STUDIES ON PHARMACOKINETICS, BIOAVAILABILITY AND …...Veterinary College, Anand and Director of Student’s Welfare, Anand Agricultural University, Anand, for providing me constant

STUDIES ON PHARMACOKINETICS,

BIOAVAILABILITY AND SAFETY OF KETOPROFEN

IN COW CALVES

BY

RATN DEEP SINGH B.V.Sc. & A.H.

DEPARTMENT OF VET. PHARMACOLOGY & TOXICOLOGY

COLLEGE OF VETERINARY SCIENCE & ANIMAL HUSBANDRY

ANAND AGRICULTURAL UNIVERSITY

ANAND

2008

STUDIES ON PHARMACOKINETICS,

BIOAVAILABILITY AND SAFETY OF KETOPROFEN

IN COW CALVES

A THESIS

SUBMITTED TO THE


IN THE PARTIAL FULFILMENT OF THE REQUIREMENTS

FOR THE AWARD OF THE DEGREE

OF

Master of Veterinary Science

IN

VETERINARY PHARMACOLOGY & TOXICOLOGY

BY

RATN DEEP SINGH B.V.Sc. & A.H.

(Reg. No. : 04-0463-2006)

DEPARTMENT OF VET. PHARMACOLOGY & TOXICOLOGY

COLLEGE OF VETERINARY SCIENCE & ANIMAL HUSBANDRY


ANAND

2008

dedicated to

my dear parents

Smt. Keshwari Devi

and

Shri Ganga Prasad Singh

Dr. S.K. BHAVSAR

M.V.Sc., Ph.D. Assistant Research Scientist,

Department of Pharmacology & Toxicology, College of Veterinary Science and Animal Husbandry,

Anand Agricultural University,

Anand- 388 001

CERTIFICATE

This is to certify that the thesis entitled “STUDIES ON

PHARMACOKINETICS, BIOAVAILABILITY AND SAFETY OF

KETOPROFEN IN COW CALVES” submitted by RATN DEEP SINGH (Reg.

No. 04-0463-2006) in partial fulfillment of the requirements for the award of the

degree of MASTER OF VETERINARY SCIENCE in the subject of

VETERINARY PHARMACOLOGY & TOXICOLOGY of the Anand

Agricultural University is record of bonafide research work carried out by him under

my guidance and supervision and the thesis has not previously formed the basis for

the award of any degree, diploma or other similar title.

Place: Anand (S. K. BHAVSAR)

Date: 09 / 05/ 2008 Major Advisor

CERTIFICATE

This is to certify that I have no objection for supplying to any scientist

only one copy of any part of this thesis at a time through reprographic process, if

necessary for rendering reference service in a library or documentation center.

Place: Anand

Date: 09/ 05/ 2008 (RATN DEEP SINGH)

(Dr. S. K. BHAVSAR)

Major Advisor

Abstract

ABSTRACT

“STUDIES ON PHARMACOKINETICS, BIOAVAILABILITY

AND SAFETY OF KETOPROFEN IN COW CALVES”

Name of Student Name of Major Advisor

Ratn Deep Singh Dr. S.K. Bhavsar

Department of Pharmacology & Toxicology

College of Veterinary Science and Animal Husbandry

Anand Agricultural University

Anand 388 001

Gujarat state, INDIA

Ketoprofen is a non steroidal antiinflammatory drug (NSAID) used for its

antiinflammatory, analgesic and antipyretic properties in Veterinary medicine. The

pharmacokinetics of ketoprofen after its single dose intravenous and intramuscular

administration was investigated in six crossbred cow calves by non compartmental

approach. The drug was administered at the dose rate of 3.0 mg.kg-1 body weight and

assayed in plasma by HPLC analysis. The present study also evaluated safety of

ketoprofen (3.0 mg.kg-1

) after repeated administration at 24 h interval for 5 days in calves.

Following intravenous and intramuscular administration of ketoprofen, values

of elimination half- life (t1/2), volume of distribution of drug at steady state [Vd(ss)],

total body clearance (ClB), area under plasma drug concentration-time curve (AUC),

and mean residence time (MRT) were 1.55 ± 0.05 and 3.40 ± 0.05 h; 0.64 ± 0.03 and

0.72 ± 0.02 L.kg-1; 4.82 ± 0.16 and 2.83 ± 0.06 ml.min-1.kg-1; 10.42 ± 0.32 and 17.72

± 0.39 µg.h.ml-1; and 1.20 ± 0.06 and 4.22 ± 0.07 h, respectively. Following

intramuscular administration peak plasma concentration (Cmax) of 6.15 0.24 g.ml-

1 was achieved at 0.50 h (Tmax). The systemic bioavailability of ketoprofen following

intramuscular administration in the calves was 77.31 per cent. Longer elimination

half- life, extensive volume of distribution at steady state and slower total body

clearance of ketoprofen following intramuscular administration as compared to

intravenous administration makes it more suitable for intramuscular use in calves.

Repeated intravenous administration of ketoprofen (3.0 mg.kg-1

body weight

repeated at 24 h interval for 5 days) in calves was found safe based on evaluation of

haematological (Hb, PCV, TLC and DLC) and blood biochemical (AKP, ACP, AST,

ALT, LDH, Total Bilirubin, Serum Creatinine, BUN, Total Serum Protein, Serum

Albumin and Blood glucose) parameters.

The present study indicate that intramuscular administration of ketoprofen at

dose rate of 3.0 mg.kg-1 in cow calves would be provide a satisfactory plasma

concentration of drug equal to its median effective concentration up to 18 h.

Therefore, ketoprofen given via intramuscular route at the dose rate of 3.0 mg.kg-1 of

body weight repeated every 18 h would be satisfactory therapeutic dosage regimen for

cow calves. However, therapeutic efficacy of the dosage remains to be evaluated in

clinical cases under field conditions.

Acknowledgement

ACKNOWLEDGEMENT

On the completion of the present study, I would like to take this opportunity to extend my deepest sense of gratitude and words of thanks

towards those, who helped me during the entire pursuit of study. Words are inadequate in the available lexicon to express my gratitude

and sincere thanks to my major advisor Dr. S. K. Bhavsar, Assistant

Research Scientist, Department of Pharmacology & Toxicology, College of Veterinary Science and Animal Husbandry, Anand Agricultural University, Anand for his scholarly guidance, prudent planning, excellent cooperation with constructive criticism, and diligent efforts throughout the pursuit of this study and preparation of this manuscript. It was indeed a valuable

opportunity to pursue my M.V.Sc. study under such a brilliant and caring man. I also thank him for introducing me to the very interesting and fascinating field of pharmacokinetics.

I express my sincere gratitude to my minor advisor Dr. B. P. Joshi, Professor, Department of Veterinary Pathology, Veterinary College, Anand for providing me spirited guidance, and critical advice as the occasions required.

I wish to express my heart-felt gratitude and thanks to Dr. A. M.

Thaker, Professor and Head, Department of Pharmacology & Toxicology, Veterinary College, Anand and Director of Student’s Welfare, Anand Agricultural University, Anand, for providing me constant guidance and valuable suggestions and for his kind supportive attitude during my entire M.V.Sc. study.

I am genuinely beholden by Dr. J. G. Sarvaiya, Ex-Director,

Information Technology Center (ITC) and Associate Research Scientist, Department of Pharmacology & Toxicology and ITC staff for providing me

superb IT & Computer amenities whenever needed during the entire study period. I extend my sincere thanks to Shri R.S. Parmar (Director, ITC) for his kind help to carry out statistical analysis of the raw data.

I am modestly thankful to Dr. R. M. Tripathi, Ex-Professor and Head,

Department of Pharmacology & Toxicology, College of Veterinary Science and Animal Husbandry, S. D. Agricultural University, S.K. Nagar for building my subject base and generating my interest in pharmacology.

I am extremely thankful to the Principal and Dean Dr. J. V. Solanki,

College of Veterinary Science and Animal Husbandry, Anand, for providing Research facilities and an opportunity to pursue my higher studies from such an esteemed institute of Gujarat state.

I am thankful to Dr. K. N. Wadhvani, Associate Professor, Instructional Farm, Anand Agricultural University, Anand for providing animals, infrastructural facility and necessary help for the research purpose.

I want to extend my fervent thanks to Dr. D. S. Nauriyal, Associate

Professor, Department of Vet. Medicine and Dr. D. M. Patel, Head, Teaching Vet. Clinics Service Complex, Veterinary College, Anand for providing facility of Blood Autoanalyzer and Serum Biochemical Autoanalyzer, respectively.

I am highly thankful to our departmental staff, Rajubhai, Parmar kaka and Bhupatbhai and all the college library staff for their kind co-operation throughout the course of study.

I extend my affable thanks to my lovely departmental friends Jatin,

Sanjay, Rasesh, Paresh, Sandip and Amar for their timely and unreserved help, support, wholehearted co-operation and continuous motivations during

the entire course of study. I feel lucky to have friends like them because friends in need are the best friend indeed.

I am especially thankful to Dr. Urvesh D. Patel for his precious

suggestions and constructive discussion that help me in a multitude of

ways. I also wish to express my sincere thanks to all my respectable seniors specially Dr. (s), Deepak Barot, LaxmiNarayan, Haresh, Jayesh, Nilesh, Himanshu, and Vaibhavi for their love, affection and unreserved help during my study and research period. I also thanks to Dr. Amit Verma for helping me out during haematological analysis.

Especial words of thanks are due to Dr. Satish Bhai and my dear

juniors Dr. (s) Anil, Kamlesh, V.C., Anirudh and S.K. Nagar internees for their timely help, love and affections. Thanks are also due to them for never

taking serious of any harsh word, if any I said.

I desperately thank to all my friends and seniors outside the department and my forever U.G. friends from S.K. Nagar for all the good as well as hard times we shared.

A distant thanks is due to my cousin Narendra Kumar Singh,

Department of Pharmaceutics, IT, BHU, Varanasi for making me available some literature resources.

My vocabulary utterly fails in expressing my admiration to my dearest Mother and Father who brought me to this stage. I am indebted to the pain they bear for my knowledge gain. I take this opportunity to dedicate this work to them. I would remain ever grateful to my brothers Amardeep and

Pradeep, sister Pooja and Saroj bhabhi for tolerating the inconvenience I might have created being away from home during this period.

I fall short of words in expressing thanks to Sarita, the wise part of myself and very special to me. She always stays on my side with patience,

dedication and love. Much of what I am, and a large amount of all I have done are on her account.

There are many, many who have helped in various ways but whose

names I have missed out. They know who they are, and I hope they will accept my sincere thanks for all that they did.

Finally, I would like to thank the Almighty Goddess Amba Maa, for it is under her grace that we live, learn and flourish.

Place : Anand (Ratn Deep Singh) Date : 09/05/ 2008

CONTENTS

CHAPTER NO.

TITLE PAGE

NO.

I INTRODUCTION 1

II REVIEW OF LITERATURE 4

III MATERIALS AND METHODS 58

IV RESULTS 73

V DISCUSSION 106

VI SUMMARY AND CONCLUSIONS 119

REFERENCES I - XIII

LIST OF TABLES

Table No. Title Page

No.

1. IC50 cyclooxygenase (COX-1: COX-2) inhibition ratio of NSAIDs in human whole blood assay.

8

2. Important pharmacokinetic parameters of ketoprofen in various species of domestic animals.

30-33

3. Recommended dosage of ketoprofen in animals. 45

4. Experimental schedule to study pharmacokinetics and safety

of ketoprofen in crossbred calves.

59

5. Scheme of preparation of ketoprofen standards in plasma. 63

6. Intraday and Interday precision and accuracy of ketoprofen in calf plasma by HPLC - UV detection.

63

7. Methods used for the determination of haematological and serum biochemical parameters in safety study.

72

8. Plasma concentrations of ketoprofen in calves following intravenous administration of ketoprofen at the dose rate of 3

mg.kg-1 body weight.

76

9. Pharmacokinetic parameters of ketoprofen in calves

following intravenous administration at the dose rate of 3 mg.kg-1 body weight.

79

10. Plasma concentrations of ketoprofen in calves following intramuscular administration of ketoprofen at the dose rate of

3 mg.kg-1 body weight.

81

11. Pharmacokinetic parameters of ketoprofen in calves following intramuscular administration at the dose rate of 3 mg.kg-1 body weight.

84

12. Effect of multiple dose intravenous administration of ketoprofen (3 mg.kg-1 of body weight, repeated for 5 days at

24 h intervals) on hemoglobin.

88

13. Effect of multiple dose intravenous administration of ketoprofen (3 mg.kg-1 of body weight, repeated for 5 days at 24 h intervals) on packed cell volume.

88

14. Effect of multiple dose intravenous administration of

ketoprofen (3 mg.kg-1 of body weight, repeated for 5 days at 24 h intervals) on total leukocyte count.

90

15. Effect of multiple dose intravenous administration of ketoprofen (3 mg.kg-1 of body weight, repeated for 5 days at 24 h intervals) on neutrophil count.

90


ketoprofen (3 mg.kg-1 of body weight, repeated for 5 days at 24 h intervals) on lymphocyte count.

91

17. Effect of multiple dose intravenous administration of ketoprofen (3 mg.kg-1 of body weight, repeated for 5 days at 24 h intervals) on basophil count.

91

Table No. Title Page

No.


24 h intervals) on eosinophil count.

92


ketoprofen (3 mg.kg-1 of body weight, repeated for 5 days at 24 h intervals) on monocyte count.

92


24 h intervals) on serum alkaline phosphatase.

94


ketoprofen (3 mg.kg-1 of body weight, repeated for 5 days at 24 h intervals) on serum acid phosphatase.

94


24 h intervals) on serum aspartate aminotransferase.

96


ketoprofen (3 mg.kg-1 of body weight, repeated for 5 days at 24 h intervals) on serum alanine aminotransferase.

96


24 h intervals) on serum lactate dehydrogenase.

98


ketoprofen (3 mg.kg-1 of body weight, repeated for 5 days at 24 h intervals) on total serum bilirubin.

98


24 h intervals) on serum creatinine.

100

27. Effect of multiple dose intravenous administration of ketoprofen (3 mg.kg-1 of body weight, repeated for 5 days at 24 h intervals) on blood urea nitrogen.

100


24 h intervals) on total serum protein.

102

29. Effect of multiple dose intravenous administration of ketoprofen (3 mg.kg-1 of body weight, repeated for 5 days at 24 h intervals) on serum albumin.

102


ketoprofen (3 mg.kg-1 of body weight, repeated for 5 days at 24 h intervals) on blood glucose.

104

LIST OF FIGURES

Figure

No. Title

Page

No.

1. General Structure of Non steroidal antiinflammatory drugs. 5

2. (a) General structure of propionic acid NSAIDs. 11

(b) Structure of ketoprofen.

(c) Structure of ketoprofen enantiomers [R(+) and S (-)].

3. Standard curve of ketoprofen in calf plasma. 64

4. Representative chromatograms of drug free plasma (A) and

ketoprofen (KTP) standard (25 µg.ml-1) (B).

74

5. Representative chromatograms of ketoprofen (KTP) plasma samples.

75

6. Semilogarithmic plot of ketoprofen concentration in plasma versus time following single dose intravenous administration at

the dose rate of 3.0 mg.kg-1 of body weight in calf.

76

7. Semilogarithmic plot of ketoprofen concentration in plasma

versus time following single dose intramuscular administration at the dose rate of 3.0 mg.kg-1 of body weight in calf.

82

8. Semilogarithmic plot of ketoprofen concentration in plasma versus time following single dose intravenous and intramuscular

administrations at the dose rate of 3.0 mg.kg-1 of body weight in calf.

85

9. Effect of multiple dose intravenous administration of ketoprofen (3 mg.kg-1 of body weight, repeated for 5 days at 24 h intervals)

on hemoglobin.

89

10. Effect of multiple dose intravenous administration of ketoprofen

(3 mg.kg-1 of body weight, repeated for 5 days at 24 h intervals) on packed cell volume.

89


on total leukocyte count.

93


(3 mg.kg-1 of body weight, repeated for 5 days at 24 h intervals) on differential leukocyte count.

93


on serum alkaline phosphatase.

95


(3 mg.kg-1 of body weight, repeated for 5 days at 24 h intervals) on serum acid phosphatase.

95


on serum aspartate aminotransferase (AST/ SGOT).

97


(3 mg.kg-1 of body weight, repeated for 5 days at 24 h intervals) on serum alanine aminotransferase (ALT/ SGPT).

97

Figure

No. Title

Page

No.


(3 mg.kg-1 of body weight, repeated for 5 days at 24 h intervals) on serum lactate dehydrogenase (LDH).

99


on total serum bilirubin.

99


ketoprofen (3 mg.kg-1

of body weight, repeated for 5 days at 24 h intervals) on serum creatinine.

101


ketoprofen (3 mg.kg-1

of body weight, repeated for 5 days at 24 h intervals) on blood urea nitrogen (BUN).

101


on total serum protein.

103


(3 mg.kg-1 of body weight, repeated for 5 days at 24 h intervals) on serum albumin.

103


on blood glucose.

105

ABBREVIATIONS

% Per Cent

α Distribution rate constant

Elimination rate constant

µg.h.ml-1 Microgram hour per milliliter

g.h2.ml-1 Microgram square hour per milliliter

μg.L-1 Microgram per Liter

μg.ml-1 Microgram per milliliter

µl Microliter

A‟ Zero-time intercept of absorption phase

A Zero-time intercept of distribution phase

ACP Acid phosphatase

AKP Alkaline phosphatase

ALT Alanine aminotransferase

AST Aspartate aminotransferase

AUC Area under curve

AUMC Area under first moment of curve

B Zero-time intercept of elimination phase

b.i.d. bis in die (twice a day)

BUN Blood urea nitrogen

°C Degree centigrade

CBC Complete Blood Count

ClB Total body clearance

Cmax Maximum drug concentration

cmm Cubic millimeter

COX Cyclooxygenase

Cp Plasma concentration

Cp0 Theoretical concentration of drug in plasma at zero-time

C.V. Co-efficient of variance

DLC Differential leukocyte count

EC50 Median Effective Concentration

e Base of natural logarithm

e.g. exempli gratia (for example)

et al. et alii (and others)

F Bioavailability of drug

g Gram

x g times gravity

g/dl Gram per deciliter

h Hour

h-1 Per hour

Hb Haemoglobin

HPLC High Performance Liquid Chromatography

IC50 Median Inhibitory Concentration

i.e. id est (that is)

I.M. Intramuscular administration

I.V. Intravenous administration

IU/L International unit per litre

K12 Rate constant of transfer of drug from central to tissue

compartment

K21 Rate constant of transfer of drug from tissue to central

compartment

Ka Absorption rate constant

Kel Elimination rate constant from central compartment

KTP Ketoprofen

L.h-1.kg-1 Liter per hour per kilogram

L.kg-1 Liter per kilogram

L.min-1.kg-1 Liter per minute per kilogram

LDH Lactate dehydrogenase

M Molar

MAT Mean absorption time

mg Milligram

mg/dl Milligram per deciliter

mg.h.ml-1 Milligram hour per milliliter

mg.h.L-1 Milligram hour per liter

mg.h2.L-1 Milligram square hour per liter

mg.kg-1 Milligram per kilogram

mg.ml-1 Milligram per milliliter

min. Minute

ml Milliliter

ml.kg-1 Milliliter per kilogram

ml.h-1.kg-1 Milliliter per hour per kilogram

ml.min-1.kg-1 Milliliter per minute per kilogram

mm Millimeter

mM Millimolar

MRT Mean residence time

ng.ml-1 Nanogram per milliliter

NSAIDs Non steroidal antiinflammatory agents

P Level of significance

PCV Packed Cell Volume

PG Prostaglandins

PGE2 Prostaglandin E2

PGF2α Prostaglandin F2α

pH Negative logarithm of H+ ion concentration

pKa Dissociation constant

PK/PD Pharmacokinetic – Pharmacodynamic relationship

P.O. Per os (oral route)

R(–) Rectus (right) enantiomer

S(+) Sinister (left) enantiomer

S.E. Standard error

SGOT Serum glutamic-oxaloacetic transaminase

SGPT Serum glutamic-pyruvic transaminase

SPSS Statistical Package for the Social Sciences

t Time

t1/2 Distribution half life

t1/2 Elimination half life

t1/2Ka Absorption half life

t.i.d. ter in die (3 times a day)

TLC Total leukocyte count

Tmax Time of maximum observed concentration in plasma

TxB2 Thromboxane B2

USP United States Pharmacopoeia

Vd Volume of distribution

Vd(area) Apparent volume of distribution

Vd(ss) Volume of distribution at steady state

v/v volume by volume

viz. Videlicet (namely)

Introduction

CHAPTER I

INTRODUCTION

Inflammation is the complex biological response of vascular tissues to harmful

stimuli, such as pathogens, damaged cells or irritants. It is a protective mechanism by

the body to remove the injurious stimuli as well as initiate the healing process for the

tissue. However, inflammation which runs unchecked can also lead to host of

diseases, such as atherosclerosis and rheumatoid arthritis. The mediators released

during the inflammatory process perpetuate the inflammatory response and are

responsible for the clinical signs associated with inflammation, including pain and

fever (Vane and Botting, 1987). Administration of antiinflammatory agents to

alleviate signs of inflammation is a standard therapeutic approach. The main anti-

inflammatory agents are the glucocorticoids and the non-steroidal antiinflammatory

drugs (NSAIDs).

