 and 8

hours post dosing. Plasma was immediately separated by centrifugation and all

samples stored at -20º C until analysis.

2.2 Volunteers:

Six healthy volunteers participated in a randomized sequential design

clinical study to evaluate and compare the pharmacokinetics of ibuprofen after a

single oral dose under head-down-bed-rest position and normal position.

The experiment details and a written informed consent were provided to

volunteers for their approval. However, the study was started after IRB and JFDA

approvals. (Appendices 1, 2&3)

Each candidate was subjected to a screening examination including

medical history and physical examination, complete blood count & clinical blood

chemistry.

Subjects were non-smokers, non-obese, with no history of migraine,

recurrent headaches, clotting disorders, no history of gastric or duodenal ulcers or

drug allergies, and in good general health as accessed by the physical examination

and laboratory results. Also, subjects were tolerant to HDBR position.

2.2.1 Selection of Subjects:

Six subjects, who fulfilled the inclusion criteria (Section 2.2.1.1), do not

present any of the exclusion criteria (Section 2.2.1.2), had been entered into the

study.

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Drug assays and statistical analysis of the data had been performed on all

plasma samples of the subjects who completed the study according to the

protocol. Subjects had been recruited from the volunteer pool of Jordan Center

for Pharmaceutical Research.

The criteria were set to ensure a homogeneous subject population without

accompanying diseases interfering with the conduct and scientific evaluation of

the study. Additionally, the criteria had been selected to minimize risk to the

subjects’ well-being.

2.2.1.1 Inclusion Criteria:

Only subjects presenting all of the following criteria had been enrolled in

the trial:

• Subject agrees to eat during HDBR position.

• Age between 18 and 50 years.

• Physically and mentally healthy as judged by means of a medical and standard

laboratory examination.

• Weight not less than 60 kg.

• No allergic history to ibuprofen.

• Body weight within ±10% of the ideal body weight in relation to height

according to the Body Mass Index: (BMI = Weight (kg) / Height (m) 2, (17.1

to 28.6)).

2.2.1.2 Exclusion Criteria:

Subjects presenting any of the following had not been included in the

trial:

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• Subjects with a baseline pressure less than 110/60 mm Hg before dosing while

in HDBR position.

• Any clinically significant allergic disease.

• Any history or presence of Ibuprofen or other NSAIDS induced peptic ulcer.

• History of allergic response to Ibuprofen or other NSAIDS.

• Presence or a history of clinically significant cardiovascular, renal, hepatic,

pulmonary, metabolic, endocrine, hematological, gastrointestinal, neurological,

psychiatric or other major disease.

• Clinically significant illness within 4 weeks before the start of the study.

• Intake or administration of any prescribed systemic or topical medication

within 2 weeks prior to the start of the study.

• Concomitant intake or administration of any systemic or topical drugs.

• Treatment with any investigational drug (i.e. drug not yet approved) in the last

3 months before beginning of the trial.

• Participation in another clinical trial within the last 2 months.

• Treatment with drugs known to alter the major metabolic systems such as

barbiturates, phenothiazines, cimetidine, omeprazole etc. within the last 30

days.

• Major surgery of the gastrointestinal tract except for appendectomy.

• Donation of blood or plasma within the last two months.

• Supine blood pressure, after resting for 5 min, higher than 140 /90 or lower

than 90/60 mmHg.

• Supine pulse, after resting of 5 min, outside the range of 60-90 beats /min.

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• Smoking.

• Consumption of more than 4 cups of Coffee or equivalent per day.

• Vegetarian.

• Knowledge to have a hepatitis B infection or carrier of the respective antigens

(HBs Ag, positive).

• Evidence of an uncooperative attitude.

• Alcohol Abuse.

2.3 Medication:

The study medication was manufactured by The Boots Company PLC

Nottingham, England, according to GMP-requirements.

2.3.1 Reference drug:

• Name: Brufen® oral tablet 600 mg.

Each tablet containing 600 mg of Ibuprofen as active ingredient.

• Mode of administration: oral administration of one tablet in the morning

with 240 ml tap water.

• Batch-No: 21V

• Expiry date: 06/2011

2.3.2 Blinding:

No blinding was planned.

2.4 Dosage and Duration of Treatment:

After an overnight fast of 10 hours, all subjects received a single oral dose

of 600 mg Ibuprofen in the morning of each period of the trial, given as one tablet

of the reference preparation, under HDBR body position during first period of the

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study and under normal body position during second period of the study. The first

period of the trial has been followed after a wash-out of seven days before the

second period of the trial.

Before administration of study medication on clinic days, the Principal or

the Clinical Investigator checked each unit and label against the randomization

schedule and also ensured that the subject number on the label corresponds to the

number allocated to each recipient.

2.4.1 Randomization List:

Table 1: Randomization List

Subject No. Period I Period II

01 HDBR N

02 HDBR N

03 HDBR N

04 HDBR N

05 HDBR N

06 HDBR N

HDBR: Head down bed rest body position N: Normal body position

2.4 Benefit – Risk Evaluation:

The efficacy and safety of Ibuprofen as an active ingredient has

been proved in clinical trials. The Brufen® oral tablet 600 mg Ibuprofen is

produced by Knoll.

A healthy subject had taken two doses each containing 600 mg Ibuprofen

of one products with 7 days interval was very small. Mild and minimal adverse

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35

events when occurred, from the study medications or study procedures were

expected in this study.

The planned procedures within the present trial also represent no special

risk to the volunteers. All volunteers had been subjected to safety examinations.

The entry examination had been carried out 14 days before the beginning of the

trial and the final examination on the day of last sampling thereafter. Blood

samples (total number of 28 blood samples for 2 periods, 5 ml each) for drug

analysis had been taken at the following times: pre-dose, 0.25, 0.5, 0.75, 1, 1.33,

1.66, 2, 2.5, 3, 4, 5, 6 and 8 hours post dosing. A total blood loss of approximately

150 ml (including laboratory examinations) per volunteer was to be expected.