NSAIDs include a variety of different agents of different chemical classes

having antiinflammatory, analgesic and antipyretic effects. In general, all of these

effects are related to the primary action of the drugs i.e. inhibition of arachidonate

cyclooxygenase and thus inhibition of the production of prostaglandins and

thromboxanes. There are two main types of cyclooxygenase enzymes, namely COX-1

and COX-2. COX-1 is a constitutive enzyme involved in tissue homeostasis whilst

COX-2 is responsible for the production of the prostanoid mediators of inflammation

(Vane et. al., 1998).

Ketoprofen (KTP) is an aryl propionic acid derivative, non-selective COX

inhibitor NSAID. Ketoprofen is a strong inhibitor of cyclooxygenase; it has powerful

antiinflammatory, analgesic and antipyretic properties (Boothe, 2001). Ketoprofen

contains an asymmetrical carbon atom and exists in two enantiomeric forms like S(+)

Ketoprofen and R(–) Ketoprofen (Sweetman, 2002). Differences in pharmacokinetics

and pharmacodynamics between the two enantiomers occur in animals and can also

vary significantly among species. In veterinary practice, ketoprofen is used to lower

body temperature in animals with fever, to relieve respiratory signs in calf and piglet

pneumonias, and to relieve pain in conditions as diverse as equine colic and joint

diseases of the horse and dog, as well as for the control of traumatic and postoperative

pain in all species (Lees et. al., 2004).

As per the figures of 2003 livestock census, India has 187.38 million cattle

population which is about 15 per cent of the world cattle population. India ranks first

in cattle population. Out of the 187.38 million cattle, 22.63 million were crossbred,

which is 12.07 per cent of the total cattle population. Between 1997 and 2003,

crossbred population increased by 12.6 per cent. Ban on some NSAIDs like

Diclofenac sodium had been put on, to be used in bovines and other domestic animals,

due to increased extinction of vultures. Under this condition ketoprofen and other

NSAIDs have emerged as a newer therapeutic substitute for treating musculoskeletal

disorders (as antiinflammatory) and painful conditions (as analgesic) in cow calves

and adult cattle.

Ketoprofen pharmacokinetic have been reported in some domestic species like

Cattle (Landoni and Lees, 1995a; De Graves et al., 1996), Sheep (Landoni et al.,

1999; Arifah et al., 2001), Goat (Musser et al., 1998; Arifah et al., 2003; Pravin et al.,

2005; Pranvendra et al., 2005) and in Horses (Jaussaud et al., 1993; Sams et al., 1995;

Landoni and Lees, 1995b; Landoni and Lees, 1996; De graves et al., 1998). However,

data on pharmacokinetics of ketoprofen in cattle are very sparse, hence the study was

undertaken.

In Veterinary therapeutics, Ketoprofen is given by oral and parenteral routes in

cattle, cats, dogs and horses. However, it is recommended that ketoprofen treatment

should be limited to a maximum of five consecutive days to reduce the risk of

gastrointestinal effects. Ketoprofen can cause gastrointestinal irritation that may lead

to ulceration. Other side effects, including hepatopathies and renal disease, have been

reported in animals (Thompson, 2006). Most of the safety and toxicity studies are

conducted in laboratory animals following oral administration. As the drug is used

parenterally in domestic animals, safety data following its repeated administrations

are needed.

Materializing that there is lack of literature on pharmacokinetics and safety of

ketoprofen in the target species like cattle (calves), the present study was planned with

the following objectives:

1. To optimize and standardize the method for detection of ketoprofen in

plasma of cow calves by High Performance Liquid Chromatography

(HPLC).

2. To study the pharmacokinetics of single dose intravenous administration

of ketoprofen (3 mg.kg-1 body weight) in crossbred cow calves.

3. To study the pharmacokinetics and bioavailability of single dose

intramuscular administration of ketoprofen (3 mg.kg-1 body weight) in

crossbred cow calves.

4. To assess safety of ketoprofen in cow calves following multiple

intravenous doses given at rate of 3 mg.kg-1 of body weight repeated at 24

hour intervals for 5 days.

Review of Literature

CHAPTER II

REVIEW OF LITERATURE

2.1 Introduction to NSAIDs:

Salicylic acid was first synthesized in 1859 and marketed in 1878 and was

the first drug with recognized antipyretic, antiinflammatory and analgesic activities.

Numerous drugs were discovered after the development of aspirin (acetyl salicylic

acid); these drugs came to be known as “Aspirin like drugs”. The term NSAIDs was

first applied to phenylbutazone after its introduction into clinical practice in 1949

(Hart and Huskisson, 1984). NSAIDs principally act as antiinflammatory agents and

are used to treat arthritis, other inflammatory diseases, injuries and in the

management of different post-operative conditions (Lombardino and Wiseman,

1982; Hawkey, 1999). NSAIDs produce a mild degree of analgesia, which is much

less than the analgesia produced by opioid analgesics such as morphine. Many of the

NSAIDs cause the excretion of uric acid (uricosuric effect), and thus are useful in the

treatment of gout (Altman et al., 1988).

The importance of pain management and the use of nonsteroidal anti-

inflammatory drugs (NSAID) in animals have recently increased significantly.

NSAID have the potential to relieve pain and inflammation without the

immunosuppressive and metabolic side effects associated with corticosteroids.

NSAIDs, which possess analgesic, antiinflammatory and antipyretic properties, are a

heterogeneous group of substances without any uniform chemical properties, but

share the same therapeutic and side effects (Steinmeyer, 2000).

The first veterinary use of a synthetic NSAID, the sodium salt of salicylic acid

was reported in 1875. Currently, several NSAIDs designated for human use are being

investigated for possible veterinary application in the expectation that they may be

more effective and without the risks of adverse effects seen with previous NSAIDs.

Phenylbutazone, salicylic acid, meloxicam, ketoprofen and some other NSAIDs are

used as therapeutic measures for pain, inflammation and fever in clinical veterinary

medicine. Modern veterinary uses of NSAIDs are to lower body temperature in

animals with fever, to relieve respiratory signs in calf and piglet pneumonias, and to

relieve pain in conditions as diverse as equine colic and joint diseases of the horse and

dog, as well as the control of traumatic and postoperative pain in all species (Lees et.

al., 2004).

2.2 Structure of NSAID‟s:

In general, NSAIDs structurally consist of an acidic moiety (carboxylic acid,

enols) attached to a planar, aromatic functionality. Some analgesics also contain a

polar linking group, which attaches the planar moiety to an additional lipophilic group

(De Ruiter, 2002). Figure 1 represents general structure of NSAIDs.

Figure 1: General Structure of Non steroidal antiinflammatory drugs.

As a result, the NSAIDs are characterized by the following chemical/

pharmacologic properties:

(1) All are relatively strong organic acids with pKa in the 3-5 range. Most, but not all,

are carboxylic acids. Thus, salts forms can be generated upon treatment with base

and all of these compounds are extensively ionized at physiological pH. The

acidic group is essential for COX inhibitory activity.

(2) The NSAIDs differ in their lipophilicities based on the lipophilic character of their

aryl groups and additional lipophilic moieties and substituents.

(3) The acidic group in these compounds serves a major binding group (ionic binding)

with plasma proteins. Thus all NSAIDs are highly bound by plasma proteins.

(4) The acidic group also serves as a major site of metabolism by conjugation. Thus a

major pathway of clearance for many NSAIDs is glucuronidation (and

inactivation) followed by renal elimination.

2.3 Classification of NSAID‟s:

Depending on their chemical structures, NSAIDs are broadly divided into two

major classes like non selective COX inhibitors and selective COX-2 inhibitors

(Roberts and Morrow, 2001).

(1) Non-Selective COX Inhibitors:

i. Salicylic acid derivatives: e.g. Aspirin, Sodium salicylate.

ii. Para-amino phenol derivatives : e.g. Acetaminophen

iii. Indol and Indane acetic acids: e.g. Indomethacin, Sulindac

iv. Heteroaryl acetic acids: e.g. Tolmetin, Diclofenac, Keterolac

v. Aryl propionic acids: e.g. Ibuprofen, Naproxen, Flurbiprofen,

Ketoprofen, Fenoprofen, Oxaprofen

vi. Anthranilic acids: e.g. Mefenamic acid, Meclofenamic acid

vii. Enolic acids (Oxicams): e.g. Piroxicam, Meloxicam, Tenoxicam,

Isoxicam

viii. Alkanones: e.g. Nabumetone

(2) Selective COX-2 Inhibitors :

i. Indole acetic acids: e.g. Etodolac

ii. Sulfonanilides: e.g. Nimesulide

iii. Diaryl substituted furanones: e.g. Rofecoxib

iv. Diaryl substituted pyrazoles: e.g. Celecoxib

2.4 Mechanism of action of NSAID‟s:

Unlike corticosteroids, which inhibit numerous pathways, NSAIDs act

primarily to reduce the biosynthesis of prostaglandins (PG) by inhibiting

cyclooxygenase (COX) enzymes (Smith and Willis, 1971; Vane, 1971). A sub-group

of the eicosanoid group of inflammatory mediators is of special interest in relation to

mechanism of action of NSAID. These are formed from cell membrane phospholipid,

which in response to tissue damage releases short chain fatty acids such as the 20

carbon compound arachidonic acid (5, 8, 11, 14 - eicosatetraenoic acid), under the

influence of phospholipase A2. Arachidonic acid can serve as a substrate for four

groups of enzymes, cyclooxygenase (COX), 5- lipoxygenase, 12- lipoxygenase and 15-

lipoxygenase (Lees et al., 2004).

The discovery of the 2 isoforms of COX (COX-1 and COX-2) has advanced

understanding of the mechanism of action (Vane et al., 1998). COX-1, expressed in

virtually all tissues of the body, catalyzes the formation of constitutive PG, which

mediates a variety of normal physiologic effects including hemostasis, gastrointestinal

mucosal protection, and protection of the kidney from hypotensive insult. In contrast,

COX-2 is activated in damaged and inflamed tissues and catalyzes the formation of

inducible PG, including PGE2, associated with intensifying the inflammatory response.

COX-2 is also involved in thermoregulation and the pain response to injury.

Therefore, COX-2 inhibition by NSAIDs is thought to be responsible for the

antipyretic, analgesic, and antiinflammatory actions of NSAIDs (Thompson, 2006).

NSAIDs appear to inhibit both COX-1 and COX-2. The amount of drug

necessary to inhibit each of the two isoforms provides a basis for assessing the

relative safety and efficacy of each drug. The ratio of COX-1 to COX-2 describes the

amount of drug necessary to inhibit the respective isoforms of the cyclooxygenase

enzyme in an experimental environment. A COX-1/COX-2 ratio of more than 1 is

desirable since a drug, that inhibits COX-2 (inducible) prostaglandins at lower

concentrations than that necessary to inhibit COX-1 (constitutive) prostaglandins, is

probably safer. IC50 inhibition ratio (COX-1: COX-2) of some NSAIDs is given in

Table 1.

Table: 1. IC50 inhibition ratio (COX-1: COX-2) of NSAIDs in human whole blood

assay (Lees et al., 2004).

Drugs COX-1 WBA: COX-2 WBA

IC50 ratio1

COX-1 WBA: COX-2 WBMA

IC50 ratio2

COX-2 WBA: COX-2 WBMA

IC50 ratio3

Diclofenac 1.97 3.75 1.90

Meloxicam 2.71 24.78 9.13

Nimesulide 5.26 25.64 4.87

Salicylate 0.14 10.28 71.73

Ketoprofen 0.02 0.20 12.10

Etodolac 5.45 12.77 2.34

Carprofen 0.02 ---- ----

1Whole blood assay (WBA) for both COX-1 and COX-2

2Whole blood assay for COX-1, William Harvey modified whole blood assay

(WHMA) for COX-2

3Ratio of whole blood: William Harvey modified whole blood assays for COX-2

The hypothesis that COX-1 inhibition is associated with side-effects of

NSAIDs whilst COX-2 inhibition is responsible for the therapeutic effects still holds

true, but it has been modified by the following developments:

i. Evidence from several sources has indicated a role for central actions of

NSAIDs as analgesics and antiinflammatory agents in addition to their

peripheral actions (Smith et al., 1998; Dolan et al., 2003).

ii. Existence of a third COX isoform, COX-3, was postulated (Willoughby et al.,

2000) and then identified in the CNS of the dog (Chandrasekharan et al.,

2002); inhibition of this isoform which is a spliced COX-1 variant may

account, in part, for the central analgesia produced by NSAIDs; some

compounds such as paracetamol and the pyrazolone derivative dipyrone

(metamizole) being possibly COX-3 preferential.

iii. Selective COX-2 inhibitors, whilst being more gastrotolerant than COX-1

inhibitors, are unlikely to be entirely free of side-effects; concerns have been

expressed that wound healing may be delayed, fracture healing may be

impaired or delayed and COX-2 inhibitors may have potentially damaging

side-effects on the kidney and some aspects of reproductive function, because

COX-2 is also a constitutive enzyme in these tissues. Also, there is

controversy concerning whether COX-2 inhibitors produce a small increase in

adverse cardiovascular events in human subjects (Mengle-Gaw & Schwartz,

2002).

iv. Both COX-1 and COX-2 may contribute to the generation of prostaglandins at

sites of inflammation (Smith et al., 1998; Wallace et al., 1998).

v. There is mounting evidence that COX-2 not only produces proinflammatory

prostaglandins in the initial stages of acute inflammation but also anti-

inflammatory prostaglandins such as 15 deoxy ∆12-14 PGJ2 in the resolution

phase (Gilroy et al., 1999).

vi. A recent investigation in the rat indicated that low doses of selective COX-2

inhibitors induced hypoalgesia in acute inflammation, whereas higher doses

associated with COX-1 inhibition did not induce hypoalgesia but produced

suppression of experimental hyperalgesia (Francischi et al., 2002).

2.5 Ketoprofen:

Ketoprofen (KTP) is an aryl propionic acid derivative non-selective COX

inhibitor NSAID. It was synthesized by Rhone-Poulenc Research Laboratories, Paris

in 1967 and was first approved for clinical use in France and the United Kingdom in

1973 (Vavra, 1987). Ketoprofen is a potent inhibitor of COX and bradykinin and may

also inhibit some lipoxygenases. Its efficacy is comparable to that of opioids in the

management of pain following orthopedic and soft-tissue surgery in dogs. As with

other NSAID, ketoprofen is metabolized in the liver to inactive metabolites that are

eliminated by renal excretion (Thompson, 2006).

2.5.1 Chemistry and Structure:

Ketoprofen is a white or almost white, crystalline, odourless powder with a

sharp bitter taste (Sweetman, 2002). It is prepared by chemical synthesis as a

racemate and contains not less than 99.0% and not more than the equivalent of

100.5% of (2RS)-2-(3-benzoylphenyl) propanoic acid, calculated with reference to the

dried substance (The British Pharmacopoeia, 2002). Structures of propionic acid

NSAIDs in general, ketoprofen and its enantiomers are shown in figure 2 (a, b and c).

Its empirical formula is C16H14O3, with molecular weight of 254.28.

Ketoprofen is a weak monocarboxylic acid and has reported dissociation constant

values, pKa, of 4.60 in water.

Figure 2(a): General structure of propionic acid NSAIDs.

Figure 2(b): Structure of ketoprofen.

Figure 2(c): Structure of ketoprofen enantiomers [R(+) and S (-)].

Adjusting the pH to a higher value can solubilize ketoprofen, as solubility

increases at pH values above its pKa (Sridevi and Diwan, 2002). It has a pKa of 5.94

in methanol:water (3:1) and an n-octanol:water partition coefficient of 0.97 (buffer pH

of 7.4). Ketoprofen is practically insoluble in water, freely soluble in acetone, ethanol

and methylene chloride (The British Pharmacopoeia, 2002). Ketoprofen has been

reported to melt in the range of 93°C to 95°C (Liversidge, 1981) and 94°C to 97°C

(Sweetman, 2002). Ketoprofen is highly lipophilic. Ketoprofen is stable below 25°C.

It must be protected from light and moisture (Liversidge, 1981).

2.5.1.1 Stereochemistry

The presence of at least one asymmetric carbon atom in a chemical entity

results in the existence of stereoisomers. Ketoprofen has one asymmetric carbon, also

referred to as a chiral centre, which gives rise to two enantiomers viz. S(+) ketoprofen

and R(–) ketoprofen (Sweetman, 2002). Currently, commercially available products

contain a racemic (50:50) mixture of the two enantiomers, S(+) ketoprofen and R(–)

ketoprofen. It has been shown that S(+) aryl propionates are many times more potent

than their R(-) enantiomers (Evans, 1992). In vitro studies on the relative anti-

inflammatory activity of individual ketoprofen enantiomers have shown that their

effect on cyclooxygenase is due to the S(+) enantiomer (Caldwell et al., 1988; Evans,

1992). Differences in pharmacokinetics and pharmacodynamics between the two

enantiomers occur in animals and can also vary significantly among species (Aberg et

al., 1995).

Chiral inversion is a characteristic metabolic pathway for various profens and,

in the case of KTP, the R(-) enantiomer readily undergoes biotransformation to the

S(+) enantiomers (Soraci et al., 1996). The R(-) enantiomer of the ketoprofen was

administered orally at 20 mg.kg-1 to a series of 8 animal species. In all species, a

highly significant degree of inversion occurred after 1 h which varied from 27%

(gerbil) to 73% (dog) and persisted or increased in plasma samples obtained 3 h after

drug administration. Percentage of total plasma ketoprofen identified as the S(+)

enantiomer 3 hours after administration of 20 mg of R(–) ketoprofen per kg in

different species was as follow: gerbil (33%), mouse (44%), monkey (45%), guinea

pig (47%), rabbit (51%), rat (74%), hamster (81%), dog (91.9%) (Aberg et al., 1995).

The chiral inversion of R(–) to S(+) ketoprofen after an intravenous dose of

R(-) ketoprofen is reported to be 31.7% in calves, 5.9 to 13.8% in sheep, 15% in goat,

48.8% in horses, 22.4% in cats and 8.9% in man. (Lees et al., 2004). Chiral inversion

of R(-) ketoprofen to S(+) ketoprofen also differs in cattle at different ages and in

different physiological situations (Igarza et al., 2002). Inversion was higher in

newborn calves than in early lactation or cows in gestation (50.5%, 33.3% and 26%,

respectively). For ketoprofen chiral inversion mechanism is almost unidirectional,

from R(-) to S(+) inversion (Evans, 1992; Jamali et al., 1997; Landoni et al.,

1999).The reverse process, conversion of S(+) to R(–) ketoprofen, has only been

demonstrated in mice (Jamali et al., 1997).

The pharmacokinetics of ketoprofen enantiomers also varies in volume of

distribution, elimination rate and plasma protein binding. In some species (rat, dog,

horse, monkey and rabbit), S(+) ketoprofen was the predominant enantiomer in

plasma after administration of the racemic ketoprofen (Abas and Meffin, 1987; Foster

and Jamali, 1988; Jaussaud et al., 1993). In humans and calves, on the other hand,

concentration of the two enantiomers were similar (Geisslinger et al., 1995; Landoni

and Lees, 1995a; Landoni et al., 1995), while R(-) ketoprofen predominated in plasma

of goat and sheep (Landoni et al., 1999; Arifah et al., 2001).

2.5.2 Pharmacological actions:

Ketoprofen is an effective antiinflammatory and analgesic drug in clinical

practice and is used in the treatment of rheumatoid arthritis and osteoarthritis. It is as

effective in clinical trials as other NSAIDs such as naproxen from both the efficacy

and side effect point of view (Dollery, 1991). Like most NSAIDs, ketoprofen is

advantageous because it lacks addictive potential and does not result in sedation or

respiratory depression. In addition ketoprofen also exerts analgesic and antipyretic

pharmacological properties (Green, 2001).

2.5.2.1 Anti-inflammatory effects

In several animal models (rats, mice, rabbits, guinea pigs and pigeons)

ketoprofen displayed potent activity against acute inflammation (increased vascular

permeability, oedema and erythema), sub acute inflammation (pleurisy, abscess and

granuloma formation) and chronic inflammation (experimental arthritis and synovitis)

(Kantor, 1986). Its antiinflammatory activity is 20 times more potent than ibuprofen,

80 times more potent than phenylbutazone and 160 times more potent than aspirin

(Dollery, 1991).

Ghezzi et al. (1998) compared the antiinflammatory effects of R(-) and S(+)

ketoprofen in vitro and in vivo. S(+) ketoprofen efficiently inhibited carrageenan

induced edema formation, but it could also amplify the lipo-polysaccharide induced

production of the inflammatory cytokines, tumor necrosis factor and interleukin-1 , in

close correlation with its ability to inhibit prostaglandin synthesis. Because these

inflammatory cytokines are among the factors involved in carrageenan- induced

inflammation and also are possibly involved in gastric damage, enhanced cytokine

production could partially mask the analgesic effect of S(+) ketoprofen, and it can be

associated with the clinical evidence of its gastric toxicity. On the other hand, R(-)

ketoprofen contributes to the overall activity of the racemate, by playing the main role

in ketoprofen- induced analgesia. Unlike the S(+) isomer, R(-) ketoprofen did not

induce a significant increase of cytokine production even at cyclooxygenase-blocking

concentrations. It is concluded that the R(-) isomer directly contributes to the anti-

inflammatory effects of ketoprofen, being more analgesic, and because it does not

amplify inflammatory cytokine production.

2.5.2.2 Analgesic effects:

The action of NSAIDs, such as ketoprofen, in the relief of pain is believed to

be the interruption of mediators of inflammation, such as bradykinin, and prevention

of their effect on peripheral pain endings.

NSAIDs also reduce the hyperalgesia that

occurs in inflammation (Owens et al., 1995b; Johnston and Fox, 1997; Arrioja, 2002).

Ketoprofen was shown to be a potent, peripherally acting analgesic in two

classical animal models of pain. It was also shown to be equivalent to indomethacin,

slightly more potent than naproxen and 30 times more potent than aspirin in pain

management. Like other NSAIDs, ketoprofen is inactive in assays measuring centrally

mediated analgesia (Kantor, 1986).

Owens et al. (1995b) employed a combination of subjective and objective

methods to compare the effects of ketoprofen and phenylbutazone on chronic hoof

pain and lameness in horses. They found that ketoprofen at 1.65 times more than the

recommended dose significantly reduced chronic pain and lameness when compared

to phenylbutazone.

2.5.2.3 Antipyretic effects:

Antipyretic effect of ketoprofen is believed to be due to inhibition of

prostaglandins synthesis in the hypothalamus (Arrioja, 2002).

Glew et al. (1996) investigated the antipyretic effectiveness of ketoprofen in

pyretic cats with a variety of bacterial and viral infections. Cats were randomly

assigned to receive a broad-spectrum antibiotic or a broad-spectrum antibiotic plus

ketoprofen. Body temperature was monitored 3 times daily, and attitude and appetite

were evaluated once daily. Mean temperatures were significantly different during the

4 and 8 h post-treatment observations, with a reduction to normal temperatures in the

ketoprofen group compared with no change in the group treated with antibiotics

alone. The antipyretic effect of ketoprofen was rapid and persisted for at least 8 h, but

for less than 24 h. The overall recovery period from pyrexia, depression, and

inappetance was also shorter in cats treated with antibiotics and ketoprofen (3 days)

than in cats treated only with antibiotics (5 days). Ketoprofen was a useful adjunct in

the treatment of pyretic cats.

2.5.3 Mechanism of action

Ketoprofen is one of the most powerful inhibitors of cyclooxygenase at

concentrations well within the range of therapeutic plasma concentrations (EC50: 2

μg.L-1) (Kantor, 1986). The primary mechanism of action for ketoprofen is considered

to be inhibition of the cyclooxygenase pathway of arachidonic acid metabolism,

leading to decreased production of prostaglandins (Lees et al., 1990; Johnston and

Fox, 1997). It produces reversible COX inhibition by competing with the substrate,

arachidonic acid, for the active site of the enzyme (Vane and Botting, 1998).

Normally, cell membrane damage triggers arachidonic acid metabolism and

production of short- lived endoperoxidases by action of cyclooxygenase (Johnston and

Fox, 1997). The endoperoxidases are converted by other enzymes to thromboxane,

prostaglandin E2, prostaglandin F

2α, prostacyclin and oxygen radicals (Kalpravidh et

al., 1984).

Inhibition of cyclooxygenase prevents the formation of these

prostaglandins and the inflammation they cause.

This inhibition results in a reduction in the tissue production of prostaglandins

such as PGE2 and PGF2α. In addition to its effects on cyclooxygenase, ketoprofen

inhibits the lipoxygenase pathway of the arachidonic acid cascade. This pathway

produces non-cyclized monohydroxyl acids and leukotrienes. Of these, only

leukotrienes (B4, C4, and D4) are thought to increase vascular permeability, however,

both monohydroxyl acids and leukotrienes synthesised within leukocytes are active in

promoting leukocyte migration and activation. It has been suggested that

lipoxygenase inhibitors may attenuate cell-mediated inflammation and thus retard the

progression of tissue destruction in inflamed joints.

Ketoprofen is a powerful inhibitor of bradykinin, an important chemical

mediator of pain and inflammation. It also stabilizes lysosomal membranes against

osmotic damage and prevents the release of lysosomal enzymes that mediate tissue

destruction in inflammatory reactions (Kantor, 1986; Dollery, 1991).

2.5.3.1 Pharmacodynamic of ketoprofen in calves.

Ketoprofen (KTP) pharmacodynamics was evaluated by determining the

effects on serum thromboxane (TxB2), exudate prostaglandin (PGE2), leukotriene

(LTB4), β- glucuronidase and bradykinin induced oedematous swelling (Landoni et

al., 1995). Effect-concentration inter-relationships were analyzed by PK/PD

modeling. KTP did not affect exudate LTB4, but inhibition of the other variables was

statistically significant. The mean EC50 values for inhibition of serum TxB2, exudate

PGE2, β- glucuronidase, and bradykinin induced swelling were 0.118, 0.086, 0.06 and

0.00029 μg.ml-1, respectively. The data indicate that KTP exerted an inhibitory action

not only on eicosanoids (TxB2 and PGE2) synthesis but also on exudate β-

glucuronidase and bradykinin induced oedema. The EC50 values for these actions

indicate that they are likely to contribute to the overall antiinflammatory effects of

KTP in calves. COX-1 is a constitutive enzyme, involved in the synthesis of

eicosanoids related to „house keeping functions‟ while COX-2 is an inducible

isoenzyme, involved in the production of eicosanoids related to the inflammatory

response. Therefore, reduction in serum TxB2 is a measure of inhibition of COX-1,

whilst decreased exudate PGE2 synthesis indicates inhibition of COX-2. The IC50

ratio (serum TxB2 : exudates PGE2) was 1.37. The greater the concentration required

to inhibit TxB2 might indicate a lesser likelihood of toxic reactions after ketoprofen

administration in cattle compared to NSAIDs with lower ratios.

2.6 General Pharmacokinetics

Pharmacokinetics is concerned with the study and characterization of the time

course of drug absorption, distribution, metabolism and excretion. It studies the

relationship of these processes with regards to the intensity and duration of

characteristic effects of drugs (Baggot, 1995). It is mainly concerned with the passage

of drug to its site of action and maintenance of adequate concentration to exer t its

therapeutic, diagnostic or toxicological effect. The pharmacokinetic study of drugs

provides an important tool to achieve the effective dosage regimens.

2.6.1 Drug absorption and Bioavailability

A drug can be given either orally or by a parenteral route. A variable degree of

drug absorption takes place from these sites of administration. The extent of

absorption depends largely on the formulation of the administered preparation and on

the drug itself. A low degree of ionization and high lipid solubility of the non- ionized

moiety are properties favouring absorption (Baggot, 1995). Bioavailability is defined

as the rate and extent to which a drug, administered as particular dosage form, enters

the systemic circulation intact. Bioavailability of drug can be adequately characterized

by determining three parameters from the plasma drug concentration-time profile viz.

peak plasma concentration (Cmax), time taken to reach peak concertration (Tmax) and

area under the curve (AUC).

2.6.2 Rate of drug movement

Pharmacokinetics is described as mathematical description of changes in drug

concentration in the body. These changes can obey first order or zero order rates. In

first order process, the actual rate of the process varies in direct proportion to the mass

of the compound. In zero order processes, the rate of drug movement is fixed and thus

independent of the amount of compound available (Riviere, 1999). First order kinetics

is typical in most of the drug studies. At low drug concentration, the drug follows first

order kinetics. When the elimination processes become gradually more saturated, the

drug follows mixed order kinetics, eventually discharging into zero order kinetics, at

high drug concentrations (Shargell and Yu, 1993).

2.6.3 Disposition curve and compartment models

Following an I.V. injection of a single dose of the drug, the decline in plasma

concentration of the drug is expressed graphically by the disposition curve. This

curve, plotted on semilogarithmic graph paper, is either monophasic or biphasic.

The study of pharmacokinetics is based on creation of mathematical models to

describe the concentration changes of drugs with time in animal body. A common

approach to study pharmacokinetics of a drug is to depict the animal body as a system

of distribution compartments (Riegelman et al., 1968a). These compartments are

mathematical entities that have no physiological meaning but are useful in describing

disposition kinetics of a drug. The purpose of pharmacokinetics is to study the time

course of drugs and metabolite concentration in various body fluids, tissues and

excreta and to interpret such data based on suitable models (Wagner, 1968).

The body eliminates all drugs at widely varying rates, and is therefore

designated as an open system. The drug is said to follow one compartment open

model when the distribution is instantaneous between blood and tissues. Any change

in blood drug level reflects directly the quantitative changes in its tissue

concentration. Baggot (1977) reported that the rate of drug elimination from the body

is proportional to the concentration in blood. If the plotted data of log blood

concentrations of the drug versus time after a rapid intravenous injection shows a

straight line, it indicates the application of one compartment open model (Sams,

1978). In this model, the plasma drug level declines monoexponentially according to

equation 1:

Cp = Be -t -------- Equation 1

Where B is the zero time intercept of the terminal elimination phase line and

is overall elimination rate constant, t is the time elapsed after drug administration and

„e‟ represents the base of natural logarithm. One compartment model with no

absorption phase has been employed for describing the pharmacokinetics o f

ketoprofen in Japanese quail after I.M. and P.O. administration (Graham et al., 2005).

The rate of change of the amount of drug in the body is a function of both the

absorption rate and the elimination rate. When the absorption rate is greater than the

elimination rate, the amount of drug in the body and the drug concentration in the

plasma increases with time. On the contrary, when the amount of drug remaining at

the absorption site is sufficiently small so that the elimination rate exceeds the

absorption rate, the amount of drug in the body and the drug concentration in the

plasma decrease with time. The peak concentration after drug administration occurs at

the moment when the absorption rate equals the elimination rate. The faster a drug is

absorbed, the higher is the peak concentration in plasma after a given dose and the

shorter is the time after administration when the peak is observed (Gibaldi and Perrier,

1982).

Majority of the drugs appear to be absorbed in a first order from the site of

administration. The rate of decline in their plasma concentration is monoexponential

as noted after intravenous administration. The disposition kinetics of such drugs have

been reported to follow a one compartment open model with first order absorption

where the plasma concentration is expressed according to equation 2 given below.

Cp = Be -t - A‟e -Kat ------- Equation 2

Where Cp is the concentration of drug in plasma at time t, A‟ and B are the

zero time plasma drug concentration intercepts for the absorption and elimination

components, is the first order rate constant of the elimination phase, Ka is the first

order absorption rate constant after drug administration and e represents the base of

natural logarithm. The disposition kinetics of phenylbutazone (I.M.) in camel (Kadir

et al., 1997), phenylbutazone (P.O.) in calves (Arifah and Lees, 2002), nimesulide

(P.O.) in goats (Rao et al., 2007) and meloxicam (P.O. and I.M.) in vultures (Naidoo

et al., 2008) has been best described by one compartment open model with first order

absorption.

The disposition kinetics of many drugs fit well to two compartment

open model which is generally adjudged by a pronounced departure from longevity in

the semilog plot during early time period following drug administration (Sams, 1978).

In this model, the drug distributes instantaneously into the central compartment

(consisting of blood and other readily accessible tissues like liver and kidney), and

then relatively slowly into the peripheral compartment (the remainder body space).

The two compartment open model also specifies that after intravenous administration

the elimination of a drug take place exclusively from the central compartment. The

distribution and elimination processes are assumed to follow the first order kinetics.

The rate of decline in plasma concentration following intravenous administration of

the drug in this model is biexponential and can be expressed by equation 3 given

below.

Cp = Ae -t + Be -t ------- equation 3

Where Cp is the concentration of drug in plasma, A and B are zero time

intercepts of the initial and terminal phases of the plasma concentration time curve

with the concentration expressed as g.ml-1, and are respectively the distribution

and elimination rate constants expressed as min-1 or h-1 and e represents the base of

natural logarithm.

In a two compartment open model the initial steep decline in plasma drug

concentration is mainly due to distribution of drug from central to peripheral

compartment. Once apparent distribution is established, the rate of decline in plasma

drug concentration is determined mainly by irreversible elimination of drug from the

central compartment, appropriately termed as or elimination phase. The straight

portion of the curve has a slope defined as -/2.303 and an extrapolated zero time

intercept B. Subtraction of extrapolated values from corresponding experimental

plasma values on the semilog plot yields a series of residual concentrations. These

residuals yield a second linear segment called the alpha () or distribution phase with

a slope equal to -/2.303 and a zero time intercept A with units in concentration

(Gibaldi et al., 1969). These experimental constants (A, B, and ) are used to

calculate the actual pharmacokinetic rate constants (K12, K21, Kel) associated with the

two-compartment model.

The disposition kinetics of ketoprofen, phenylbutazone and sodium

meclofenamate in dairy cattle (De Graves et al., 1996; Arifah and Lees, 2002; Picco

et al., 2004), phenylbutazone in camel (Kadir et al., 1997), indomethacin in sheep

(Vinagre et al., 1998), ketorolac in dogs (Pasloske et al., 1999) ketoprofen and

carprofen in horses (Verde et al., 2001; Lees et al., 2002), ibuprofen in broiler

chickens (Vermeulen and Remon, 2001), ketoprofen and nimesulide in goats (Arifah

et al., 2003; Rao et al., 2007), flunixin in cats (Horii et al., 2004) and flunixin-

meglumine in angora rabbits and swine (Elmas, et al., 2005; Burr et al., 2006)) have

been best described by two compartment open model.

Some drugs after extravascular administration (oral or intramuscular) are

absorbed in a first order fashion but their plasma concentration time profiles show a

biexponential decline during the elimination phase. The pharmacokinetics of these

drugs is well fitted to a two compartment open model where the plasma concentration

is described by equation 4 given below.

Cp = Ae -t + Be -t – A‟e -Kat ------- equation 4

Where Cp is the concentration of drug in plasma at time t. A‟, A and B are the

zero time plasma drug concentration intercepts for the absorption, distribution and

elimination phases, respectively. Ka, and are the respective first order rate

constants and e is the base of natural logarithm. The disposition kinetics of

indomethacin (I.M.) in sheep (Vinagre et al., 1998), ibuprofen (P.O.) in broiler

chickens (Vermeulen and Remon, 2001), sodium meclofenamate (I.V.) in cattle

(Picco et al., 2004), ketoprofen (P.O.) in dogs (Montoya et al., 2004) and etodolac

(P.O.) in horses (Davis et al., 2007) have been best described by a two compartment

open model with first order absorption.

The disposition kinetics of some drugs may follow three-compartment open

model, where the plasma concentrations after single intravenous administration are

described by a triexponential expression (equation 5).

Cp = Ae -t + Be -t + Pe -t ------- equation 5

The additional constants P and are estimated by the method of residuals.

The rate constants for drug entry into and out of the third component i.e. K13 and K31,

respectively, can be calculated from the above equation (Baggot, 1977; Gibaldi and

Perrier, 1982). The disposition kinetics of ketoprofen (I.V.) in mares (Sams et al.,

1995), ketorolac (P.O.) in dogs (Pasloske et al., 1999) and sodium meclofenamate

(I.M.) in cattle (Picco et al., 2004) have been reported to fit well into three

compartment open model.

Baggot (1977) has stated that overall elimination rate constant () is the most

important pharmacokinetic parameter as it is a part of the equations used to calculate

the elimination half- life, volume of distribution by area method, total body clearance ,

microconstants for multicompartment models and dosage intervals of multiple dose

regimen. Mercer et al. (1977) further indicated that it might be helpful in predicting

the withdrawal periods for drug residues in tissues.

2.6.4 Half –life, Volume of distribution, Clearance and Mean residence time

The rate of drug elimination is determined by the elimination mechanisms.

The half- life of elimination of a drug is defined as the time required for the body to

eliminate one-half of the drug. If the drug obeys first order kinetics, the half- life value

is independent of the dose administered. When drug absorption from the

gastrointestinal tract or an injection site is rapid, the half- life is independent of the

route of administration. When drug obeys zero order kinetics, the half- life becomes

progressively longer as the dose increased (Baggot, 1995). The half- life is inversely

proportional to the overall elimination rate constant.

Another parameter, apparent volume of distribution [Vd(area)] is hypothetical

volume of body fluids that could be required to dissolve the total amount of drug at

the same concentration as that found in blood. This value serves as a proportionality

constant relating the total amount of drug in the body at any time to the pla sma

concentration of a drug after pseudo-distribution equilibrium has been attained. It is

the volume of fluid that would be required to contain the amount of drug in the body

if it were uniformly distributed at a concentration equal to plasma. The calculated

value of apparent volume of distribution [Vd(area)] is not dependent upon the method

used for its calculation, if the drug distributes truly according to one compartment

(Riegelman et al., 1968b) or multicompartment open model (Notari, 1973). The

apparent volume of distribution indicates the extent or magnitude of distribution of

drug without providing any clue whether the drug is uniformly distributed or

restricted to certain body tissues. This kinetic parameter is most helpful for computing

the dosage regimen that must be administered to maintain the desired plasma

concentration (Baggot, 1977). .