In general, blood loss could provoke faintness, dizziness, thirst, sweating,

weak and rapid pulse, rapid respiration, orthostatic hypotension and eventual

decrease in some laboratory parameters such as red blood cells and hemoglobin.

Having in mind that the blood loss in the present trial had taken place over a

period of two weeks, the risk for the volunteers to experience one of the upper

side effects was relatively low.

2.6 Study Procedure:

2.6.1 General Procedure:

The entry examination was carried out before the beginning of the trial by

14 days and the final examination on the day of last sampling. All laboratory tests

were carried out at Al Mowasah Hospital laboratories.

The subjects attended the clinic at 7:00 pm at night on the day before dosing

(day 0 in each study period), and remained there for 8 hours after dosing.

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36

In the morning of day one the volunteers received the trial medication under

fasting conditions. The volunteers had to fast from food for 10 hours before and

until 4 hours after administration of study medication on day 1.

At the end of period II, standard clinical examination performed for each and

every subject.

2.6.2 Special Procedures:

All subjects had been at HDBR body position for the whole duration of

period I (around 18 hours), while they had been in normal body position during

period II.

2.6.3 Daily Activities in Each Period of the Trial:

2.6.3.1 Entry Examination:

The entry examination carried out before the beginning of the trial by 14

days. Following parameters were documented and /or investigated:

• Thorough explanation of the study medication from the Investigators.

• Date of inclusion (date of signing the informed consent).

• Date of examination.

• Inclusion criteria (according to protocol).

• Exclusion criteria (according to protocol).

• Informed consent.

• Demographic data: date of birth, ethnic group, sex, height, weight, BMI.

• Additional information concerning: illness within the last 4 weeks prior

start of the trial, last participation in any clinical trial, last donation of

blood or plasma, last administration of any medication (including OTC or

`

37

topical medication), last administration of drugs known to alter the major

organs or systems as well as specification of the drug name, dosage, start

and end of treatment.

• Vital signs: registration of body temperature, measurements of supine

heart rate (HR) and blood pressure (BP) after 5 minutes supine rest.

• Physical examination.

• Laboratory examination of blood which includes Blood hematology,

Blood chemistry and check for presence of Hepatitis B, C, and HIV.

• Urinalysis which includes, pH, protein and glucose (semi quantitatively)

and evaluation of sediment.

A blood sample of 10 ml was taken for laboratory screening before the

study. Both drug abuse test and alcohol test had been done before the two periods

of the study. ECG had been done before the study.

2.6.3.2 Period I of the Trial:

Day 0: This day is the day before administration of study medication, for this

purpose the subjects had to attend to the clinic at 7:00 pm.

The following procedures were performed and documented in case report form

(CRF) on this day:

• Urine drug screen

• Alcohol test in saliva

• Check of exclusion criteria, adverse events and concomitant medication,

• Evening snack not later than 7.30 p.m.,

• Overnight stay in the clinic.

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38

• They started the HDBR body position directly after the snack in day 0.

Day 1: The following procedures carried out for HDBR body position on this

day:

• A venous cannula was inserted and 5 ml blood sample was drawn before the

morning dose.

• Measurement of blood pressure

• Administration in the morning of day 1 of an oral single dose of one tablet

of the reference drug Brufen® with 240 ml tap water while seating on bed in

the upright position then return to HDBR body position after 5 minutes of

the dose.

• Saliva and blood sampling was continued at the following times: pre-dose,

and 0.25, 0.5, 0.75, 1, 1.33, 1.66, 2, 2.5, 3, 4, 5, 6 and 8 hours.

• Breakfast 4 hours post dose (volunteers had to eat up the meals while

seating on beds in the upright position during period one of the study,

volunteers had to eat their meals in 15 minutes then returned to HDBR body

position directly).

• Standard lunch 8 hours post dose (after last blood sample) was given.

• Discharged from hospital 8 hours post dose.

2.6.3.3 Wash Out:

7 days from Day 1

2.6.3.4 Period II of the Trial:

It was exactly the same as period I while at normal body position.

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39

2.6.3.5 Final Examination:

The final examination carried after the end of period II. The following

parameters were documented and /or investigated:

• Date of examination.

• Check of exclusion criteria.

• Physical examination.

• Registration of vital signs.

• Registration of adverse events

2.6.4 Restrictions

Volunteers were requested not to undertake vigorous exercise beginning 2

days before the entry screening laboratory test until after the final examination.

The chewing of chewing gum was not allowed on the days of dosing. Smoking

was restricted. Foods and beverages containing xanthines or grapefruit were

prohibited 7 days before dosing until the final sampling time. Alcohol

consumption was prohibited 48 hours before dosing until the final sampling time.

The volunteers had not take any prescribed medicine or over the counter

medications at least 2 weeks before the beginning of the study. The subjects were

fast from food and beverages other than water, for at least ten hours, from 8:00

pm on the evening before dosing (Day 0) until breakfast on the following day

(Day 1). Volunteers were requested to restrict their movements as much as

possible while at HDBR body position during first period of the study. Volunteers

were allowed to lie, sit or walk after administrating the dose except the first 4

`

40

hours after the dose where the volunteer should stay seated on chair in the upright

position while at normal body position during second period of the study.

2.6.5 Drug Administration

The precise instructions for drug administration were given to subjects by the

investigators.

All subjects received in each period of the trial a single dose of 600 mg Ibuprofen of

the reference drug Brufen®. The drug administrations were separated by a washout

period of 7 days. Volunteers swallowed the tablet of the reference preparation with

240 ml of water while seating on bed in the upright position then return to HDBR

body position after 5 minutes of the dose during period one of the study.

2.7 Assay Method of Ibuprofen:

All samples analyzed by HPLC method, Mean of Ibuprofen concentration

versus time curves generated in Microsoft Excel for plasma, in both normal phase

and HDBR phase.

The procedure and analysis was done at Jordan Center for Pharmaceutical

Research.

2.7.1 Instrumentation:

A Dionex HPLC auto-sampler system was used and composed of the

following:

• A constant solvent delivery system (P580).

• A 100 µL fixed volume injector (Rheodyne 7125).

• UV Detector (UVD340S).

• Autosampler (ASI-100).

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41

• Computer System, Windows2000, Chromeleon Chromatography

Management System.

• Thermo Electron Corporation, BDS Hypersil C18 Column (150 x 4.6 mm),

5 µ.

2.7.2 Reagents:

• Methanol and Acetonitrile of HPLC grade (ACR S)

• Glacial acetic acid.

• Deionized Water (Nanopure)

• Human Plasma (harvested from donors)

2.7.3 Standards and Stock Solutions:

• Ibuprofen, a concentrated stock solution was prepared in Methanol (2000

g/ml).

• Glibenclamide, a concentrated stock solution was prepared in Methanol.

(1000 g/ml).

2.7.3.1 Preparation of Glibenclamide, (Internal Standard):

10 mg of Glibenclamide was dissolved in 10 ml of Methanol; solution (A),

this contains 1000 g/ml. 0.3 ml of solution (A) was taken and diluted to 100 ml of

Methanol; solution (B), which contains 3.0 µg/ml. Solution (B) is considered to be

working solution contains 3.0 µg/ml of Glibenclamide.

2.7.3.2 Preparation of Ibuprofen in Plasma:

Spiked plasma samples were prepared by taking 20 mg of Ibuprofen in 10 ml of

Methanol, (solution A), which contains 2000 g/ml of Ibuprofen. 1.0 ml from solution

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42

(A), which contains 2000 g/ml was taken and diluted to 20 ml of Plasma, solution (B),

which contains 100 g/ml of Ibuprofen (solution B).

From solution B, which contains 100.0 g/ml the following dilutions were

prepared:

0.05 ml to 10 ml plasma ‡ 0.5 g/ml ….. STD 1







Samples of the standard curve in plasma were prepared using seven

concentrations, not including zero these concentrations are:

0.5, 1.0, 2.0, 5.0, 10.0, 20.0 AND 30.0 g /ml.

Each concentration of the plasma sample was divided to 0.20 ml in an eppendorf

tube and kept at – 20 0C, standard samples were given daily to gather with the

quality control samples.

2.7.3.3 Preparation of Quality Control Samples (Q.C) in Plasma:

Spiked plasma samples were prepared by taking 20 mg of Ibuprofen in 10 ml of

Methanol, (solution A), which contains 2000 g/ml of Ibuprofen. 1.0 ml from solution

(A), which contains 2000 g/ml was taken and diluted to 20 ml of Plasma, solution (B),

which contains 100 g/ml of Ibuprofen (solution B).

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43

From solution B, which contains 100.0 g/ml the following dilutions were

prepared:

0.30 ml to 20 ml plasma‡ 1.5 g/ml ….. (QC Low)

3.00 ml to 20 ml plasma‡ 15.0 g/ml ….. (QC Mid)

5.00 ml to 20 ml Plasma‡ 25.0 g/ml ….. (QC High)

Quality Control Samples were kept at - 20 0C until required for subsequent

analysis.

2.7.4 Description of Method:

2.7.4.1 Sample Preparation:

A 0.20 ml of plasma was add to 0.35 ml of 3.0 g/ml Glibenclamide prepared in

Methaol in an Eppendorf, the mixture was vortex-mixed for 30 seconds, then

centrifuged for 15 minutes at 14000 rpm. 200 l was transferred to flat bottom insert, 30

l was injected on BDS, C-18 Column. Ibuprofen and the internal standard were

separated from endogenous substances.

2.7.4.2 Chromatographic Conditions:

• Column: Thermo Electron Corporation, BDS Hypersil, C18, (150 X 4.6 mm

i.d).

• Solvent system: 65 % Acetonitrile, 35 % Water, 150 l of Glacial acetic acid

per one liter of mobile phase, pH = 4.35

• Detection: UV Detector = 230 nm.

• Injection volume: 30 microliters.

• Flow Rate: 1.0 ml/min.

• Retention time for Ibuprofen: 4.0 – 4.5 minutes.

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44

• Retention time for Glibenclamide: 3.0 – 3.5 minutes (Internal Standard)

(Retention times dependent upon HPLC conditions).

2.7.5 Method Validation:

To guarantee reliability and reproducibility of the assay for quantitation of

Ibuprofen in human plasma, the method was validated in concordance with the FDA

guideline requirements in Jordan Center for Pharmaceutical Research (JCPR). The

method was evaluated for linearity, sensitivity, selectivity, accuracy, precision, and

stability.

The method was sensitive for Ibuprofen. The variability in concentrations at

LLOQ of 0.5 µg/ml was within the acceptable limits of precision (4.72%) and

accuracy (100.94%), and LLOQ intensities were 5 times of the blank in all

samples. (US FDA, Guidance for Industry: Bioanalytical Method Validation, 2011).

2.8 Data Analysis:

Pharmacokinetic parameters were determined by standard non-

compartmental estimation method using Winnonlin software V 5.2 . They include

the following parameters:

• AUC: Area under the curve.

• Cmax: Maximum concentration of drug in plasma.

• Tmax: Time to achieve Cmax.

• Kel: Elimination rate constant.

• t0.5: Half life.

• V: Volume of distribution.

• CL: Clearance.

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45

• MRT: Mean residence time.

• AUMC: Area under moment curve.

Statistical comparisons for the above parameters were performed by t-test

using Excel program. Friedman design was applied for Tmax comparison using

Kinetica. 0.05 level of significance was used for all comparisons. For P values

less than 0.05, significant difference was concluded.