Total body clearance (ClB) of a drug is important as it gives the sum of

clearance from each elimination organ, primarily liver and kidneys. It is defined as the

volume of plasma cleared of the drug by various elimination processes per unit of

time (Shargel and Yu, 1993). Unlike and t½ which are dependent upon K12, K21 and

Kel, the blood clearance changes exactly in proportion to Kel (Jusko and Gibaldi, 1972;

Rowland et al., 1973).

After the administration of dose of a drug, a large number of drug molecules

distribute throughout the body. These molecules will stay in the body for various time

periods. Some drug molecules will leave the body almost immediately after entering,

where as other drug molecules will leave the body at later time periods. The term

mean residence time (MRT) describes the average time for all the drug molecules to

reside in the body. MRT may also be considered as the mean transit t ime.

2.6.5 Non-compartmental technique

As discussed earlier, compartmental models were the primary approach to

pharmacokinetics. Only in the last two decades has there been an indication of a

migration in pharmacokinetics to non-compartmental methods. Non-compartmental

models were first developed and applied to radiation decay analysis and remain

dominant in the physical and biological science literature for general applications.

Since their first application to problems in pharmacokinetics by Yamaoka et al. in

1978, non-compartmental methods have grown steadily in use. This relatively new

approach is for the most part based on classical statistical moment theory.

A moment in statistic is simply a mathematical description of a discrete

distribution of data. In pharmacokinetics, a moment is a true estimate of

pharmacokinetic function describing the entire time profile of plasma drug

concentration (Cp) (Yamaoka et al., 1978). It reveals overall properties of time course

of its residence in a body or in a body compartment. The primary task of model-

independent or non-compartment methods is the direct estimation of the moments

form data. Statistical moments analysis simply done as SHAM (slops, height, areas

and moments) analysis to stress that these are the only data requirements for solution

of these models.

Many non-compartmental methods have been propounded for

pharmacokinetic data analysis, especially for estimation of summary parameters like

clearance and volume of distribution. Amongst the popular ones are linear system

analysis, recirculatory models and the statistical moment approach. Linear system

analysis tends to describe the entire time course of plasma drug concentration in body

in a relatively more comprehensive and dynamic manner (Veng-Pedersen, 1991). This

approach may be exploited for prediction or simulation of systemic drug

concentration by convolution, estimation of time course of drug absorption by

deconvolution, and can even be used for establishing in vivo correlations. The

recirculatory models tend to describe drug disposition in terms of repeated cycle

through the body circulatory system (Cutler, 1978). The statistical moment approach

is doubtlessly the most indisputable non-compartmental approach for pharmacokinetic

data analysis. The technique of statistical moments yields simplistic modeling and has

less restrictive, easily verifiable and realistic assumptions (Chan and Gibaldi, 1982).

This approach is applicable to virtually any linear body model and can be used to

detect nonlinearity, if any, in the data.

Non-compartmental methods can be used to determine certain

pharmacokinetic parameters without fitting data in any compartmental model. The

basic calculations are based on the area under the plasma concentration verses time

curve (Zero moment) and the first moment curve (AUMC). Only the zero to second

order moments have been employed in pharmacokinetics, as higher moments are

prone to unacceptable limits of computational error. The area under curve (AUC) and

area under moment curve (AUMC) can be calculated by using the trapezoidal rule

without making any assumption concerning the number of compartments. Non-

compartmental technique has been used to determine pharmacokinetics of ketoprofen

in horse, camel and cat (Landoni and Lees, 1995b; Alkatheeri et al., 1999; Castro et

al., 2000), tiaprofenic acid in horse (Delbeke et al., 1998), ketorolac in sheep (Santos

et al., 2001) and meloxicam in dogs (Montoya et al., 2004).

2.7 Pharmacokinetics of ketoprofen

2.7.1 General Pharmacokinetics

The absorption of ketoprofen was rapid and almost complete on oral

administration while its bioavailability was 71–96% and 73–93% following I.M. and

rectal administration, respectively, in humans (Jamali and Brocks, 1990). Substantial

concentrations of the drug are attained in synovial fluid, the proposed site of action of

NSAIDs in case of rheumatoid arthritis. Ketoprofen binds to plasma albumin,

apparently in a stereoselective manner. Plasma protein binding of ketoprofen was

extensive; the mean plasma protein binding of ketoprofen was 92.8% at 5 g.ml-1 and

91.6% at 10.0 g.ml-1 (Jamali and Brocks, 1990). Generally, volume of distribution is

relatively low; attributable to a high degree of binding to plasma protein. In case of

horse the plasma protein binding was 92% (Landoni and Lees, 1996).

Ketoprofen is completely metabolized in liver and excreted through kidneys.

Metabolism of ketoprofen consists of three stages of biotransformation: (1) inversion

of R(-) to S(+) enantiomers, (2) hydroxylation on the aromatic ring of the benzoic

group and (3) conjugation to an acylglucuronide (Mauleon et al., 1996). It is

eliminated following extensive biotransformation to inactive glucuronide conjugated

metabolite (Jamali and Brocks, 1990). The process of glucuronidation is the major

transformation in all the species studied while the relevance of other two types of

biotransformation varies considerable between species, according to their age and

different physiological situation (Aberg et al., 1995; Igarza et al., 2002). A ketoprofen

metabolite, hydroxyl ketoprofen, resulting from reduction of keto group has been

identified in the horse (Sams et al., 1995).

Marked species differences occur in some pharmacokinetic parameters,

notably terminal half- life and clearance (Landoni and Lees, 1996). Total plasma

clearance was attributed to renal excretion of ketoprofen and metabolism of

ketoprofen to baseline conjugate, which was also excreted in the urine. Renal

clearance of ketoprofen was attributed to renal tubular secretion, as rena l clearance

was grater than filtration clearance (Sams et al., 1995). Urinary recovery of

ketoprofen during first 7 h post administration accounted for 26.4% of the dose as

unconjugated ketoprofen and 29.8% of the dose as a base- labile conjugate of

ketoprofen. Thus, total urinary recovery was 56.2% of the dose (Jamali and Brocks,

1990).

2.7.2 Pharmacokinetics of ketoprofen: In various species

Ketoprofen pharmacokinetics have been reported in following main species:

1. Cattle (Landoni and Lees, 1995a; Landoni et.al., 1995; De Graves et al.,

1996; Igarza et al., 2004).

2. Sheep (Landoni et al., 1999).

3. Goat (Musser et al., 1998; Arifah et al., 2003; Pranvendra et al., 2005;

Pravin et al., 2005).

4. Horse (Jaussaud et al., 1993; Landoni and Lees, 1995b; Owens et al.,

1995a; Sams et al., 1995; Landoni and Lees, 1996; Corveleyn et al., 1996;

Anfossi et al., 1997; De Graves et al., 1998; Wilcke et al., 1998; Verde et

al., 2001).

5. Dog (Schmitt and Guentert, 1990; Montoya et al., 2004).

6. Cat (Lees et al., 2003).

7. Camel (Alkatheeri et al., 1999; Alkatheeri et al., 2000).

8. Japanese quail (Graham et al., 2005).

Important Pharmacokinetic parameters of ketoprofen in various species of

domestic animals are compared in Table 2.

Table 2: Important pharmacokinetic parameters of ketoprofen in various species of domestic animals.

Animal Spp.

Dose (mg.kg

-1)

Route of Administ

-ration

Enanti-omer

Pharmacokinetic Parameters

References t1/2 (h)

Vd (area) (L.kg

-1)

Vd (ss) (L.kg

-1)

ClB (ml.min

-1.kg

-1)

AUC

(g.h.ml-1

)

Cmax

(g.ml-1

) Tmax (h)

MRT (h)

F (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14

Cattle including

calves

1.5 IV

S(+) 2.19

± 0.42

---- 0.26

± 0.04

1.98 ± 0.20 ---- ---- ---- ---- ---- Landoni and Lees,

1995(a) R(-)

1.30 ±

0.27 ----

0.19 ± 0.03

2.51 ± 0.60 ---- ---- ---- ---- ----

3.0

IV

S(+) 0.42

± 0.08

0.22 ±

0.06 ----

5.50 ± 0.67

9.94 ± 1.09 ---- ---- 2.67

± 0.79

----

Landoni et al., 1995

R(-) 0.42

± 0.09

0.20 ±

0.06 ----

5.50 ± 0.50

9.49 ± 0.84 ---- ---- 1.69

± 0.25

----

3.3 IV ---- 0.49 ---- 0.11 2.8 ---- ---- ---- ---- ---- De Graves et al.,

1996

0.5

IV (in gesta-

tion)

S(+) 0.26 ---- 0.28 ---- 0.67 ---- ---- 0.38 ----

Igarza et al., 2004

R(-) 0.31 ---- 0.33 ---- 0.87 ---- ---- 0.45 ----

IV (Early

lactation)

S(+) 0.69 ---- 0.19 ---- 2.78 ---- ---- 0.99 ----

R(-) 0.87 ---- 0.25 ---- 2.93 ---- ---- 1.25 ----

IV (New born)

S(+) 1.71 ---- 0.25 ---- 5.15 ---- ---- 2.47 ----

R(-) 0.52 ---- 0.30 ---- 3.24 ---- ---- 2.02 ----

Table 2: continued.

1 2 3 4 5 6 7 8 9 10 11 12 13 14

Sheep

3 IV

S(+) 0.86

± 0.08

---- 0.26

± 0.02

5.85 ± 0.83

0.36 ± 0.05

----- ---- 0.79

± 0.11

----

Landoni et al., 1999

R(-) 0.87

± 0.10

---- 0.17

± 0.02

3.27 ± 0.53 0.20 ± 0.03 ---- ---- 0.95

± 0.13

----

1.5 IV

S(+) 3.23

± 1.56

---- 0.47

± 0.14

4.77 ± 0.98 0.29 ± 0.06 ---- ---- 1.90

± 0.72

----

R(-) 1.63

± 0.34

---- 0.34

± 0.05

3.87 ± 0.50 0.23 ± 0.03 ---- ---- 1.47

± 0.20

----

Goat

2.2 IV ---- 0.32

± 0.14

---- 0.23

± 0.05

12.33 ± 2.0 3.05 ± 0.052

---- ---- 0.31

± 0.06

---- Musser et al., 1998

3 IV

S(+) 1.79

± 0.24

---- 0.39

± 0.07

5.0 ± 0.05 5.15 ± 0.46 ---- ---- 1.28

± 0.20

----

Arifah et al., 2003

R(-) 0.87

± 0.28

---- 0.29

± 0.05

3.83 ± 0.02 6.81 ± 0.66 ---- ---- 1.36

± 0.29

----

3 IV ---- 0.81 0.67 ---- 9.53 5.31 ---- ---- 0.95 ---- Pravin et al., 2005

3 IV ---- 1.21 0.93 ---- 8.81 5.95 ---- ----- ---- ----

Pranvendra et al., 2005 IM ---- 0.46 0.43 ---- 14.43 3.97 ----- ---- ---- 80.15

Table 2: continued.

1 2 3 4 5 6 7 8 9 10 11 12 13 14

Horse

2.2

P.O. (paste)

S(+) 5.69

± 1.69

---- ---- ---- 0.99

± 0.10

0.28 ±

0.08

0.50 ±

0.07

8.12 ±

2.43

5.75 ±

1.48

Landoni and Lees, 1995

R(-) 5.64

± 0.60

---- ---- ---- 0.85

± 0.02

0.23 ±

0.03

0.50 ±

0.60

8.35 ±

0.88

2.67 ±

0.43

P.O. (powder capsule)

S(+) 3.18

± 0.93

---- ---- ---- 6.75

± 0.81

2.73 ±

0.49

0.92 ±

0.14

4.40 ±

1.28

54.17 ±

9.90

R(-) 3.15

± 0.99

---- ---- ---- 4.13

± 0.58

2.19 ±

0.44

0.83 ±

0.05

3.93 ±

1.20

50.50 ±

10.95

2.2 IV ----

1.02 ±

0.47

0.28 ±

0.14

0.14 ±

0.05

3.08 ± 0.38

12.06 ±

1.64 ---- ----

0.76 ±

0.25 ----

Owens et al., 1995a 0.63

± 0.29

0.20 ±

0.08

0.13 ±

0.03

3.66 ± 0.39

10.10 ±

1.00 ---- ----

0.62 ±

0.13 ----

2.2 IV

(Mare) ----

1.63 0.68 0.16 4.81 7.44 378 6.54 0.54 ---- Sams et al., 1995 1.60 0.64 0.18 4.80 7.63 362 4.64 0.63

1.1 IV

S(+) 1.09 0.64 ---- 6.62 2.74 ---- ---- ---- ---- Landoni and Lees, 1996 R(-) 1.98 0.53 ---- 5.77 2.73 ---- ---- ---- ----

Dog 100 mg

PO ---- ---- ---- ---- ---- ---- --- 0.83

± 0.61

---- 0.90

± 0.10

Schmitt and Guentert, 1990

Table 2: continued.

1 2 3 4 5 6 7 8 9 10 11 12 13 14

Cat

2 IV S(+) 1.52 ----- 0.10 0.88 20.25 ----- ----- 1.73 -----

Lees et al., 2003

R(-) 0.56 ----- 0.14 4.95 4.09 ----- ----- 0.59 -----

1 P.O. S(+) 0.87 ----- ----- ----- 6.36 2.47 0.95 ----- 112

R(-) 0.60 ----- ----- ----- 1.83 1.22 0.57 ----- 85

1 IV S(+) 1.18 ----- 0.22 2.17 10.02 6.31 ----- 1.70 -----

Camel

2 IV --- 4.16 0.32 0.13 1.00 ---- ---- ---- ---- ----- Alkatheeri et al.,

1999 IM 3.28 0.27 ---- 0.99 44.0 12.2 1.5 3.77 121.1

2

IV

(Female)

S(+) 1.83 0.13 0.10 50.6 19.8 ---- ---- 2.08 ----

Alkatheeri et al.,

2000

R(-) 1.88 0.11 0.09 44.6 22.4 ---- ---- 2.13 ----

IV

(Male)

S(+) 2.33 0.22 0.15 69.6 14.4 ---- ---- 2.20 ----

R(-) 2.11 0.21 0.15 62.8 16.0 ---- ---- 2.17 ----

2.7.2.1 Cattle

Landoni and Lees (1995a) studied pharmacokinetics and pharmacodynamics

of S(+) and R(-) ketoprofen (KTP) enantiomers in calves after intravenous

administration of each enantiomer at a dose of 1.5 mg.kg-1. Chiral inversion of R(-) to

the S(+) antipode occurred. The R:S ratio in plasma was 33:1 at 5 min after

administration, decreasing to 1:1 at 8 h. The calculated extent of inversion was 31 ±

7%. The R:S ratio in inflammatory exudate was of the order 3:1 at all the sampling

times and the ratio in transudate was approximately 2:1 at 6 h, declining to 1:1 at 30

h. Only S(+) KTP was detected in biological fluids after administration of this

enantiomer. Elimination half- life was longer for the S(+) (2.19 h) than the R(-)

enantiomer (1.30 h) and volume of distribution was slightly higher for the S(+)

enantiomer. They concluded that effects of R(-) KTP were obtained as a hybrid, since

they potentially reflect the actions of both enantiomers.

Landoni et al. (1995) studied pharmacokinetics and pharmacodynamics of

ketoprofen (KTP) in calves following intravenous administration of the drug racemate

at a dose rate of 3 mg.kg-1. No differences were observed between disposition curves

of KTP enantiomers in plasma, exudates or transudate. This indicates that in calves

KTP pharmacokinetics is not enantioselective. S(+) and R(-) KTP each had a short

elimination half- life (t1/2β) of 0.42 ± 0.08 h and 0.42 ± 0.09 h, respectively. The

volume of distribution were 0.20 ± 0.06 and 0.22 ± 0.06 L.kg-1 for R(-) and S(+) KTP,

respectively. Body clearance (ClB) was high i.e. 0.33 ± 0.03 and 0.32 ± 0.04 L.h-1.kg-1

for R(-) and S(+) KTP, respectively, correlating with the short elimination half- life.

De Graves et al. (1996) determined plasma and milk concentration-time

profiles and pharmacokinetic variables after I.V. administration of ketoprofen to

lactating dairy cows. Apparent volume of distribution at steady state was 0.11 L.kg-1,

elimination half- life was 0.49 h and total clearance was 0.17 L.h-1.kg-1. Ketoprofen

was detected in some milk samples, 10 to 120 minutes after administration, but all

concentrations were below the limit of quantification.

Igarza et al. (2004) studied pharmacokinetic of ketoprofen in three categories

of cattle following intravenous administration at a dose of 0.5 mg.kg-1. Significant

differences between the three categories of animal (i.e. cows in gestation, early

lactation and newborn calves, respectively) were obtained in elimination half- life

(t1/2β) (0.31, 0.87, 1.52 and 0.26, 0.69, 1.71 h), mean residence time (MRT) (0.45,

1.25, 2.20 and 0.38, 0.99, 2.47 h), and area under the plasma concentration-time curve

(AUC) (0.87, 2.93, 3.24, and 0.67, 2.78, 5.13 µg.h.ml-1) for the R(-) and S(+)

enantiomer, respectively.

2.7.2.2 Sheep

Landoni et al. (1999) established pharmacokinetic and pharmacodynamic

parameters for the R(-) and S(+) enantiomers of ketoprofen. Each enantiomers was

administered separately (1.5 mg.kg-1) and in a racemic mixture (3 mg.kg-1)

intravenously to a group of eight sheep in a four-way, four-period cross-over study

using a tissue cage model of inflammation. Plasma disposition of each KTP

enantiomer was similar following separate administration of the pure compounds

compared to administration of the racemic mixture. Following I.V. administration of

racemic ketoprofen, values of distribution rate constant, elimination rate constant,

distribution half life, elimination half life, volume of distribution at steady state, total

body clearance, area under curve and mean resident time were 5.23 h-1, 0.85 h-1, 0.14

h, 0.86 h, 0.27 L.kg-1, 0.351 L.h-1.kg-1, 4.74 µg.h.ml-1 and 0.79 h, respectively for S(+)

KTP and 9.28 h-1, 0.86 h-1, 0.13 h, 0.87 h, 0.17 L.kg-1, 0.196 L.h-1.kg-1, 8.73 µg.h.ml-1

and 0.95 h, respectively for R(−) KTP.

2.7.2.3 Goat

Musser et al. (1998) reported pharmacokinetic parameters and milk

concentrations of ketoprofen in six lactating goats after single intravenous bolus dose

(2.2 mg.kg-1) administration. Blood and milk samples were collected prior to and for

24 h after drug administration. Drug concentrations in serum and milk were

determined by high performance liquid chromatography. Elimination half- life, total

body clearance and volume of distribution at steady state were 0.32 ± 0.14 h, 0.74 ±

0.12 L.h-1.kg-1 and 0.23 ± 0.051 L.kg-1, respectively. Ketoprofen was not detectable by

the employed method in all milk samples.

Arifah et al. (2003) investigated pharmacokinetics and pharmacodynamics of

ketoprofen (KTP) using a tissue cage model of acute inflammation. In this study,

ketoprofen was administered intravenously to goats as the racemate (3.0 mg.kg-1 total

dose) and as the single enantiomers, S(+) KTP and R(−) KTP (1.5 mg.kg-1 of each).

Following I.V. administration of racemic ketoprofen, values of distribution rate

constant, elimination rate constant, distribution half life, elimination half life, volume

of distribution at steady state, total body clearance, area under curve and mean

resident time were 3.9 h-1, 0.45 h-1, 0.18 h, 1.79 h, 0.39 L.kg-1, 0.30 L.h-1.kg-1, 5.15

µg.h.ml-1 and 1.28 h, respectively for S(+) KTP and 3.75 h-1, 0.42 h-1, 0.19 h, 1.87 h,

0.29 L.kg-1, 0.23 L.h-1.kg-1, 6.81 µg.h.ml-1 and 1.86 h, respectively for R(−) KTP. The

pharmacokinetics of both KTP enantiomers was characterized by rapid clearance,

short mean residence time (MRT) and low volume of distribution. The penetration of

R(−) KTP into inflamed (exudate) and noninflamed (transudate) tissue cage fluids

was delayed but area under the curve values were only slightly less than those in

plasma, whereas MRT was much longer. The S(+) enantiomer of KTP penetrated less

readily into exudate and transudate. Unidirectional inversion of R(−) to S(+) KTP

occurred.