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46

Chapter 3:

Results and Discussion

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47

3. Results and Discussion:

3.1 Plasma Concentrations for All the Volunteers in Normal (N) and HDBR

Positions:

In the first volunteer the Tmax at HDBR was faster than it at normal position,

(Tmax at HDBR was 0.75 hr, while Tmax at normal position was 1.66 hr) and Cmax

was higher at HDBR than it at normal position, (Cmax at HDBR was 48.316 µg/ml,

while the Cmax for normal position was 39.242 µg/ml. (Fig 2, Table 2, Table 3)

05

10152025303540455055

0 2 4 6 8 10Time (hr)

Con

c (u

g/m

l)

1 N1 HDBR

Fig 2: Ibuprofen plasma concentrations of volunteer 1 in normal (N) and HDBRpositions

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48

In the second volunteer the Tmax at HDBR was faster than it at normal

position, (Tmax at HDBR was 0.50 hr, while Tmax at normal position was 2.50 hr)

and Cmax was slightly higher at HDBR than it at normal position, (Cmax at HDBR

was 38.149 µg/ml, while the Cmax for normal position was 39.096 µg/ml). (Fig 3,

Table 2, Table 3)

05

10152025303540455055

0 2 4 6 8 10Time (hr)

Con

c (u

g/m

l)

2 N2 HDBR


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49

In the third volunteer the Tmax at HDBR was also faster than it at normal

position, (Tmax at HDBR was 0.75 hr, while Tmax at normal position was 1.33 hr)

and Cmax was slightly higher at HDBR than it at normal position, (Cmax at HDBR

was 35.631 µg/ml, while the Cmax for normal position was 36.553 µg/ml). (Fig 4,

Table 2, Table 3)

05

10152025303540455055

0 2 4 6 8 10Time (hr)

Conc

(ug

/ml)

3 N3 HDBR


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50

In the fourth volunteer the Tmax for both HDBR and normal position were the

same, (Tmax for HDBR and for normal position were 1.00 hr) while Cmax was lower

at HDBR than it at normal position, (Cmax at HDBR was 43.145 µg/ml, while the

Cmax for normal position was 48.654 µg/ml). (Fig 5, Table 2, Table 3)

05

10152025303540455055

0 2 4 6 8 10Time (hr)

Con

c (u

g/m

l)

4 N4 HDBR


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51

In the fifth volunteer the Tmax at HDBR was more than normal position,

(Tmax for HDBR was 1.33 hr, and for normal position was 1.00 hr) while Cmax was

lower at HDBR than it at normal position, (Cmax at HDBR was 42.704 µg/ml, while

the Cmax for normal position was 49.097 µg/ml). (Fig 6, Table 2, Table 3)

05

10152025303540455055

0 2 4 6 8 10Time (hr)

Con

c (u

g/m

l)

5 N5 HDBR


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52

In the sixth volunteer the Tmax at HDBR was more than normal position,

(Tmax for HDBR was 2.50 hr, and for normal position was 0.75 hr) while Cmax was

lower at HDBR than it at normal position, (Cmax at HDBR was 24.768 µg/ml, while

the Cmax for normal position was 28.088 µg/ml). (Fig 7, Table 2, Table 3)

05

10152025303540455055

0 2 4 6 8 10Time (hr)

Conc

(ug

/ml)

6 N6 HDBR


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53

Volunteers average and also median Tmax at HDBR was faster than normal

position, (average and median Tmax values for HDBR were 1.138 hr and 0.875 hr,

and for normal position were 1.373 hr and 1.165 hr). However, average Cmax at

HDBR was slightly lower than it at normal position, (average Cmax at HDBR was

38.785 µg/ml, while the average Cmax for normal position was 40.121 µg/ml).

(Fig 8)

This changes in the Tmax and Cmax are due to changes in the body fluid

concentration in HDBR position, as it is known during the HDBR position the fluids

shift from the lower part of the body to the upper part of the body causing increasing

of the blood concentration in the upper part of the body and in gastro intestinal tract.

Hence the disintegration and dissolution of Ibuprofen in GIT was faster, leading to

an increase in absorption.

As it is shown in figure number 8, the therapeutic concentrations of

Ibuprofen, 20 to 30 µg/ml, were achieved.

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54

0

5

10

15

20

25

30

35

40

45

50

55

0 2 4 6 8 10Time (hr)

Con

c (u

g/m

l)

N HDBR

Fig 8: Ibuprofen plasma concentrations of 6 volunteers average in normal (N) andHDBR positions

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55

Normal Phase:Table 2: Ibuprofen plasma concentrations (µg/ml) after 600 mg oral dose in 6volunteers in normal position, the Cmax for each volunteer is bolded.

Timing

(hr)

Vol. # 1

Conc.

(µg/ml)

Vol. # 2

Conc.

(µg/ml)

Vol. # 3

Conc.

(µg/ml)

Vol. # 4

Conc.

(µg/ml)

Vol. # 5

Conc.

(µg/ml)

Vol. # 6

Conc.

(µg/ml)

0.00 0.000 0.000 0.000 0.000 0.000 0.000

0.25 0.426 0.000 0.906 1.574 2.139 11.622

0.50 4.220 0.835 10.917 10.437 12.617 26.126

0.75 8.163 3.387 21.149 41.728 38.950 28.088

1.00 13.732 23.636 33.900 48.654 49.097 26.957

1.33 36.058 29.289 36.553 45.008 45.138 26.127

1.66 39.242 32.176 29.605 42.801 38.307 27.853

2.00 35.070 36.452 24.710 34.747 33.406 27.843

2.50 27.774 39.096 18.251 26.801 29.247 24.608

3.00 26.119 29.731 14.280 20.695 21.857 18.595

4.00 14.974 19.377 8.874 15.005 15.100 11.489

5.00 9.502 13.412 5.220 10.457 9.300 6.811

6.00 5.203 8.435 3.198 6.726 6.605 4.362

8.00 2.583 4.522 1.403 3.303 3.715 1.893

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56

HDBR Phase:Table 3: Ibuprofen plasma concentrations (µg/ml) after 600 mg oral dose in 6volunteers in HDBR position, the Cmax for each volunteer is bolded.

Timing

(hr)

Vol. # 1

Conc.

(µg/ml)

Vol. # 2

Conc.