Pravin et al. (2005) studied the pharmacokinetics of ketoprofen in five female

goats following intravenous administration. The peak concentrations of 9.87 ± 0.48

and 106.15 ± 7.40 μg.ml-1 were obtained at 0.042 and 0.75 h in plasma and urine,

respectively. The drug was detectable up to 6 h in plasma and 12 h in urine. The

distribution half- life (t1/2α) and elimination half- life (t1/2β) were 0.15 ± 0.02 h and 0.81

± 0.02 h, respectively. The shorter t1/2α denoted the faster distribution of ketoprofen in

goats. Area under plasma concentration time curve (AUC), area under first moment

curve (AUMC), mean resident time and apparent volume of distribution [Vd(area)] were

5.31 ± 0.22 mg.h.L-1, 5.06 ± 0.21 mg.h2.L-1, 0.95 ± 0.02 h, and 0.67 ± 0.04 L.kg-1,

respectively. Higher value of total body clearance (ClB) of 9.53 ± 0.39 ml.min-1.kg-1

denoted faster excretion of ketoprofen in goats.

Pranvendra et al. (2005) studied the pharmacokinetics of ketoprofen following

single dose (3.0 mg.kg-1 body weight) administration through I.V. and I.M. routes in

healthy goats. A two-compartment and one-compartment open model adequately

described plasma concentration time profile of ketoprofen in healthy goats following

single dose I.V. and I.M. administration, respectively. Peak concentration of 18.64

μg.ml-1 was obtained at 0.033 h, following I.V. administration of ketoprofen in goats,

which declined to 0.25 μg.ml-1 at 6 h. The value of distribution half life (t1/2α) and

elimination half life (t1/2β) were, 0.1 h and 1.21 h, respectively, while the values of

volume of distribution (Vd), clearance (Cl) and area under curve (AUC) were 932.03

ml.kg-1, 529.1 ml.h-1.kg-1 and 5.95 μg.h.ml-1, respectively following I.V.

administration. Absorption half- life (t1/2Ka) and elimination half- life (t1/2β) were 0.08 h

and 0.46 h, respectively following I.M. administration. The values of volume of

distribution (Vd), clearance (ClB) and area under curve (AUC) were 430.27 ml.kg-1,

866.75 ml.h-1.kg-1 and 3.97 μg.h.ml-1, respectively following I.M. administration.

Intramuscular bioavailability of ketoprofen was 80.15 % in goats.

2.7.2.4 Horse

Jaussaud et al. (1993) calculated pharmacokinetic parameters of ketoprofen in

horses after single dose administration (2.2 mg.kg.-1). The elimination half life, area

under the curve, clearance and volume of distribution were in range of 21.7-28.7 min.,

221.9-319.9 µg.h.ml-1, 1.5 -2.3 L.min-1.kg-1 and 59.7-96.1 L.kg-1, respectively.

Landoni and Lees (1995b) studied influence of formulation on the

pharmacokinetics and bioavailability of racemic ketoprofen in horses after oral

administration. Two oral formulations were studied, an oil-based paste containing

micronised rac-KTP (racemic form of ketoprofen) and powder from the same source

in hard gelatin capsules, each at a dose rate of 2.2 mg.kg-1. After intravenous

administration of rac-KTP, S(+) enantiomer concentrations exceeded those of the R(-)

enantiomer. For S(+) KTP, elimination half life, total body clearance and volume of

distribution at steady state were 0.99 ± 0.14 h, 0.56 ± 0.09 L.h.kg-1 and 0.61 ± 0.10

L.kg-1, respectively. For R(-) KTP, elimination half life, total body clearance and

volume of distribution at steady state were 0.70 ± 0.13 h, 0.92 ± 0.20 L.h.kg-1 and

0.53 ± 0.11 L.kg-1, respectively. The study demonstrates that rac-KTP had a modest

bioavailability when administered as a micronised powder in hard gelatin capsules to

horses with restricted access to food. When powder from the same source was

administered as an oil-based paste, bioavailability was very poor regardless of the

feeding schedule.

Owens et al. (1995a) studied pharmacokinetics of single intravenous dose of

ketoprofen (2.2 mg.kg-1) in healthy and experimentally acute synovitis induced

horses. The plasma harmonic mean half- life in healthy horses (0.88 h) was longer

than in horses with synovitis (0.55 h). In horses with synovitis, synovial fluid

concentrations approximated plasma concentrations by 1 h. Synovial fluid

concentrations of ketoprofen in horses with synovitis were 6.5 times higher than those

in healthy horses at 1 h. The area under the synovial fluid concentration curve for

horses with synovitis was greater than in healthy horses. These data suggest that the

inflamed joint serves as a site of sequestration for ketoprofen. Furthermore, these

results indicate that plasma pharmacokinetics may be altered by inflammation in

peripheral compartments such as the joint.

Sams et al. (1995) studied pharmacokinetics and urinary excretion of

ketoprofen after multiple intravenous doses to mares. The harmonic mean of the

terminal elimination half- life of ketoprofen after the first and last dose was 98.2 and

78.0 min, respectively. The median values of the total plasma clearance and the renal

clearance after the first dose were 4.81 and 1.93 ml.min-1.kg-1, respectively.

Landoni and Lees (1996) establish pharmacokinetic and pharmacodynamic

parameters for enantiomers of ketoprofen, each administered separately at a dose level

of 1.1 mg.kg-1 to geldings. For both S(+) and R(-) KTP, penetration into tissue cage

fluid (transudate) and inflamed tissue cage fluid (exudate) was rapid, and clearances

from exudate and transudate were much slower than from plasma. AUC values were,

therefore, higher for exudate and, to a lesser degree, transudate than for plasma.

Unidirectional chiral inversion of R(-) to S(+) KTP was demonstrated. Administration

of both enantiomers produced marked, time-dependent inhibition of synthesis of

serum thromboxane B2 and exudate prostaglandin E2, indicating non-selective

inhibition of cyclo-oxygenase (COX) isoenzymes COX-1 and COX-2, respectively.

Corveleyn et al. (1996) administered three different formulations in horses,

each containing 1 g of ketoprofen, in a fatty and hydrophilic suppository base and

liquid suspension per rectal. An average elimination half- life of 1.3 h was calculated.

The average value for the total plasma clearance (ClB), volume of distribution

[Vd(area)] and the mean residence time (MRT) were 131.9 ml.min-1.kg-1, 255 ml.kg-1

and 0.47 h, respectively. After rectal administration, the mean maximal plasma

ketoprofen concentrations were 1.6, 1.1 and 1.6 g.ml-1 for the fatty suppository, the

hydrophilic suppository and the liquid suspension, respectively. The absolute

bioavailability of ketoprofen was relatively low with an inter- individual variability

(24.5-31.3 %).

Anfossi et al. (1997) studied the intramuscular bioavailability of ketoprofen,

administered as the lysine salt in horses treated at the dose rate of 2.2 mg.kg-1. The

absorption rate of ketoprofen administered as the lysine salt was rather low; the mean

residence time increase from 31.7 min. following intravenous administration to 128.9

min. following intramuscular administration and the bioavailability was high (92.4%).

De Graves et al. (1998) determine intravascular and intrasynovial

pharmacokinetics of the R(-) and S(+) enantiomers of ketoprofen after I.V. and I.M.

administration to horses. Mean S(+) to R(-) serum concentration ratios after I.V. and

I.M. administrations were 1.36 ± 0.214 and 1.34 ± 0.245, respectively. Intrasynovial

concentrations of the S(+) and R(-) enantiomer of ketoprofen could be measured for

only the first 3 hours after I.V. administration. Concentrations were less than the limit

of quantification by 4 hours after I.V. administration and at all times after I.M.

administration. Extent of protein binding of the R(-) enantiomer was not significantly

different from extent of protein binding of the S(+) enantiomer, extent of protein

binding did not appear to be concentration dependent. Mean free S(+) to free R(-)

serum concentration ratios, adjusted for protein binding, after I.V. and I.M.

administration were 1.58 and 1.56, respectively.

Wilcke et al. (1998) determine pharmacokinetic variables that describe

disposition of ketoprofen in healthy foals less than 24 hours old. Ketoprofen was

administered I.V. to foals at a dosage of 2.2 mg.kg-1 of body weight. Plasma

concentration versus time profiles were best described, using a two-compartment

open model. Clearance (normalized for body weight) was significantly lower than that

determined for adult horses. Volume of distribution (normalized for body weight) was

larger than that determined for adult horses. Mean (harmonic) plasma half- life for

healthy foals less than 24 hours old was 4.3 hours.

Verde et al. (2001) reported pharmacokinetic of ketoprofen in plasma and

synovial fluid of horse with acute synovitis after I.V. administration at the dose rate of

2.2 mg.kg -1 body weight. Various Pharmacokinetic parameters like distribution half

life, elimination half life, area under curved, volume of distribution at steady state,

body clearance and mean residence time were 0.20 ± 0.03 and 0.13 ± 0.01 h, 1.14 ±

0.18 and 1.87 ± 0.63 h, 5.54 ± 0.98 and 3.13 ± 0.50 mg.h.ml-1, 0.10 ± 0.01 and 0.12 ±

0.01 L.kg-1, 0.23 ± 0.04 and 0.40 ± 0.07 ml.h-1.kg-1, and 0.93 ± 0.13 and 1.40 ± 0.51 h

for S(+) and R(-) enantiomer, respectively.

2.7.2.5 Dog

Schmitt and Guentert (1990) conducted pharmacokinetic of ketoprofen in dogs

following oral administration. The parameters observed were time required to attain

maximum concentration (t max) of 0.83 ± 0.61 h and bioavailability of 0.90 ± 0.10.

Montoya et al. (2004) compared pharmacokinetic of meloxicam and

ketoprofen following oral administration to healthy dogs. Pharmacokinetics of

ketoprofen (KTP) and meloxicam were studied in dogs following a single oral dose of

1 mg.kg-1 and 0.2 mg.kg-1, respectively. There were differences between the

disposition curves of the KTP enantiomers, confirming that the pharmacokinetics of

KTP is enantioselective in dogs. S(+) KTP was the predominant enantiomer. The S:R

ratio in the plasma increased from 2.58 ± 0.38 at 15 min to 5.72 ± 2.35 at 1 h. The

area under the concentration–time curve (AUC) of S(+) KTP was approximately 6

times greater than that of R(–) KTP. The mean pharmacokinetic parameters like Tmax,

Cmax, t1/2β, AUC, Vd/F and Cl/F for ketoprofen and meloxicam were 0.76 ± 0.19 and

8.5 ± 1.91 h, 2.02 ± 0.41 and 0.82 ± 0.29 µg.ml-1, 1.65 ± 0.48 and 12.13 ± 2.15 h, 6.06

± 1.16 and 15.41 ± 1.24 mg.h.ml-1, 0.39 ± 0.07 and 0.23 ± 0.03 L.kg-1 and 170 ± 39

and 10 ± 1.4 ml.h-1.kg-1, respectively. These results indicated a significant

pharmacokinetic difference between meloxicam and ketoprofen after therapeutic

doses in dogs.

2.7.2.6 Cat

Lees et al. (2003) studied pharmacokinetic of racemate ketoprofen

administered intravenously and orally at the dose rate of 2 and 1 mg.kg-1, respectively,

in cats. The absorption of both S(+) and R(-) enantiomers was rapid after oral dosing.

Enantioselective pharmacokinetics was demonstrated by the predominance of S(+)

KTP, as indicated by plasma AUC of 20.25 mg.h.ml-1 for S(+) KTP and 4.09

mg.h.ml-1 for R(-) KTP following I.V. administration and 6.36 mg.h.ml-1 for S(+)

KTP and 1.83 mg.h.ml-1 for R(-) KTP following oral dosing of ketoprofen.

2.7.2.7 Camel

Alkatheeri et al. (1999) studied the pharmacokinetics of ketoprofen following

I.V. and I.M. administration (2.0 mg.kg-1). Following I.V. administration elimination

half- life (t1/2β), volume of distribution at steady state [Vd(ss)], volume of distribution

[Vd(area)] and total body clearance (ClB) were 4.16 h, 130.2 ml.kg-1, 321.5 ml.kg-1 and

1.00 ml.min-1.kg-1, respectively. Following I.M. administration, the drug was rapidly

absorbed with peak serum concentration of 12.2 mg.ml-1 at 1.50 h. The elimination

half- life was 3.28 h.

Alkatheeri et al. (2000) studied pharmacokinetics of racemic ketoprofen

enantiomers in ten female and eight male camels after a single intravenous dose (2.0

mg.kg-1). The areas under the curve to infinity (AUC) (μg.h.ml-1) were 22.4 (13.5-

29.7) and 19.8 (13.8-22.1) for R(-) and S(+) KTP, respectively, in female camels

while the corresponding values in male camels were 16.0 (12.9-22.4) and 14.4 (11.0-

19.3). In both sexes, the AUC for the R(-) enantiomer was significantly larger than

that of the S(+) enantiomer. Total body clearances (ClB) were 44.6 (33.7-74.1) and

50.6 (45.2-72.4) ml.h-1.kg-1 for R(-) and S(+) KTP, respectively, in female camels and

were 62.8 (44.6-77.8) and 69.6 (51.8-91.1) ml.h-1.kg-1 for R(-) and S(+) KTP,

respectively, in male camels. The steady-state volumes of distribution [Vd(ss)] were

97.9 (82.8-147.2) and 102.0 (90.1-169.0) ml.kg-1 for R(-) and S(+) KTP, respectively,

in female camels and were significantly different from each other, while the

respective values in male camels were 151.5 (105.3-222.3) and 154.0 (114.7-229.0)

ml.kg-1 but were not significantly different from each other. The volumes of

distribution (area) [Vd(area)] followed a similar pattern, where the values for R(-) and

S(+) KTP in female camels were 118.5 (95.6-195.2) and 137.6 (115.8-236.2) ml.kg-1,

respectively, and the respective values in male camels were 215.6 (119.1-270.1) and

229.1 (143.3-277.4) ml.kg-1. The elimination half- lives (t1/2β) were 1.88 (1.42-2.34) h

and 1.83 (1.67-2.26) h for R(-) and S(+) KTP, respectively, in female camels and

were significantly different from each other, while the corresponding values in male

camels were 2.11 (1.50-4.20) and 2.33 (1.52-3.83) h for R(-) and S(+) KTP,

respectively, but were not significantly different from each other. The mean residence

time followed a similar pattern. All pharmacokinetic parameters for R(-) and S(+)

KTP in female camels were significantly different from their corresponding values in

male camels.

2.7.2.8 Japanese Quail

Graham et al. (2005) reported pharmacokinetic of ketoprofen in 45 Japanese

quails. The drug was given at 2 mg.kg-1 body weight intravenously (I.V.),

intramuscularly (I.M.) or orally (P.O.) in a three-period crossover design. The

bioavailability of ketoprofen following I.M. and P.O. administrations were 56 and

24%, respectively. The elimination half- life was shortest for the I.M. route (8.7 min)

compared to the I.V. (21.1 min) and P.O. routes (35 min). They proposed that short

half- life of ketoprofen in Japanese quails may be due to their rapid metabolism and

excretion of the drug.

2.8 Therapeutic efficacy:

Ketoprofen is used for musculoskeletal and joint disorders such as ankylosing

spondylitis, osteoarthritis, rheumatoid arthritis and in peri-articular disorders such as

bursitis and tendinitis. It is also used for treating postoperative pain, painful and

inflammatory conditions such as acute gout or soft tissue disorders and to reduce

fever (Dollery, 1991; Sweetman, 2002). It is also indicated for the management of

acute painful shoulder syndrome and juvenile rheumatoid arthritis (Kantor, 1986).

Ketoprofen can also be used in the following instances (Dollery, 1991).

i. In surgical and traumatic situations where analgesic action is required.

ii. In infectious diseases which require analgesic, antiinflammatory and

anti-pyretic effects.

iii. In gynaecological conditions for uterine relaxation and analgesia in

post-partum.

Recommended dosage of ketoprofen in domestic animals (USP monograph, 2004) is

shown in table 3.

Table 3: Recommended dosage of ketoprofen in animals.

Animals Route of Administration Dosages

Cattle Intravenous 3 mg kg -1 every 24 h. for 3 days.

Intramuscular 3 mg kg -1 every 24 h. for 3 days

Dog & Cat

Intravenous 2 mg kg -1 as single dose followed by maintenance dose 1 mg kg -1 24 h.

Intramuscular 2 mg kg -1 as single dose followed by

maintenance dose 1 mg kg -1 24 h.

Horse including

Foals

Intravenous 2.2 mg kg -1 every 24 h.

Intramuscular 2.2 mg kg -1 every 24 h.

Longo et al. (1990) found that Ketoprofen given at 2 mg.kg-1 I.V. daily for 1-5

days, showed good to very good results in 81% of 91 horses with different

musculoskeletal affections.

Shpigel et al. (1994) conducted two separate clinical trials in cows to evaluate

efficacy of ketoprofen for mastitis. All cows were treated with 20 g sulfadiazine and 4

g trimethoprim I.M. and a half dosage daily thereafter. The ketoprofen treatment

groups received 2 g ketoprofen I.M. once daily for the duration of the antimicrobial

therapy. They concluded that ketoprofen significantly improved recovery in gram

negative clinical mastitis in dairy cows.

Glew et al. (1996) studied the antipyretic effectiveness of Ketoprofen in

pyretic cats with a variety of bacterial and viral infections. Cats were randomly

assigned to receive broad-spectrum antibiotic or broad-spectrum antibiotic plus

ketoprofen. Body temperature was monitored 3 times daily and attitude and appetite

were evaluated once daily. The treatment groups were compared with respect to mean

body temperatures, using a one-way analysis of variance. Mean temperatures were

significantly different during the 4 and 8 h post-treatment observations, with a

reduction to normal temperatures in the ketoprofen group compared with no change in

the group treated with antibiotics alone. The antipyretic effect of ketoprofen was rapid

and persisted for at least 8 h, but for less than 24 h. The overall recovery period from

pyrexia, depression, and inappetance was also shorter in cats treated with antibiotics

and ketoprofen (3 days) than in cats treated only with antibiotics (5 days). They

concluded that ketoprofen was a useful adjunct in the treatment of pyretic cats.

Pibarot et al. (1997) compared analgesic effects of ketoprofen, oxymorphone

hydrochloride, and butorphanol when used to control postoperative pain associated

with elective orthopedic surgery. 70 dogs undergoing orthopedic surgery on a hind

limb were randomly assigned to 1 of 4 postoperative analgesic treatment groups like

ketoprofen alone, oxymorphone alone, butorphanol alone and ketoprofen-

oxymorphone combination. Drugs were given I.M. at the end of anesthesia. Pain

score, sedation score, arterial blood pressures, arterial blood gas partial pressures, and

plasma cortisol concentration were measured for 12 h after surgery. They found that

except during the first hour after surgery, dogs given ketoprofen alone after elective

orthopedic surgery had a greater level of, and longer- lasting, analgesia than did dogs

given oxymorphone or butorphanol alone.

Cabre et al. (1998a) examined the analgesic, antiinflammatory and antipyretic

activities of S(+) ketoprofen in rats and mice. Anti-nociceptive action of S(+)

ketoprofen was measured in abdominal pain models. After I.V. administration (0.5

mg.kg-1), S(+) ketoprofen inhibited 92.1 ± 2.2% of writhing in mice but I.V.

administration of the R(-) enantiomer resulted in no statistically significant activity in

a dose range of 0.15-1.0 mg.kg-1. Similar results were obtained after oral

administration in mice. In the rat, S(+) ketoprofen was more potent analgesic than

diclofenac by both intravenous and oral administration. There was no significant

difference between the analgesic effect of S(+) ketoprofen treatment and the twofold

dose of the racemic form in both the mouse and rat models. They evaluated anti-

inflammatory activity of S(+) ketoprofen using a carrageenan- induced paw edema

model in the rat. Intravenous administration of 5 mg.kg -1 of S(+) ketoprofen almost

completely inhibited edema formation. After oral administration, S(+) ketoprofen is

both more potent and effective than diclofenac. S(+) ketoprofen showed a marked

antipyretic action (ED50 = 1.6 mg.kg-1) and was the most potent of the NSAIDs tested.

Thus, S(+) ketoprofen was a potent antiinflammatory, analgesic, and antipyretic

agent in vivo, consistent with its potent anti-COX activity.

Lemke et al. (2000) studied the effect of pre-operative administration of

ketoprofen on post-operative pain in dogs after elective ovariohysterectomy. Sixty

minutes before induction of anesthesia, 11 dogs were given saline solution (control)

and another 11 dogs were given ketoprofen (2 mg.kg-1 I.M.). Thirty minutes before

induction of anesthesia, glycopyrrolate (0.01 mg.kg-1), acepromazine (0.05 mg.kg-1)

and butorphanol (0.2 mg.kg-1) were given intramuscularly. Anesthesia was induced

with thiopental (5–10 mg.kg-1 I.V.) and maintained with isoflurane (1–3%) in oxygen.

They concluded that pre-operative administration of ketoprofen reduces severity of

post-operative pain, and does not alter serum cortisol or glucose.

Faulkner and Weary (2000) investigated behavioral responses after dehorning

and a sham procedure in Holstein calves. All calves received a sedative (xylazine) and

local anaesthetic (lidocaine) before dehorning, and responses were scored over 24 h

after the procedure. The results indicated that ketoprofen mitigates pain after hot- iron

dehorning in young dairy calves.

Earley and Crowe (2002) studied the effects of the ketoprofen alone or with

local anesthesia during castration on cortisol, immune and acute phase responses in

friesian calves. They concluded that surgical castration induced a significant elevation

in cortisol secretion and the rise in cortisol was reduced to control levels by the

administration of ketoprofen but not local anaesthetic. Thus, systemic analgesia using

ketoprofen was more effective than local anesthesia during castration to alleviate the

associated stress response.

Lockwood et al. (2003) studied clinical efficacy of flunixin, carprofen and

ketoprofen as adjuncts to the antibacterial treatment (ceftiofur) of naturally occurring

bovine respiratory disease in beef cattle. They were allocated randomly to four

treatment groups. All the groups received ceftiofur for three days at a dose rate of 1·1

mg.kg-1 by I.M. injection, and three groups received, in addition, a single dose of

either flunixin (2·2 mg.kg-1 by intravenous injection) or ketoprofen (3 mg.kg-1 by

intravenous injection) or carprofen (1·4 mg.kg-1 by subcutaneous injection). During

the first 24 hours of the study, the pyrexia of the three groups treated with a NSAID

was reduced significantly more than the pyrexia of the group treated with ceftiofur

alone, and two and four hours after treatment, the reduction in pyrexia was

significantly greater in the groups treated with flunixin and ketoprofen than in the

group treated with carprofen.

Milligan et al. (2004) experimentally dehorned dairy calves of less than 2

weeks age with (20 calves) or without (20 calves) intramuscular injections of

ketoprofen. All calves received a local anesthetic (lidocaine) prior to dehorning and

were dehorned with heat cauterization. Cortisol concentration was measured via

jugular blood samples taken immediately before dehorning and at 3 and 6 hours

following dehorning. Calf behavior was recorded between 0 and 2, 3 and 5, and 6 and

8 hours following dehorning. There was no significant effect on creep feed

consumption, cortisol concentration, or any of the behavioral measures during the

time periods studied. However, the difference in cortisol concentrations from the time

of dehorning until 3 hours later was significantly lower in the ketoprofen-treated

group. These results suggest that ketoprofen may alleviate short-term pain following

dehorning in dairy calves.

Solankar and Jagtap (2005) investigated chronobiological and

chronopharmacological studies of ketoprofen and its solid dispersion form using

adjuvant arthritis model in rats. Chronobiology of rheumatoid arthritis was studied

using a standard adjuvant arthritis animal model. Chronopharmacology of ketoprofen,

and its solid dispersion forms was also studied. Temporal variations in the degree of

articular inflammation (paw volume) and progression of articular destruction were

studied by injecting Freund's Complete Adjuvant (FCA) at 08:00 and 20:00 hrs.

Temporal variations in antiinflammatory effects and ulcerogenic effect were also

studied by administration of plain ketoprofen (20 mg.kg-1) and its solid dispersion

with hydroxypropyl beta-cyclodextrin (equivalent to 20 mg.kg-1 of ketoprofen) at the

same time points (08:00 and 20:00 hrs) twice weekly for 22 days. Solid dispersion of

ketoprofen was found to be more effective in inhibiting progression of rheumatoid

arthritis. The protective effect of ketoprofen and its solid dispersion was significantly

higher when these were administered at 08:00 hrs. The incidence of ulceration was

more in 20:00 hrs group. Thus, it was observed ketoprofen and its solid dispersion

showed better protection from inflammation in the morning than in the evening.

http://lib.bioinfo.pl/pmid:15691065




2.9 Adverse effects of ketoprofen

Adverse effects including gastrointestinal upset are similar to those of other

NSAIDs. Other side effects, including hepatopathies and renal disease, have been

reported in animals. Due to potential antiplatelet effects, care should be exercised

when using ketoprofen perioperatively (Thompson, 2006).

2.9.1 Gastrointestinal effects:

The most common NSAIDs induced unwanted toxic effect is gastric or

intestinal ulceration that can sometimes be accompanied by anemia from the resultant

blood loss (Gabriel et al., 1991; Figueras et al., 1994). They inhibit gastric acid

secretion and stimulate the production of protective mucous in the stomach and

duodenum. The decrease in prostaglandins caused by NSAIDs may decrease

gastrointestinal cytoprotection, secretion of bicarbonate, and repair of tissue,

eventually leading to vascular compromise in the gastrointestinal mucosa and

subsequent tissue necrosis (Dow et al., 1990).

Collins et al. (1998) tested the effect of ketoprofen, on the upper

gastrointestinal tract (UGIT) in patients with osteoarthritis. The drug was given as

simple ketoprofen and as a slow release preparation. These formulations were

compared with indomethacin for endoscopically proven damage to the UGIT. Both

type of preparations produced similar damage to previously normal UGITs over 56

days; each formulation produced about a 50% incidence of ulceration and

inflammation. Indomethacin, by comparison produced less damage. The results

suggested that the direct action of ketoprofen (barrier breaking effect) adds little to the

mechanism of gastric cytotoxicity of this drug, which may be assumed to be

predominantly caused by a systemic effect of ketoprofen on gastric cytoprotective

mechanisms.

Jerussi et al. (1998) evaluated clinical endoscopy of the gastroduodenal

tolerance to R(-) ketoprofen, R(-) flurbiprofen, racemic ketoprofen, and paracetamol.

Seventy-two healthy male volunteers not receiving NSAIDs, alcohol, or anti-ulcer

drugs, were enrolled in a randomized, investigator-blind, placebo-controlled trial to

evaluate the gastroduodenal tolerance of R(-) ketoprofen 100 mg b.i.d., R(-)

flurbiprofen 100 mg b.i.d., racemic ketoprofen 100 mg b.i.d., and paracetamol 1,000

mg b.i.d. Gastroduodenal endoscopies at baseline and after 25 days of dosing were

used to detect newly occurring hemorrhages and erosions. The incidence of

submucosal hemorrhages was 4/16 in the R(-) ketoprofen group, 5/16 in the R(-)

flurbiprofen group, 12/16 in the racemic ketoprofen group, 1/16 in the paracetamol

group, and 1/8 in the placebo group. The incidence of erosions was 2/16 in the R(-)

ketoprofen group, 4/16 in the R(-) flurbiprofen group, 10/16 in the racemic ketoprofen

group, 0/16 in the paracetamol group, and 2/8 in the placebo group. The differences in

hemorrhages and erosions among treatments were statistically significant. At 100 mg

b.i.d., R(-) ketoprofen caused fewer gastroduodenal hemorrhages and erosions than

racemic ketoprofen. The difference between 100 mg b.i.d. R(-) ketoprofen and 100

mg b.i.d. R(-) flurbiprofen was not statistically significant. The dissociation between

analgesic and antiinflammatory properties for R(-) ketoprofen suggests that it may

represent a unique analgesic with a favorable safety profile.

Cabre et al. (1998b) examined the intestinal ulcerogenic effects of single oral

doses of S(+) ketoprofen compared with racemic ketoprofen in the small intestine and

cecum of rats. The toxicity in the small intestine was measured as the weight ratio

between portions of intestine showing lesions and the total weight of the tissue.

Toxicity in the cecum was evaluated by measuring the s ize of the ulcers. S(+)

ketoprofen had no significant ulcerogenic effect at 10 or 20 mg.kg-1. However,

racemic ketoprofen was clearly ulcerogenic in the small intestine and cecum at the 40

mg.kg-1 dose. R(-) ketoprofen at 20 mg.kg-1 does not show any effect in the cecum

and only limited ulcerogenesis was observed in the small intestine. The latter effect

may be the result of racemic inversion. Therefore, the ulcerogenic act ion of racemic

ketoprofen was interpreted as a synergism between S(+) and R(-) ketoprofen.

Donnelly et al. (2000) studied dose-dependent effects of ketoprofen on the

human gastric mucosa in comparison with ibuprofen in healthy volunteers. Each

subject took, over four separate 10-day dosing periods, ibuprofen 400 mg t.d.s.,

ketoprofen 12.5 mg t.d.s., ketoprofen 25 mg t.d.s. or ketoprofen 50 mg t.d.s. Mucosal

injury was assessed by endoscopy at baseline and on the 3rd and 10th day of each

dosing period. Ketoprofen 50 mg t.d.s. suppressed prostaglandin synthesis to a

significantly greater extent than ibuprofen and caused significantly more

gastroduodenal injury. The profile of prostaglandin synthesis and injury on ketoprofen

12.5 mg t.d.s. most closely resembled that of ibuprofen 400 mg t.d.s.. They concluded

that ketoprofen 12.5 mg t.d.s. was an appropriate dose for self-medication, which was

likely to be similar to ibuprofen 400 mg t.d.s. in its effects on the stomach and

duodenum.

Narita et al. (2005) investigated the adverse effects of long-term

administration of ketoprofen in dogs. Ketoprofen (1 mg.kg-1) was administered to five

clinically healthy beagle dogs and gelatin capsules (control group) were administered

to four clinically healthy beagle dogs for 30 days. The lesions in the stomach,

especially in the pyloric antrum, and fecal occult blood progressively worsened in the

ketoprofen group. However, the differences between the ketoprofen treated group and

the control group were not statistically significant. One dog in the ketoprofen treated

group temporarily exhibited a decrease in renal plasma flow and two dogs exhibited

enzymuria. However, these changes did not persist and the other examinations

showed no significant difference between premedication and postmedication in the

ketoprofen group. Therefore, the adverse effects of long-term administration of

ketoprofen observed in this study were not clinically important in healthy dogs.

2.9.2 Renal system:

Ketoprofen causes abnormal renal function tests, acute renal failure, interstitial

nephritis, nephrotic syndrome. Prostaglandins produce or maintain vasodilation in the

kidneys when necessary.

Animals with compromised hemodynamics become more

vulnerable to ischemia and acute renal damage from the inhibition of prostaglandins.

Compromised animals chronically administered NSAIDs may be more susceptible to

renal papillary necrosis (Johnston and Fox, 1997).

NSAIDs may decrease renal function, resulting in increased BUN, serum

creatinine, and serum electrolyte concentrations and in decreased urine volume and

urine electrolyte concentrations; however, in some cases, water retention may exceed

that of sodium, resulting in dilutional hyponatremia (Klasco, 2003).

2.9.3 Hepatic system:

Ketoprofen causes elevations of transaminase levels and sometime hepatitis.

Lactate dehydrogenase (LDH) and Transaminases values may be increased; liver

function test abnormalities may return to normal despite continued use; however, if

significant abnormalities occur, clinical signs and symptoms consistent with liver

disease develop, or systemic manifestations such as eosinophilia or rash occur

(Klasco, 2003).

Major clinical sign of NSAID toxicity related to liver damage is increase in

level of AST, ALT or ALP and presence or absence of hyperbilirubinemia and

hypoalbuminemia. Alkaline phosphatase and alanine aminotransferase concentrations

were significantly increased compared with baseline with meloxicam (0.2 mg.kg-1)

and ketoprofen (2 mg.kg-1) in dogs (Deneuche et al., 2004).

2.9.4 Hypersensitivity reactions:

Ketoprofen causes dermatological reactions such as rash, pruritis, urticaria,

angioedema, respiratory reactions such as asthmatic attack, bronchospasm and

anaphylactic reactions. Prolonged photosensitivity has been often observed in

ketoprofen- induced photocontact dermatitis long after stopping contact to the

causative agent (Fumikazu et al., 2003).

Gregoricka et al. (1990) studied the effect of the intramuscular administration

of ketoprofen in horses. It was administered intramuscularly to evaluate its potential

to induce injection site reactions at the neck and gluteal regions. Forty horses were

divided into 2 test groups; one given single doses, the other multiple doses. Horses in

both groups were observed again at 4 and 7 days following the last injection to assess

any long-term reactions. They concluded that ketoprofen administered

intramuscularly did not produce injection site heat, muscle soreness, or inappetence.

Pyorala et al. (1999) evaluated tissue irritation after intramuscular injections

of 4 nonsteroidal antiinflammatory agents in 5 lactating cows. Preparations

containing phenylbutazone, flunixin, metamizole and ketoprofen were investigated;

physiological saline was used as a control. Tissue reactions at the injection sites were

examined by palpation and by measuring serum creatine kinase. The metamizole

preparation clearly induced signs of pain in all the cows. After flunixin and

phenylbutazone injections slight reactions were observed, and ketoprofen and saline

did not cause any clinical signs. Some palpation findings after injections were found

for all the preparations except saline. Based on serum creatine kinase, the 2 most

irritating preparations were those containing flunixin and phenylbutazone. After

injections of these 2 substances, the estimated amount of damaged muscle was about

80 g. The statistical difference between flunixin and phenylbutazone and the other 2

preparations was significant. Physiological saline had no effect on serum creatine

kinase.

2.9.5 Haematological and Biochemical Safety Profile:

Jelic-Ivanovic et al. (1985) investigated the in vivo effects of acetylsalicylic

acid (2.7 to 4 g), diclofenac (75-150 mg), indomethacin (75-200 mg), ibuprofen (600-

1600 mg), and ketoprofen (150-200 mg) on the concentrations of various blood

constituents on human patients suffering from rheumatic disorders. Total protein,

glucose, calcium, and inorganic phosphate were not significantly affected by any of

these drugs. Ketoprofen had no definite influence on any constituent.

MacAllister et al. (1993) studied the relative toxicity of phenylbutazone,

flunixin meglumine, and ketoprofen in healthy adult horses. Sixteen horses were

randomly assigned to receive 10 ml of physiologic saline solution, or ketoprofen (2.2

mg.kg-1 of body weight), flunixin meglumine (1.1 mg.kg-1), or phenylbutazone (4.4

mg.kg-1) I.V., every 8 hours, for 12 days. Mean CBC values remained within normal

limits for all groups. Phenylbutazone-treated horses had a significant decrease in

serum total protein and albumin concentrations. Mean values of all other serum

biochemical assays were not different from those of the saline-treated group.

Ketoprofen causes thrombocytopenia, anaemia, agranulocytosis, bone marrow

aplasia and hypocoagulability. Ketoprofen is effective in inhibiting the production of

thromboxane B2, a platelet aggregation promoter (Landoni et al.,1999).

Nazifi et al. (2002) conducted comparative study on the effects of flunixin

meglumine and ketoprofen on haematological and some biochemical parameters of

cattle. Five male cattle were used in the study. Haematocrit decreased and the

percentage of neutrophils and monocytes increased significantly in flunixin

meglumine group compared to the ketoprofen, which showed no significant changes.

In the ketoprofen group, concentration of creatinine increased significantly. In the

flunixin meglumine group, concentrations of albumin, glucose, urea nitrogen, calcium

and activity of aspartate aminotransferase decreased significantly, and concentration

of creatinine and total globulin increased significantly.

Lemke et al. (2002) determined effects of preoperative administration of

ketoprofen on whole blood platelet aggregation, buccal mucosal bleeding time, and

hematologic indices in dogs after elective ovariohysterectomy. Sixty minutes before

induction of anesthesia, 11 dogs were given 0.9% NaCl solution (control), and 11

dogs were given ketoprofen (2 mg.kg-1, I.M.). Blood samples for measurement of

variables were collected at intervals before and after surgery. They found that in dogs

given ketoprofen, platelet aggregation was decreased 95 ± 10% and 80 ± 35%

immediately after surgery and 24 hours after surgery, respectively, compared with

preoperative values. At both times, mean values in dogs given ketoprofen differed

significantly from those in control dogs. Significant differences between groups were

not observed for mucosal bleeding time or haematologic indices.

Bleeding time may be prolonged by most NSAIDs, with ketoprofen, by 3 to 4

minutes above baseline values because of suppressed plate let aggregation. Hematocrit

or haemoglobin values may be decreased, possibly because of gastrointestinal

bleeding or microbleeding and/or hemodilution caused by fluid retention. Leukocyte

count and platelet count may be decreased. (Klasco, 2003).

Luna et al. (2007) evaluated adverse effects of long-term oral administration

of carprofen, etodolac, flunixin meglumine, ketoprofen, and meloxicam in 36 adult

dogs. Values for CBC, urinalysis, serum biochemical urinalyses, and occult blood in

faeces were investigated before and 7, 30, 60, and 90 days afte r daily oral

administration (n = 6 dogs/group) of lactose (1 mg.kg-1, control treatment), etodolac

(15 mg.kg-1), meloxicam (0.1 mg.kg-1), carprofen (4 mg.kg-1), and ketoprofen (2

mg.kg-1for 4 days, followed by 1 mg.kg-1 daily thereafter) or flunixin (1 mg.kg-1 for 3

days, with 4-day intervals). Gastroscopy was performed before and after the end of

treatment. Gastric lesions were detected in all dogs treated with etodolac, ketoprofen,

and flunixin, and one of six treated with carprofen. At 7 days, bleeding time was

significantly longer in dogs treated with meloxicam, ketoprofen, and flunixin,

compared with control dogs. They concluded that carprofen induced the lowest

frequency of gastrointestinal adverse effects, followed by meloxicam.

Materials and Methods

CHAPTER III

MATERIALS AND METHODS

3.1 Experimental animals:

The present study was carried out on six healthy crossbred (Holstein-Friesian

x Kankrej) male calves, weighing between 60 to 122 kilograms, at Instructional Farm,

College of Veterinary Science and Animal Husbandry, Anand Agricultural

University, Anand. The calves were housed in experimental calf pen having concrete

floor, two weeks before start of experiment for acclimatization to the new

environment. During this period they were kept under observation in order to rule out

the possibility of any disease. The study was undertaken during the months of

September-October when the ambient temperature was recorded between 20-35oC.

The animals were maintained on concentrate, green fodder and dry grass. Water was

provided ad libitum. All essential managemental measures were adopted to keep the

calves free from stress.