(µg/ml)

Vol. # 3

Conc.

(µg/ml)

Vol. # 4

Conc.

(µg/ml)

Vol. # 5

Conc.

(µg/ml)

Vol. # 6

Conc.

(µg/ml)

0.00 0.000 0.000 0.000 0.000 0.000 0.000

0.25 16.097 30.769 0.000 1.740 10.669 8.824

0.50 45.831 38.149 25.256 23.338 24.220 19.978

0.75 48.316 34.146 35.631 39.436 34.177 18.287

1.00 43.520 32.659 32.933 43.145 42.431 17.776

1.33 31.480 28.477 29.912 42.324 42.704 15.036

1.66 25.138 26.675 24.842 38.062 32.339 13.550

2.00 21.404 25.218 18.732 31.392 29.669 11.383

2.50 15.945 22.477 14.713 25.798 21.461 24.768

3.00 13.108 18.477 12.943 20.679 16.447 23.965

4.00 8.548 13.316 7.282 14.849 11.458 18.583

5.00 5.667 9.355 4.536 9.542 7.508 10.895

6.00 3.618 6.127 2.612 6.898 4.776 6.936

8.00 1.713 3.576 1.059 3.566 2.911 3.675

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57

3.2 Data for Maximum Concentration of Drug in Plasma (Cmax):

Cmax is maximal plasma drug concentration that was reached in plasma.

Tmax is time of maximum plasma drug concentration.

The results are shown in table number 4.

Table 4: Cmax and Tmax in both phases for all volunteers.

Position Subject Tmax (hr) Cmax (µg/ml)

HDBR 1 0.75 48.316

HDBR 2 0.50 38.149

HDBR 3 0.75 35.631

HDBR 4 1.00 43.145

HDBR 5 1.33 42.704

HDBR 6 2.50 24.768

Position Subject Tmax (hr)

Cmax

(µg/ml)

N 1 1.66 39.242

N 2 2.50 39.096

N 3 1.33 36.553

N 4 1.00 48.654

N 5 1.00 49.097

N 6 0.75 28.088

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58

3.3 Data for K elimination:

Elimination rate constant is the rate constant for removal of drugs from

the body. Kel = First order elimination rate constant associated with the

terminal (log-linear) portion of the curve. Estimated by linear regression of

log concentration versus time.


Table 5: Kel in both phases for all volunteers

Position Subject Kel (hr-1) Half life (hr)

HDBR 1 0.4027 1.7209

HDBR 2 0.3300 2.1000

HDBR 3 0.4845 1.4303

HDBR 4 0.3508 1.9755

HDBR 5 0.3411 2.0317

HDBR 6 0.3559 1.9472

Position Subject Kel (hr-1) Half life (hr)

N 1 0.4431 1.5640

N 2 0.3664 1.8914

N 3 0.4580 1.5131

N 4 0.3812 1.8179

N 5 0.3033 2.2849

N 6 0.4465 1.5521

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59

3.4 Data for Area Under the Curve ( AUC):

The area under curve, AUC, corresponds to the integral of the plasma

concentration versus an interval of definite time. In practice, the approximation is

used by application of linear trapezoidal method. One of AUC uses is to allow the

measurement of the bioavailability of a drug.

AUCinf_obs = AUC from dosing time extrapolated to infinity, based on the

last observed concentration (obs).

AUC_%Extrap_obs = Percentage of AUCinf_obs due to extrapolation from Tlast

to infinity.

The results are shown in tables number 6.a and 6.b.

Table 6.a : AUClast, AUC0-4, AUCinf_obs and AUC%Extrap in HDBR phase for allvolunteers

position Subject

AUClast

(µg/ml) hr

AUC0-4

(µg/ml)hr

AUCinf_obs

(µg/ml) hr

AUC_%Extrap

(%)

HDBR 1 107.1396 90.0586 111.3936 3.8189

HDBR 2 124.6960 95.9165 135.5327 7.9957

HDBR 3 84.6915 71.5375 86.8772 2.5159

HDBR 4 135.2551 104.3756 145.4201 6.9901

HDBR 5 119.0651 95.7531 127.5992 6.6882

HDBR 6 105.1240 70.8585 115.4508 8.9448

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60

Table 6.b : AUClast, AUC0-4, AUCinf_obs and AUC_%Extrap in normal phase forall volunteers.

position Subject

AUClast

(µg/ml) hr

AUC0-4

(µg/ml)hr

AUCinf_obs

(µg/ml) hr

AUC_%Extrap

(%)

N 1 115.2989 87.9224 121.1281 4.8124

N 2 135.4739 95.1989 147.8155 8.3493

N 3 90.5619 74.7049 93.6253 3.2720

N 4 139.1049 107.7534 147.7702 5.8641

N 5 138.4623 107.9898 150.7115 8.1276

N 6 106.9095 85.918 111.1488 3.8140

3.5 Data for Volume of Distribution ( V/F ) and Clearance (CL/F):

The volume of distribution (Vd) is the fictitious volume, expressed in liter

or in liter per kilogram, in which the drug would have been distributed by

supposing that its concentration is homogeneous, for a given drug, the knowledge

of its desirable concentration in blood and of its volume of distribution allows

evaluation of the dose to administer.

Clearance is the fraction of a theoretical volume completely purified (i.e.

no longer containing any of the drug concerned) per unit of time. Plasma

clearance is the apparent volume of plasma purified per unit of time. Total

clearance (Clt) is the fraction of the volume of distribution, Vd, which is

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61

completely purified per unit of time. The total clearance depends on the constant

of elimination and thus on T ½ and on Vd. Clearance is a constant in linear

kinetics.


Table 7: V/F and Cl/F in both phases for all volunteers.

Position Subject V/F (L) Cl/F (L/hr)

HDBR 1 13.3762 5.3863

HDBR 2 13.4156 4.4270

HDBR 3 14.2541 6.9063

HDBR 4 11.7612 4.1260

HDBR 5 13.7853 4.7022

HDBR 6 14.6037 5.1970

Position Subject V/F (L) Cl/F (L/hr)

N 1 11.1787 4.9534

N 2 11.0783 4.0591

N 3 13.9930 6.4085

N 4 10.6523 4.0604

N 5 13.1266 3.9811

N 6 12.0888 5.3982

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62

3.6 Data for (AUMClast ) and (AUMCinf_obs):

AUMClast is the area under the moment curve from the time of dosing

(Dosing time) to the last measurable concentration.

AUMCinf_obs Area under the first moment curve (AUMC) extrapolated to

infinity, based on the last observed concentration.


Table 8: AUMClast and AUMCinf_obs in both phases for all volunteers.

Position Subject AUMClast (µg/ml) hr2 AUMCinf_obs (µg/ml) hr2

HDBR 1 232.4074 277.0039

HDBR 2 324.5403 444.0741

HDBR 3 193.3673 215.3640

HDBR 4 365.5351 475.8308

HDBR 5 297.5320 390.8232

HDBR 6 340.8410 452.4741

Position Subject AUMClast (µg/ml) hr2 AUMCinf_obs (µg/ml) hr2

N 1 341.5563 401.3454

N 2 441.3853 573.8016

N 3 225.0263 256.2229

N 4 375.4078 467.4642

N 5 374.2287 512.6107

N 6 274.4322 317.8397

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63

3.7 Data for Mean Residence Time (MRT):

Mean residence time (MRT) is the average total time molecules of a given

dose spend in the body. Thus, this includes all ADME processes.

MRT = AUMC/AUC.


Table 9: MRT in both phases for all volunteers.

Position Subject MRT (hr)

HDBR 1 2.4867

HDBR 2 3.2765

HDBR 3 2.4789

HDBR 4 3.2721

HDBR 5 3.0629

HDBR 6 3.9192

Position Subject MRT (hr)

N 1 3.3134

N 2 3.8819

N 3 2.7367

N 4 3.1635

N 5 3.4013

N 6 2.8596

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64

3.8 Pharmacokinetic Parameter Means (CV %) of Ibuprofen Under Normal

(N) and Head-Down-Bed-Rest (HDBR) positions:

Table 10: Pharmacokinetic parameter means (CV %) of ibuprofen under normal (N)and Head-Down-Bed-Rest (HDBR) positions

Pk parameter N HDBR P values *

CV%

N

CV%

HDBR

Kel (hr-1) 0.399 0.377 0.317 15.099 15.375

Half life (hr) 1.770 1.867 0.348 16.717 13.367

Tmax (hr) 1.373 1.138 0.167 * * 46.290 63.580

Cmax (µg/ml) 40.121 38.785 0.510 19.711 21.010

AUC0-8 (µg/ml) hr 120.968 112.661 0.015 16.532 15.708

AUC0-4 (µg/ml) hr 93.247 88.083 0.137 14.043 15.730

AUC4-8 (µg/ml) hr 27.720 24.578 0.388 30.801 33.526

AUCinf_obs (µg/ml)hr 128.699 120.378 0.071 18.417 17.170

AUC_% Extrap_obs 5.707 6.159

V_/F_obs (L) 10.951 13.341 0.056 13.781 20.377

Cl_/F_obs (L/hr) 4.810 5.124 0.071 20.230 19.339

AUMC0-8 (µg/ml) hr2 338.672 292.370 0.158 22.953 22.770

AUMCinf_obs

(µg/ml) hr2 421.547 375.928 0.329 28.481 28.220

MRT (hr) 3.226 3.082 0.568 12.751 17.728

* P values of t test after logarithmic transformation of parameters** Friedman DESIGN for Tmax was done using Kinetica.

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65

The logarithmic transformation of parameters was used to be sure that the

distribution was normal.

The P values of t test results were not significant for all the parameters

except for AUClast, it was less than 0.05 (0.015), but the P value results of t test for

AUC0-4 was 0.137 which is not significant and for AUC4-8 was 0.388 which is

also not significant and for AUCinf_obs was 0.071 which is also not significant. As it

was mentioned above, the P values of t test for AUC after dividing it to three zones

became not significant, that is suggested to be due to the increase in difference

between both positions for AUClast, as compared to the differences in AUC0-4 and

AUC4-8. . The average AUC % extrapolated for normal phase was (5.707 %) and for

HDBR position was (6.159 %), in both cases the result is less than 10% which

indicates of enough sampling times (Guidance for Industry, Bioavailability and

Bioequivalence Studies for Orally Administered Drug, USFDA, 2003).

The average Tmax in HDBR (1.138 hr) was faster, yet not significantly

different than it in Normal phase (1.373 hr), these changes are due to changes in the

body fluid concentration in HDBR position, as it is known during the HDBR

position the fluids shift from the lower part of the body to the upper part of the body

causing increasing of the blood volume in the upper part of the body and in gastro

intestinal tract. In addition, ibuprofen is low soluble drug and hence the

disintegration rate in GIT was faster in HDBR position leading to increase of

absorption of Ibuprofen in the body (Newa M, Bhandari KH, Oh DH, et al, 2008).

Mean residence time (MRT) which indicates the average total time

molecules of a given dose spend for all ADME processes in the body, in both

`

66

phases was almost same, P value of t test result was (0.568) which is not

significant.

Half life (t0.5) which indicates the period of time required for the

concentration or amount of drug in the body to be reduced to one-half of a given

concentration or amount, in HDBR phases the average half life was 1.87 hr and in

normal phase the average half life was 1.77 hr. and the P value of t test result was

(0.348) which is not significant.

The average volume of distribution of ibuprofen at HDBR position was

13.341L and normal position was 10.951L. Yet P-value of t test result for volume

of distribution was (0.056) which is not significant. This increase in volume of

distribution at HDBR was as a result of decrease in the AUCinf and Kel at HDBR,

according to the following formula:

V/F= Dose/ (Kel * AUCinf)

The average clearance at HDBR position was 5.124 L/hr which is faster

than it at normal position which was 4.810 L/hr. The P-value of t test result for

clearance was (0.071) which is not significant. This slight increase of clearance at

HDBR is due to increase of volume of distribution at HDBR position according to

the following formula :

Cl/F=V*Kel=D/AUCinf.