3.2 Drugs and Chemicals:

Ketoprofen injection (Neoprofen, Vetnex Ranbaxy Fine Chemicals Limited,

New Delhi, India) was procured from local market. Water, Methanol, Acetonitrile and

Diethyl ether of HPLC grade and Ortho-phosphoric acid and Hydrochloric acid

(analytical grade) were purchased from Merck India Ltd., Mumbai. Sodium hydroxide

pellets and di-sodium hydrogen orthophosphate anhydrous (analytical grade) were

purchased from Qualigens fine chemicals, Mumbai. Ketoprofen technical grade

powder was obtained from Ranbaxy Fine Chemicals Limited, New Delhi.

3.3 Plan of Work:

Pharmacokinetic and safety study was undertaken in three different phases with an

interval of at least fifteen days between two successive phases. Pharmacokinetic

study was planned in a cross over design. Six calves were randomly allocated to

receive the drug by either intravenous or intramuscular injection. Entire study was

carried out as per the plan given in Table 4.

Table 4: Experimental schedule to study pharmacokinetics and safety of ketoprofen in

crossbred calves.

Phase I

Pharmacokinetic study of ketoprofen by intravenous route at the

dose rate of 3 mg.kg-1 body weight in three healthy crossbred

calves (C1, C2 and C3) and pharmacokinetic study of ketoprofen

for intramuscular route at the dose rate of 3 mg.kg-1 body weight

in another three healthy crossbred calves (C4, C5 and C6).

Phase II

Pharmacokinetic study of ketoprofen for intravenous route at the

dose rate of 3 mg.kg-1 body weight in three calves (C4, C5 and

C6) and pharmacokinetic study of ketoprofen for intramuscular

route at the dose rate of 3 mg.kg-1 body weight in others calves

(C1, C2 and C3).

Phase III

Safety study of ketoprofen in all six healthy calves (C1, C2, C3,

C4, C5 and C6) administered Ketoprofen intravenously at the

dose rate of 3 mg.kg-1 body weight repeated at 24 hour intervals

for 5 days.

3.4 Pharmacokinetics of ketoprofen following single dose intravenous and intramuscular administration:

Six male calves numbered C1, C2, C3, C4, C5 and C6 were employed to study

the pharmacokinetics of ketoprofen following single dose intravenous and

intramuscular administration at the rate of 3 mg kg-1 of body weight.

3.4.1 Dosage and administration of ketoprofen:

Ketoprofen (100 mg.ml-1) was administered at the dose rate of 3 mg.kg-1

intravenously through left jugular vein, while intramuscular injection was given in the

lateral deeper neck muscles (just anterior to leading edge of scapula through the

trapezius and serratus ventrallis) using a 20G x 25 mm needle.

3.4.2 Collection of blood samples:

Blood samples (approximately 5 ml) were collected from intravenous catheter

(venflon) fixed into the right jugular vein. Blood samples were collected at 0 time

(just before drug administration), and at 0.033 (2 minute), 0.083 (5 minute), 0.166 (10

minute), 0.25 (15 minute), 0.5 (30 minute), 0.75 (45 minute), 1, 2, 4, 8, 12, 18, and 24

h respectively after intravenous administration. Following intramuscular

administration the blood samples were collected at 0 time (before drug

administration), and at 0.083 (5 minute), 0.166 (10 minute), 0.25 (15 minute), 0.5 (30

minute), 0.75 (45 minutes), 1, 2, 4, 8, 12, 18, 24, and 36 h respectively. The blood

samples were drawn into sterile heparinised centrifuge tubes of 10 ml capacity to

separate the plasma. The blood samples were centrifuged (2000 x g for 15 minutes)

and plasma were taken into separate cryo-vials (2 ml) from all samples. Plasma

samples were stored at – 40oC and were analyzed within two weeks.

3.5 High Performance liquid Chromatography (HPLC) assay of ketoprofen

Ketoprofen concentration was analyzed in plasma as per the method developed by

Wanwimolruk et al. (1991), with minor modifications.

3.5.1 Apparatus:

The high performance liquid chromatography (HPLC) apparatus of Lab

Alliance (USA) was used. It comprised of quaternary gradient delivery pump (model

AIS 2000) and UV detector (model 500). Chromatographic separation was performed

by using reverse phase C18 column (Thermo, 5 ODS; 250 4.6 mm ID) at room

temperature. The data integration was performed using „Clarity‟ (Version 2.4.0.190)

software.

3.5.2 Chromatographic conditions:

The analysis of samples of ketoprofen was performed using mobile phase

consisting of a mixture of acetonitrile-methanol-water (25:25:50, v/v) containing 10

mM Na2HPO4, and adjusted to pH equal to 4.15 with pure orthophosphoric acid.

Mobile phase was filtered by filter assembly using 0.45 filters (PALL Life Sciences,

Mumbai, India) and pumped into column at a flow rate of 1.5 ml.min-1 at room

temperature. The effluent was monitored through UV detector operated at 258 nm

wavelength.

3.5.3 Preparation of standard curve:

Initially, stock solution was prepared by dissolving 0.5 mg of pure ketoprofen

powder in 1 ml methanol. Known concentrations of ketoprofen in plasma were

prepared by diluting the stock standard with drug-free calf plasma. The final

ketoprofen concentrations in plasma were 0.01, 0.05, 0.1, 0.25, 0.5, 1, 2.5, 5, 10, 25

and 50 g.ml-1. Scheme for preparation of standards is shown in table 5.

Calibration curve was prepared using the final dilution in plasma by plotting

the area of curve at the ordinate and the drug concentration at abscissa (Figure 3). The

sensitivity of ketoprofen assay was 0.05 g.ml-1. The assay was sensitive,

reproducible and linearity was observed from 0.05 to 50 g.ml-1. The mean

correlation coefficient (R2) was 0.99945. The lower level of detection (LOD) was

0.05 g.ml-1.

3.5.4 Validation of HPLC method:

Plasma sample (n=5) with the final concentration of 0.05, 1 and 50 μg.ml-1 for

ketoprofen were extracted according to the procedure mentioned above. To fulfill the

requirement of partial validation of modified method, intraday and interday absolute

recovery, precision and accuracy were determined. Precision C.V. % (Co-efficient of

variance) and accuracy of ketoprofen in calf plasma are presented in table 6.

The absolute recovery of recemate ketoprofen was measured by comparison

of the areas of ketoprofen after injection of the extracted sample with those obtained

after injection of the standard solution containing equivalent concentrations of the

drug. The mean recovery of ketoprofen from plasma was 81.45 % at 0.05 μg.ml-1 and

86.23 % at 50 μg.ml-1 (n=5).

Various concentrations of ketoprofen were added into blank plasma (n=5).

Intraday and interday (n=5) precision and accuracy were calculated. At all

concentration studied the precision C.V. was less than 10%. These results indicate

good precision and accuracy of the assay. It also indicates that the method is reliable,

reproducible and accurate.

Table 5: Scheme of preparation of ketoprofen standards in plasma.

0.5 mg Ketoprofen powder + 1 ml Methanol Stock solution (500 μg.ml-1)

Drug free plasma + Solution Final solution (concentration)

1800 μl + 200 μl Stock solution Standard A (50 μg.ml-1)

1000 μl + 1000 μl Standard A Standard B (25 μg.ml-1)

1500 μl + 1000 μl Standard B Standard C (10 μg.ml-1)

1000 μl + 1000 μl Standard C Standard D (5 μg.ml-1)

1000 μl + 1000 μl Standard D Standard E (2.5 μg.ml-1)

1500 μl + 1000 μl Standard E Standard F (1.0 μg.ml-1)

1000 μl + 1000 μl Standard F Sandard G (0.5 μg.ml-1)

1000 μl + 1000 μl Standard G Standard H (0.25 μg.ml-1)

1500 μl + 1000 μl Standard H Standard I (0.10 μg.ml-1)

1000 μl + 1000 μl Standard I Standard J (0.05 μg.ml-1)

800 μl + 1000 μl Standard J Standard K (0.01 μg.ml-1)

Table 6: Intraday and Interday precision and accuracy of ketoprofen in calf

plasma by HPLC - UV detection.

Spiked

concentration (μg.ml-1)

Observed

concentration (μg.ml-1)

Mean ± S.D.

Precision C.V. (%) Accuracy (%)

Intraday (n=5)

0.05 0.046 ± 0.004 8.133 92.800

1 0.964 ± 0.068 7.084 96.400

50 47.098 ± 2.241 4.758 94.196

Inter day (n=5)

0.05 0.049 ± 0.004 7.861 98.400

1 0.948 ± 0.059 6.201 94.800

50 47.636 ± 2.003 4.206 95.272

Accuracy % = (Observed concentration / Spiked concentration) X 100.

Precision C.V. % = (Standard deviation / Mean of observed concentration) X 100.

0

500

1000

1500

2000

2500

3000

3500

4000

4500

5000

5500

6000

6500

0 10 20 30 40 50 60

Ketoprofen concentration in plasma (ug/ml)

Are

a o

f P

eak

(m

Vs)

Figure 3: Standard curve of ketoprofen in calf plasma. Each point represents a mean of six replicates.

3.5.5 Sample preparation

Plasma samples (1 ml) were taken in a centrifuge tube and 100 l of 4-N-HCL

was added in each tube. The contents were then shaken with 7 ml of diethyl ether for

10 minutes. The mixture were centrifuged at 4 0C for 10 minutes at 1500 x g to

separate the phases and upper organic layer was transferred to clean centrifuge tube

containing 200 l of 0.1-M-NaOH. The mixture was shaken for 15 minutes and

centrifuged at 1500 x g at 40 C for 10 minutes. Upper organic layer was discarded by

aspiration and 50 l of 0.5-M-H3PO4 was added to the remaining aqueous phase.

Then the aqueous solution was transferred into a chromatographic vial and 20 µl

injected into HPLC column manually using microliter syringe.

3.6 Pharmacokinetic analysis:

Various pharmacokinetic parameters were calculated from plasma

concentration of ketoprofen by software PK solution (version 2.0) after its single dose

intravenous and intramuscular administration given at the rate of 3.0 mg.kg-1 of body

weight in calves. “PK Solutions 2.0” relies on the use of non-compartmental method

of analysis for the estimation of pharmacokinetic parameters.

3.6.1 Formula used by „PK Solutions 2.0‟ Software for single dose

pharmacokinetics.

3.6.1.1 General Disposition Parameters and Constant

A) Dose Amount: D

B) Exponential Summation: Expression for sum of 1 st order kinetic terms.

(for n exponential terms)

C) Y-Intercept: Coefficient of each exponential term: Cn

D) Slope:

E) Rate constant:

F) Elimination rate constant: z

G) Half- life:

3.6.1.2 Descriptive Curve Parameters

A) C initial: Initial concentration extrapolated to time zero for I.V. dose

B) Cmax (obs) = maximum concentration observed

C) Tmax (obs) = time at maximum concentration observed

3.6.1.3 Curve Area Calculation

A) AUC(0-t) (obs area):

Trapezoid calculation of AUC using observed data points only (not extrapolated

to infinity).

Where, n is the number of data points.

B) AUC (area): Total AUC computed by combining

Where, Cn is the last concentration.

3.6.1.4 Statistical Moment Calculations

A) AUMC (area):

Calculation of total area under the first-moment curve by combining trapezoid

calculation of AUMC (0-t) and extrapolated area

B) MRT (area):

Mean residence time is calculated using trapezoid area calculations extrapolated to

infinity.

Where, both area terms use trapezoidal calculations.

C) MAT (Mean Absorption Time) = MRT i.m. – MRT i.v.

3.6.1.5 Volume of Distribution Calculations

A) Vd(area):

Apparent volume of distribution based on trapezoid AUC (area) and elimination

rate. Applies mainly to I.V., but also to intramuscular if complete absorption

(F=1) is assumed.

B) Vd(area) / kg:

Apparent volume of distribution normalized by animal weight. Uses same

equation as Vd (area).

+

C) Vdss (area):

Apparent volume of distribution at steady state estimated graphically from

trapezoidal total area measurements (Applies to I.V. dose).

3.6.1.6 Systemic Clearance Calculations

A) CL (area):

Systemic clearance based on trapezoid AUC (area). Applies mainly to I.V. data

Limited to oral data only if complete absorption (F=1) is assumed.

B) CL (area) / kg:

Systemic clearance normalized by animal weight.

3.6.1.7 Two-compartment Open Model Micro-constants

A) Kel (or K10): Micro-constant calculated using exponentials (Applies to two

compartment I.V. dose data only).

B) K12: Micro-constant calculated using exponentials. (Applies to two compartment

I.V. dose data only).

C) K21: Micro-constant calculated using exponentials. (Applies to two compartment

I.V. dose data only).

3.6.2 Calculation of Bioavailability (F):

F, the fraction of drug absorbed after intramuscular administration (Wagner, 1967):

AUC (I.M.) X t1/2β (I.V.)

F (%) = X 100

AUC (I.V.) X t1/2β (I.M.)

3.7 The safety study of ketoprofen in calves following repeated intravenous administration

Six healthy male crossbred calves (C1, C2, C3, C4, C5 and C6) were employed

to assess safety of ketoprofen following multiple dose intravenous administration at

the rate of 3 mg.kg-1 of body weight repeated at 24 hour intervals for 5 days. The

animals were observed for any clinical abnormalities during the period of experiment.

Safety of repeated intravenous administration of ketoprofen was assessed by studying

the following parameters.

1. Haematology

2. Serum biochemistry

3.7.1 Collection of blood samples for safety study

Blood samples from calves were withdrawn from jugular vein into sterile

heparinized and non-heparinized test tube at 0 hour (before drug administration) and

at 24, 48, 72, 96, and 120 hours for haematological and serum biochemical analysis.

Blood smears for determination of differential leukocyte count (DLC) were prepared

from fresh blood at the time of blood collection. Blood samples (2 ml) collected in

heparinized test tubes were utilized for haematological evaluation and those collected

in non-heparinized test tubes (5 ml) were allowed to clot at room temperature. Serum

was collected and stored at -20ºC for biochemical analysis.

3.7.2 Haematological evaluation

Blood samples collected in heparinised test tubes and blood smear

prepared at predetermined time intervals during course of experiment were used to

study the following parameters.

1. Haemoglobin

2. Packed cell volume

3. Total leukocyte count

4. Differential leukocyte count

3.7.3 Serum biochemical parameters

Serum from blood samples collected at predetermined time intervals was

used to determine following parameters.

1. Serum alkaline phosphatase (AKP)

2. Serum acid phosphatase (ACP)

3. Serum aspartate aminotransferase (AST) / Serum glutamic-oxaloacetic

transaminase (SGOT)

4. Serum alanine aminotransferase (ALT) / Serum glutamic-pyruvic

transaminase (SGPT)

5. Serum lactate dehydrogenase (LDH)

6. Total serum bilirubin

7. Serum creatinine

8. Blood urea nitrogen

9. Total serum protein

10. Serum albumin

11. Blood glucose

All the biochemical parameters were estimated using standard assay kits

(Anamol Laboratories Pvt. Ltd., Palghar, India) with the help of Clinical Chemistry

Analyzer (Junior Selectra, Vital Scientific, Netherland) at Zhaveri Veterinary Clinical

Complex, Veterinary College, AAU, Anand. Haemoglobin estimation and Total

Leukocyte Count were done by Automated Hematology Analyzer (CA 620 VET,

Boule Medical, Sweden) at Department of Medicine, Veterinary College, AAU,

Anand. Packed Cell Volume and Differential Leukocyte Count were manually carried

out at the Department of Pathology, Veterinary College, AAU, Anand. The methods

employed for determination of various haematological and biochemical indices are

presented in Table 7.

3.7.4 Statistical analysis

The data generated from the safety profile study were compared by Least

Square Difference test using SPSS software (version 12.0.1). All the data are

presented as mean ± S.E.

Table 7: Methods used for the determination of haematological and serum biochemical parameters in safety study.

Sr.

No. Parameter Method

Expression of

result (Unit)

Haematology

1 Haemoglobin Colorimetric Photometer System

(Automated hematology analyzer) g/dl

2 Packed Cell Volume Wintrobe‟s tube method Per cent

3 Total Leukocyte Count Electronic impedance principle

(Automated hematology analyzer) Per cmm

4 Differential Leukocyte

Count Whole blood smear counting method Per cent

Blood Biochemistry

5 Serum Alkaline

Phosphatase (AKP) Kinetic Method using p-nitro

phenylphosphate IU/L

6 Serum Acid Phosphatase

(ACP) Kinetic method using -

Napthylphosphate IU/L

7 Serum Aspartate Aminotransferase

(SGOT/ AST) UV kinetic method IU/L

8 Serum Alanine

Aminotransferase (SGPT /ALT)

UV kinetic method IU/L

9 Serum Lactate

Dehydrogenase (LDH) IFCC method IU/L

10 Total Serum Bilirubin Jendrassik & Grof method mg/dl

11 Serum Creatinine Alkaline picrate initial rate method mg/dl

12 Blood Urea Nitrogen

(BUN) Enzymatic UV-kinetic initial rate

method mg/dl

13 Total Serum Protein Biuret Method gm/dl

14 Serum Albumin BCG Method gm/dl

15 Blood glucose GOD/POD Enzymatic Method mg/dl

Results

CHAPTER IV

RESULTS

The present study was conducted to investigate pharmacokinetics of

ketoprofen in crossbred calves following single intravenous and intramuscular

administration at the dose rate of 3 mg.kg-1 of body weight and to evaluate safety of

repeated administration of ketoprofen. The plasma samples were assayed for

ketoprofen concentration using validated High Performance Liquid Chromatography

(HPLC) assay. Calibration curve prepared from plasma s tandards was used to

determine the concentration of ketoprofen in plasma samples obtained from calves for

kinetic study. Representative chromatograms of drug free plasma and ketoprofen

standard are shown in figure 4 and that of plasma samples after intrave nous and

intramuscular administration of drug are shown in figure 5.

4.1 Plasma levels and pharmacokinetics of ketoprofen following

single dose intravenous administration (3.0 mg.kg-1

of body weight) in crossbred calves.

4.1.1 Plasma levels:

The Plasma levels of ketoprofen as a function of time in healthy calves after

its single intravenous administration at the dose rate of 3 mg.kg-1 of body weight are

given in Table 8. The graphical representation of mean plasma values of ketoprofen is

presented in Figure 6. The mean peak plasma ketoprofen level of 31.63 ± 1.71 g.ml-1

was observed at 0.033 h, which rapidly declined to 4.16 ± 0.19 g.ml-1 at 0.5 h.

Thereafter, the drug concentration in plasma diminished gradually and was not

detectable after 8 h.

(A) (B)

Figure 4: Representative chromatograms of drug free plasma (A) and ketoprofen (KTP) standard (25 µg.ml-1) (B).

Time (min) Time (min)

Voltag

e (m

v.)

Voltag

e (m

v.)

10.4

13, K

TP

(A) (B)

Figure 5: Representative chromatograms of ketoprofen (KTP) plasma samples.

(A) Plasma sample collected at 1 h post intravenous administration of ketoprofen at the dose rate of 3 mg.kg-1 body weight in calf No. 4. (B) Plasma sample collected at 1 h post intramuscular administration of ketoprofen at the dose rate of 3 mg.kg-1 body weight in calf No. 1.

10.4

63, K

TP

10.4

77, K

TP

Time (min) Time (min)

Voltag

e (m

v.)

Voltag

e (m

v.)

Table 8: Plasma concentrations of ketoprofen in calves following intravenous administration of ketoprofen at the dose rate of 3 mg.kg-1 body weight.