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67

Chapter 4:

Conclusions

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68

4. Conclusions:

AUCs were similar in both positions. However, slight differences in Cmax

and Tmax were observed between HDBR position and normal positions due to faster

drug dissolution in HDBR position, yet such differences were not significant.

This indicates that relative bioavailability is similar and hence no need for

dose adjustment of ibuprofen for crew members of space flight.

However, this can not be generalized to other drugs, especially highly

soluble medications.

Ibuprofen was not detected in all saliva samples, suggesting that Ibuprofen

is not excreted in saliva.

`

69

References

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A. Pavy-Le Traon . M. Heer . M. V. Narici . J. Rittweger . J. Vernikos, Eur J ApplPhysiol ; 2007. From space to earth: advances in human physiology from 20 years ofbed rest studies (1986–2006). 101:143–194, DOI 10.1007/s00421-007-0474-z.

Benet LZ; 1988. Pharmacokinetic considerations of space flight. ed., Houston, TX:Lunar and Planetary Institute - NASA. p 15-16.

C. H. Kleinbloesem et al.,Arzneimittelforschung; 1995, 45, 1117–1121

Cintron NM, Putcha L, Chen YM, Vanderploeg JM; 1987. Inflight salivarypharmacokinetics of scopolamine and dextroamphetamine. Results of the lifesciences DSOs conducted aboard the space schuttle 1981-1986, Houston, TX:NASA. p 25-29.

Edgar L. Schuck, PhD, Maria Grant, MD and Hartmut Derendorf, PhD, FCP, fromthe department of pharmaceutics; 2004. Effect of simulated microgravity on thedisposition and tissue penetration of Ciprofloxacin in healthy volunteers. Universityof florida, Gainesville.

Fitts RH, Riley DR, Widrick JJ; 2000. Physiology of a microgravity environmentinvited review: microgravity and skeletal muscle. J Appl Physiol 89(2):823-839.

Graebe A, Schuck EL, Lensing P, Putcha L, Derendorf H; 2004. Physiological,pharmacokinetic, and pharmacodynamic changes in space. J Clin Pharmacol44(8):837-853.

Guidance for Industry, Bioavailability and Bioequivalence Studies for OrallyAdministered Drug, Products — General Considerations, U.S. Department ofHealth and Human Services, Food and Drug Administration, Center for DrugEvaluation and Research (CDER); March 2003.

Harm DL, Jennings RT, Meck JV, Powell MR, Putcha L, Sams CP, Schneider SM,Shackelford LC, Smith SM, Whitson PA; 2001. Invited review: gender issuesrelated to spaceflight: a NASA perspective. J Appl Physiol 91(5):2374-2383.

Merrill AH, Jr., Hoel M, Wang E, Mullins RE, Hargrove JL, Jones DP, Popova IA;1990. Altered carbohydrate, lipid, and xenobiotic metabolism by liver from ratsflown on cosmos 1887 [published erratum appears in FASEB J 1990May;4(8):2539]. Faseb J 4(1):95-100.

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70

Newa M, Bhandari KH, Oh DH, et al; 2008. Enhancement of solubility, dissolutionand bioavailability of ibuprofen in solid dispersion systems. Chem PharmBull.56(4):569-574.

Nicogossian AE, Sawin CF, Huntoon CL; 1994. Overall physiologic response tospace flight. In Nicogossian AE, Huntoon CL, Pool SL, editors. Space physiologyand medicine, Phildelphia: Lea & Febiger. p 213-227.

Pool SL, Nicogossian A; 1983. Biomedical results of the space shuttle orbital flighttest program. Aviat space environ Med 54(12 Pt 2):S41-49.

Putcha L, Berens KL, Marshburn TH, Ortega HJ, Billica RD; 1999. Pharmaceuticaluse by U.S. astronauts on space shuttle missions. Aviat space environ Med70(7):705-708.

Santy PA, Kapanka H, Davis JR, Stewart DF; 1988. Analysis of sleep on shuttlemissions. Aviat space environ Med 59(11 Pt 1):1094-1097.

Stewart JJ, Wood MJ, Wood CD, Mims ME; 1994. Effects of motion sickness andantimotion sickness drugs on gastric function. J Clin Pharmacol 34(6):635-643.

US Food and Drug Administration (US FDA) Centre for Drug Evaluation andResearch; 2001. Guidance for Industry: Bioanalytical Method Validation, Rockville,MD, Retrieved from http://www.fda.gov/cder/guidance/4252fnl.pdf

Wang DS, Ren W, Xiang QL, Sun L, Liu ZX, Su SN; 2001. [Changes of arterialblood pressure during various stages in spaceflight activities]. Space Med Eng(Beijing) 14(1):66-69.

Watenpaugh DE; 2001. Fluid volume control during short-term space flight andimplications for human performance. J Exp Biol 204(Pt 18):3209-3215.

Zhang LF; 2001. Vascular adaptation to microgravity: what have we learned? J ApplPhysiol. 91(6):2415-2430.

http://www.fda.gov/cder/guidance/4252fnl.pdf

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71

Appendix # 1

Institutional Review Board (IRB)

Approval

`

73

Appendix # 2

Jordan Food and Drug Administration

(JFDA) Approval

`

75

Appendix # 3

Informed Constant Form

`

88

Appendix # 4

Winnonlin Output

`

89

Input File: Workbook - [C:\PROGRAM FILES\PHARSI...\ABRN.PWO]position=ABRSubject=1

Date: 10/04/2010 Time: 13:57:23

WINNONLIN NONCOMPARTMENTAL ANALYSIS PROGRAM Version 5.2 Build 200701231637 Core Version 18Sept2006

Settings--------Model: Plasma Data, Extravascular AdministrationNumber of nonmissing observations: 14Dose time: 0.00Dose amount: 600.00Calculation method: Linear Trapezoidal with Linear InterpolationWeighting for lambda_z calculations: Uniform weightingLambda_z method: User-specified lambda_z range, Log regressionUser's lambda_z bounds: 4.00, 8.00

Summary Table------------- Time Conc. Pred. Residual AUC AUMC Weight hr ug/ml ug/ml ug/ml hr*ug/ml hr*hr*ug/ml------------------------------------------------------------------------------- 0.0000 0.0000 0.0000 0.0000 0.2500 16.10 2.012 0.5030 0.5000 45.83 9.753 3.871 0.7500 48.32 21.52 11.26 1.000 43.52 33.00 21.23 1.330 31.48 45.38 35.32 1.660 25.14 54.72 49.12 2.000 21.40 62.63 63.49 2.500 15.95 71.97 84.16 3.000 13.11 79.23 104.0 4.000 * 8.548 8.422 0.1263 90.06 140.7 1.000 5.000 * 5.667 5.630 0.03683 97.17 172.0 1.000 6.000 * 3.618 3.764 -0.1459 101.8 197.0 1.000 8.000 * 1.713 1.682 0.03079 107.1 232.4 1.000

*) Starred values were included in the estimation of Lambda_z.

Final Parameters---------------Rsq 0.9985Rsq_adjusted 0.9977Corr_XY -0.9992No_points_lambda_z 4Lambda_z 1/hr 0.4027Lambda_z_lower hr 4.0000Lambda_z_upper hr 8.0000HL_Lambda_z hr 1.7213Tlag hr 0.0000Tmax hr 0.7500Cmax ug/ml 48.3160Cmax_D ug/ml/mg 0.0805Tlast hr 8.0000Clast ug/ml 1.7130AUClast hr*ug/ml 107.1396AUCall hr*ug/ml 107.1396AUCINF_obs hr*ug/ml 111.3936AUCINF_D_obs hr*ug/ml/mg 0.1857AUC_%Extrap_obs % 3.8189Vz_F_obs ml 13376.2132Cl_F_obs ml/hr 5386.3043AUCINF_pred hr*ug/ml 111.3172AUCINF_D_pred hr*ug/ml/mg 0.1855AUC_%Extrap_pred % 3.7528

`

90

Vz_F_pred ml 13385.4024Cl_F_pred ml/hr 5390.0046AUMClast hr*hr*ug/ml 232.4074AUMCINF_obs hr*hr*ug/ml 277.0039AUMC_%Extrap_obs % 16.0996AUMCINF_pred hr*hr*ug/ml 276.2022AUMC_%Extrap_pred % 15.8561MRTlast hr 2.1692MRTINF_obs hr 2.4867MRTINF_pred hr 2.4812AUC0_4 hr*ug/ml 90.0586AUC4_8 hr*ug/ml 17.0810

`

91


Date: 10/04/2010 Time: 13:57:24





Final Parameters---------------Rsq 0.9900Rsq_adjusted 0.9851Corr_XY -0.9950No_points_lambda_z 4Lambda_z 1/hr 0.3300Lambda_z_lower hr 4.0000Lambda_z_upper hr 8.0000HL_Lambda_z hr 2.1005Tlag hr 0.0000Tmax hr 0.5000Cmax ug/ml 38.1490Cmax_D ug/ml/mg 0.0636Tlast hr 8.0000Clast ug/ml 3.5760AUClast hr*ug/ml 124.6960AUCall hr*ug/ml 124.6960AUCINF_obs hr*ug/ml 135.5327AUCINF_D_obs hr*ug/ml/mg 0.2259AUC_%Extrap_obs % 7.9957Vz_F_obs ml 13415.5564Cl_F_obs ml/hr 4426.9758AUCINF_pred hr*ug/ml 135.1207AUCINF_D_pred hr*ug/ml/mg 0.2252AUC_%Extrap_pred % 7.7151Vz_F_pred ml 13456.4634

`

92

Cl_F_pred ml/hr 4440.4746AUMClast hr*hr*ug/ml 324.5403AUMCINF_obs hr*hr*ug/ml 444.0741AUMC_%Extrap_obs % 26.9175AUMCINF_pred hr*hr*ug/ml 439.5295AUMC_%Extrap_pred % 26.1619MRTlast hr 2.6027MRTINF_obs hr 3.2765MRTINF_pred hr 3.2529AUC0_4 hr*ug/ml 95.9165AUC4_8 hr*ug/ml 28.7795

`

93


Date: 10/04/2010 Time: 13:57:24





Final Parameters---------------Rsq 0.9985Rsq_adjusted 0.9978Corr_XY -0.9993No_points_lambda_z 4Lambda_z 1/hr 0.4845Lambda_z_lower hr 4.0000Lambda_z_upper hr 8.0000HL_Lambda_z hr 1.4306Tlag hr 0.2500Tmax hr 0.7500Cmax ug/ml 35.6310Cmax_D ug/ml/mg 0.0594Tlast hr 8.0000Clast ug/ml 1.0590AUClast hr*ug/ml 84.6915AUCall hr*ug/ml 84.6915AUCINF_obs hr*ug/ml 86.8772AUCINF_D_obs hr*ug/ml/mg 0.1448AUC_%Extrap_obs % 2.5159Vz_F_obs ml 14254.1174Cl_F_obs ml/hr 6906.2965AUCINF_pred hr*ug/ml 86.8366AUCINF_D_pred hr*ug/ml/mg 0.1447AUC_%Extrap_pred % 2.4703Vz_F_pred ml 14260.7836

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94

Cl_F_pred ml/hr 6909.5264AUMClast hr*hr*ug/ml 193.3673AUMCINF_obs hr*hr*ug/ml 215.3640AUMC_%Extrap_obs % 10.2138AUMCINF_pred hr*hr*ug/ml 214.9553AUMC_%Extrap_pred % 10.0430MRTlast hr 2.2832MRTINF_obs hr 2.4789MRTINF_pred

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