Time after drug

adminis-tration

(h)

Plasma concentration (µg.ml-1)

Calf Number Mean

S.E. (n = 6)

C1 C2 C3 C4 C5 C6

0.0333 26.20 28.80 36.90 30.10 36.10 31.70 31.63 ± 1.71

0.0833 14.60 15.50 27.20 21.60 23.60 21.40 20.65 ± 1.97

0.1666 10.20 8.30 14.40 8.90 10.70 13.40 10.98 ± 1.00

0.25 7.20 4.88 6.60 4.84 7.00 6.50 6.17 ± 0.43

0.5 4.36 3.56 4.40 3.60 4.46 4.60 4.16 ± 0.19

0.75 2.74 2.58 2.86 3.12 2.80 2.84 2.82 ± 0.07

1 1.62 1.49 1.45 1.94 1.47 1.69 1.61 ± 0.08

2 0.74 0.68 0.72 0.76 0.78 0.85 0.76 ± 0.02

4 0.34 0.31 0.26 0.35 0.33 0.24 0.31 ± 0.02

8 0.095 0.103 0.091 0.094 0.117 0.088 0.098 ± 0.004

12 ND ND ND ND ND ND ND

ND = Not detectable; under level of detection.

31.63

20.65

10.98

6.17

4.16

2.82

1.61

0.76

0.31

0.098

0.0

0.1

1.0

10.0

100.0

0 1 2 3 4 5 6 7 8 9 10

Time (h)

Pla

sma

ket

opro

fen

con

cen

trat

ion

(u

g/m

l)

Figure 6: Semilogarithmic plot of ketoprofen concentration in plasma versus time following single dose intravenous

administration at the dose rate of 3.0 mg.kg-1

of body weight in calf. Each point represents mean ± S.E of six calves.

4.1.2 Pharmacokinetic studies:

Observed plasma levels of ketoprofen were analyzed by non-compartmental

approach. The detailed pharmacokinetic parameters calculated for calves given single

intravenous dose of 3 mg.kg-1 body weight of ketoprofen are presented in Table 9.

The distribution rate constant () varied from 5.41 to 10.71 h-1 with a mean

of 7.50 ± 0.86 h-1. The distribution half- life (t1/2 ) ranged between 0.07 and 0.13 h

with a mean of 0.10 h. The range of elimination rate constant () was 0.40 to 0.50 h-1

with a mean of 0.45 ± 0.01h-1. The elimination half- life (t1/2 ) ranged from 1.39 to

1.73 h with a mean of 1.55 ± 0.05 h. The elimination rate from the central

compartment (Kel) was calculated to be 3.26 ± 0.24 h-1. The average rates of transfer

of drug from central to the tissue compartment (K12) and tissue to the central

compartment (K21) were calculated to be 3.66 ± 0.60 and 1.04 ± 0.09, respectively.

The mean values of apparent volume of distribution [Vd (area)] and volume of

distribution of drug at steady-state [Vd(ss)]were calculated to be 0.64 ± 0.03 and 0.35 ±

0.02 L.kg-1, respectively. The mean value of total body clearance (ClB) was 4.82 ±

0.16 ml.min-1.kg-1 with a range of 4.45 to 5.43 ml.min-1.kg-1. The average values for

area under plasma drug concentration-time curve (AUC) and area under first moment

of curve (AUMC) were 10.42 ± 0.32 g.h.ml-1 and 12.37 ± 0.38 g.h2.ml-1. The

average value of mean residence time (MRT) was 1.20 ± 0.06 h.

Table 9: Pharmacokinetic parameters of ketoprofen in calves following intravenous administration at the dose rate of 3 mg.kg-1 body weight.

Pharmaco-kinetic

Parameter

Unit

Calf Number Mean

S.E. (n = 6)

C1 C2 C3 C4 C5 C6

Cp0 µg.ml-1 28.40 37.30 36.30 27.40 31.40 44.00 34.13 ± 2.57

A µg.ml-1 25.29 34.56 34.00 24.58 29.04 40.69 31.36 ± 2.53

B µg.ml-1 3.15 2.73 2.33 2.83 2.32 3.32 2.78 ± 0.17

α h-1 7.25 10.71 5.74 6.59 5.41 9.31 7.50 ± 0.86

β h-1 0.47 0.45 0.44 0.45 0.40 0.50 0.45 ± 0.01

t ½ α h 0.10 0.07 0.12 0.11 0.13 0.07 0.10 ± 0.01

t ½ β h 1.47 1.56 1.59 1.54 1.73 1.39 1.55 ± 0.05

AUC µg.h.ml-1 9.90 9.20 11.20 10.20 11.10 10.90 10.42 ± 0.32

AUMC µg.h2.ml-1 12.50 12.10 11.50 12.80 13.90 11.40 12.37 ± 0.38

Kel h-1 2.80 3.99 3.23 2.74 2.81 4.00 3.26 ± 0.24

K12 h-1 3.70 5.97 2.18 3.22 2.23 4.65 3.66 ± 0.60

K21 h-1 1.22 1.20 0.78 1.09 0.77 1.17 1.04 ± 0.09

Vd(area) L.kg-1 0.64 0.73 0.61 0.65 0.68 0.55 0.64 ± 0.03

Vd(ss) L.kg-1 0.38 0.43 0.27 0.37 0.34 0.29 0.35 ± 0.02

ClB ml.min-1. kg-1 5.05 5.43 4.45 4.91 4.51 4.57 4.82 ± 0.16

MRT h 1.30 1.30 1.00 1.30 1.30 1.00 1.20 ± 0.06

4.2 Plasma levels and pharmacokinetics of ketoprofen following single dose intramuscular administration (3 mg.kg

-1 of body

weight) in calves.

4.2.1 Plasma levels:

The plasma levels of ketoprofen in calves at different time intervals after its

single dose intramuscular administration at the dose rate of 3 mg.kg-1 of body weight

are presented in Table 10. The graphical representation of mean plasma levels of

ketoprofen is shown in Figure 7. The mean peak plasma concentration (Cmax) of

ketoprofen (6.15 0.24 g.ml-1) was achieved at 0.50 h (Tmax) which declined rapidly

to 3.31 0.08 g.ml-1 at 1 h. Thereafter, the drug concentration in plasma declined

gradually to 2.13 0.14 g.ml-1 at 2 h, 1.37 0.06 g.ml-1 at 4 h, 0.54 0.04 g ml-1

at 8 h, 0.27 0.01 g.ml-1 at 12 h and 0.090 ± 0.005 g.ml-1 at 18 h. ketoprofen

concentration was not detectable in Plasma samples beyond 18 h.

Table 10: Plasma concentrations of ketoprofen in calves following intramuscular administration of ketoprofen at the dose rate of 3 mg.kg-1 body weight.

Time after drug

administration

(h)

Plasma concentration (µg.ml-1)

Calf Number Mean

S.E. (n = 6)

C1 C2 C3 C4 C5 C6

0.0833 3.10 2.64 2.55 2.70 2.36 2.43 2.63 ± 0.11

0.1666 4.50 4.64 3.60 5.40 3.34 4.26 4.29 0.30

0.25 6.40 5.56 4.73 6.45 5.20 4.87 5.54 0.30

0.5 6.96 5.82 5.40 6.68 6.10 5.92 6.15 0.24

0.75 4.74 4.63 4.54 4.44 4.48 4.24 4.51 0.07

1 3.48 3.28 3.24 3.04 3.23 3.56 3.31 0 .08

2 1.94 2.05 2.15 1.86 1.98 2.78 2.13 0.14

4 1.22 1.32 1.57 1.18 1.38 1.52 1.37 0.06

8 0.43 0.48 0.66 0.46 0.59 0.61 0.54 0.04

12 0.29 0.26 0.31 0.28 0.24 0.22 0.27 0.01

18 0.089 0.073 0.08 0.106 0.093 0.097 0.090 0.005

24 ND ND ND ND ND ND ND

ND = Not detectable; under level of detection.

2.63

4.29

5.546.15

4.51

3.31

2.13

1.37

0.54

0.27

0.090

0.0

0.1

1.0

10.0

0 2 4 6 8 10 12 14 16 18 20

Time (h)

Pla

sma

ket

opro

fen

con

cen

trat

ion

(u

g/m

l)

Figure 7: Semilogarithmic plot of ketoprofen concentration in plasma versus time following single dose intramuscular

administration at the dose rate of 3.0 mg.kg-1

of body weight in calf. Each point represents mean ± S.E of six calves.

4.2.2 Pharmacokinetic studies:

Observed plasma levels of ketoprofen were analyzed by non-compartmental

approach. The detailed pharmacokinetic parameters calculated for calves given single

intramuscular dose of 3 mg.kg-1 body weight of ketoprofen are presented in Table 11.

The mean elimination rate constant () was 0.20 ± 0.00 h-1. The

corresponding elimination half- lives were in the range of 3.27 to 3.65 h with an

average of 3.40 ± 0.05 h. The mean area under the plasma concentration-time curve

(AUC), mean area under first moment of curve (AUMC), mean residence time (MRT)

and mean absorption time (MAT) were 17.72 ± 0.39 g.h.ml-1, 74.93 ± 2.02

g.h2.ml-1, 4.22 ± 0.07 h and 3.02 ± 0.10 h, respectively. The volume of distribution

at steady state [Vd(ss)] was 0.72 ± 0.02 L.kg-1. The bioavailability of the ketoprofen

following single dose intramuscular administration ranged from 69.80% to 85.65%

with an average of 77.31 ± 2.23 %. Figure 8 shows the comparative plasma levels of

ketoprofen following single dose intravenous and intramuscular administration at the

dose rate of 3 mg.kg-1 in calves.

Table 11: Pharmacokinetic parameters of ketoprofen in calves following intramuscular administration at the dose rate of 3 mg.kg-1 body weight.

Pharmaco-kinetic

Parameter

Unit

Calf Number Mean

S.E. (n = 6)

C1 C2 C3 C4 C5 C6

β h-1 0.20 0.21 0.21 0.19 0.21 0.21 0.20 ± 0.00

t ½ β h 3.41 3.36 3.36 3.65 3.38 3.27 3.40 ± 0.05

Cmax µg.ml-1 7.00 5.80 5.40 6.70 6.10 5.90 6.15 ± 0.24

Tmax h 0.50 0.50 0.50 0.50 0.50 0.50 0.50 ± 0.00

AUC µg.h.ml-1 17.20 17.00 18.60 16.90 17.40 19.20 17.72 ± 0.39

AUMC µg.h2.ml-1 70.70 68.90 82.00 73.90 75.10 79.00 74.93 ± 2.02

Vd(ss) L.kg-1 0.71 0.71 0.71 0.78 0.74 0.65 0.72 ± 0.02

ClB ml.min-1. kg-1 2.90 2.94 2.69 2.96 2.87 2.61 2.83 ± 0.06

MRT h 4.10 4.00 4.40 4.40 4.30 4.10 4.22 ± 0.07

MAT h 2.80 2.70 3.40 3.10 3.00 3.10 3.02 ± 0.10

F % 75.03 85.65 78.43 69.80 80.35 74.57 77.31 ± 2.23

0.0

0.1

1.0

10.0

100.0

0.0 2.0 4.0 6.0 8.0 10.0 12.0 14.0 16.0 18.0 20.0

Time (hr)

Pla

sma

ket

opro

fen

con

cen

trat

ion

(u

g/m

l)

Intravenous

Intramuscular

Figure 8: Semilogarithmic plot of ketoprofen concentration in plasma versus time following single dose intravenous and

intramuscular administrations at the dose rate of 3.0 mg.kg-1

of body weight in calf. Each point represents mean ±

S.E of six calves.

4.3 Studies on the safety assessment of ketoprofen following multiple dose intravenous administration at the dose rate of 3.0

mg.kg-1

of body weight repeated at 24-hour intervals for five days in crossbred calves.

4.3.1 Effect on haematological parameters

Values of haemoglobin, packed cell volume, total leukocyte count and

differential leukocyte count (neutrophil, lymphocyte, basophil, eosinophil a nd

monocyte) for calves under safety study are presented in tables 12 to 19 and same has

been presented graphically in figures 9 to 12.

The mean values of haemoglobin, packed cell volume, total leukocyte count,

and differential leukocyte count observed in treated animal (24-120 h) do not differ

significantly (P < 0.05) from the corresponding values observed in control (0 h)

animals.

4.3.2 Effect on biochemical parameters

4.3.2.1 Effect on serum enzymes

The values of serum alkaline phosphatase, acid phosphatase, aspartate

aminotransferase, alanine aminotransferase and lactate dehydrogenase of calves under

safety study are presented in tables 20 to 24, respectively and same has been

presented graphically in figures 13 to 17, respectively.

The mean values of serum alkaline phosphatase, acid phosphatase, aspartate

aminotransferase, alanine aminotransferase and lactate dehydrogenase observed at 0 h

and during treatment period (24-120 h) do not differ significantly (P < 0.05) from the

corresponding values observed in control (0 h) animals.

4.3.2.2 Effect on other blood biochemical parameters:

The values of total serum bilirubin, serum creatinine, blood urea nitrogen,

total serum protein, serum albumin and blood glucose of calves under safety study are

presented in tables 25 to 30, respectively and same has been presented graphically in

figures 18 to 23, respectively.

The mean values of total serum bilirubin, serum creatinine, blood urea

nitrogen, total serum protein, serum albumin and blood glucose observed in treatment

animals (24-120 h) did not differ significantly (P < 0.05) from the values of these

blood biochemical parameters observed in control (0 h) animals.

Table 12: Effect of multiple dose intravenous administration of ketoprofen (3 mg.kg-1 of body weight, repeated for 5 days at 24 h intervals) on hemoglobin.

Time of treatment

(hours)

Haemoglobin (g/dl)

Calf Number Mean ± S.E.

(n = 6) C1 C2 C3 C4 C5 C6

0 11.2 10.5 10.7 11.8 11.4 10.1 10.95 ± 0.26

24 11.2 10.8 10.4 11.3 11.2 10.3 10.87 ± 0.18

48 11.3 11.1 10.8 11.6 10.8 10.5 11.02 ± 0.16

72 10.9 10.7 11.2 11.6 11.2 10.3 10.98 ± 0.19

96 10.8 10.6 10.8 12.2 10.7 9.8 10.82 ± 0.32

120 11.1 10.9 10.6 11.9 10.5 10.2 10.87 ± 0.24

Table 13: Effect of multiple dose intravenous administration of ketoprofen (3 mg.kg-1 of body weight, repeated for 5 days at 24 h intervals) on packed cell volume.

Time of treatment (hours)

Packed Cell Volume (%)


(n = 6) C1 C2 C3 C4 C5 C6

0 35 34 34 36 35 34 34.67 ± 0.33

24 34 34 33 35 35 34 34.17 ± 0.31

48 36 35 35 36 34 35 35.17 ± 0.31

72 34 34 35 36 36 35 35.00 ± 0.37

96 34 34 34 37 35 34 34.67 ± 0.49

120 35 35 34 36 35 34 34.83 ± 0.31

0

2

4

6

8

10

12

0 24 48 72 96 120

Treatment hour

Haem

og

lob

in C

on

cen

trati

on

(mg

/dl)

Figure 9: Effect of multiple dose intravenous administration of ketoprofen (3 mg.kg-1 of body weight, repeated for 5 days at 24 h intervals) on hemoglobin.

0

6

12

18

24

30

36

0 24 48 72 96 120

Treatment hour

PC

V (

Perc

en

tag

e)

Figure 10: Effect of multiple dose intravenous administration of ketoprofen (3 mg.kg-1

of body weight, repeated for 5 days at 24 h intervals) on packed cell volume.

Table 14: Effect of multiple dose intravenous administration of ketoprofen (3 mg.kg-1 of body weight, repeated for 5 days at 24 h intervals) on total leukocyte

count.

Time of

treatment (hours)

Total Leukocyte Count (per cmm)


(n = 6) C1 C2 C3 C4 C5 C6

0 7600 8800 9200 7800 8200 8400 8333.33 ± 245.85

24 7700 8700 9100 8200 8500 8400 8433.33 ± 192.64

48 7400 8800 9400 8100 8200 8300 8366.67 ± 276.49

72 7600 9000 9300 8200 8200 8200 8416.67

± 253.53

96 7600 9100 9500 8300 7900 8400 8466.67

± 292.88

120 7700 8900 9500 8300 8000 8500 8483.33

± 263.84

Table 15: Effect of multiple dose intravenous administration of ketoprofen (3 mg.kg-1

of body weight, repeated for 5 days at 24 h intervals) on neutrophil count.


Neutrophil (%)


(n = 6) C1 C2 C3 C4 C5 C6

0 24 21 23 25 24 22 23.17 ± 0.60

24 24 24 22 26 24 20 23.33 ± 0.84

48 21 19 24 23 26 23 22.67 ± 0.99

72 23 21 20 24 24 19 21.83 ± 0.87

96 21 23 21 20 23 21 21.50 ± 0.50

120 23 19 22 22 23 21 21.67 ± 0.61

Table 16: Effect of multiple dose intravenous administration of ketoprofen (3 mg.kg-1 of body weight, repeated for 5 days at 24 h intervals)on lymphocyte count.

Time of treatment

(hours)

Lymphocyte (%)


(n = 6) C1 C2 C3 C4 C5 C6

0 74 74 73 70 69 75 72.50 ± 0.99

24 73 72 76 70 72 75 73.00 ± 0.89

48 76 76 71 73 71 72 73.17 ± 0.95

72 72 76 75 72 72 78 74.17 ± 1.05

96 76 74 73 77 74 76 75.00 ± 0.63

120 73 77 76 75 73 74 74.67 ± 0.67

Table 17: Effect of multiple dose intravenous administration of ketoprofen (3 mg.kg-1 of body weight, repeated for 5 days at 24 h intervals) on basophil count.

Time of

treatment (hours)

Basophil (%)


(n = 6) C1 C2 C3 C4 C5 C6

0 0 1 0 1 2 1 0.83 ± 0.31

24 0 0 1 1 1 0 0.50 ± 0.22

48 1 0 1 0 0 1 0.50 ± 0.22

72 0 1 1 1 0 1 0.67 ± 0.21

96 1 0 2 1 1 0 0.83 ± 0.31

120 0 1 0 0 1 1 0.50 ± 0.22

Table 18: Effect of multiple dose intravenous administration of ketoprofen (3 mg.kg-1 of body weight, repeated for 5 days at 24 h intervals) on eosinophil count.

Time of treatment

(hours)

Eosinophil (%)


(n = 6) C1 C2 C3 C4 C5 C6

0 1 2 3 2 3 1 2.00 ± 0.37

24 2 3 1 1 3 3 2.17 ± 0.40

48 2 4 3 3 2 3 2.83 ± 0.31

72 3 2 3 2 3 1 2.33 ± 0.33

96 1 2 2 0 1 1 1.17 ± 0.31

120 2 2 1 2 2 2 1.83 ± 0.17

Table 19: Effect of multiple dose intravenous administration of ketoprofen (3 mg.kg-1 of body weight, repeated for 5 days at 24 h intervals) on monocyte count.

Time of

treatment (hours)

Monocyte (%)


(n = 6) C1 C2 C3 C4 C5 C6

0 1 2 1 2 2 1 1.50 ± 0.22

24 1 1 0 2 0 2 1.00 ± 0.37

48 0 1 1 1 1 1 0.83 ± 0.17

72 2 0 1 1 1 1 1.00 ± 0.26

96 1 1 2 2 1 2 1.50 ± 0.22

120 2 1 1 1 1 2 1.33 ± 0.21

0

1000

2000

3000

4000

5000

6000

7000

8000

9000

0 24 48 72 96 120

Treatment hour

TL

C (

per

cu

bic

mil

imete

r)


of body weight, repeated for 5 days at 24 h intervals) on total leukocyte count.

0

10

20

30

40

50

60

70

80

0 24 48 72 96 120

Treatment hour

DL

C (

Perc

en

tag

e) Neutrophil

Lymphocyte

Basophil

Eosinophil

Monocyte


of body weight, repeated for 5 days at 24 h intervals) on differential

leukocyte count.

Table 20: Effect of multiple dose intravenous administration of ketoprofen (3 mg.kg-1 of body weight, repeated for 5 days at 24 h intervals) on serum alkaline

phosphatase.

Time of

treatment (hours)

Serum Alkaline Phosphatase (IU/L)


(n = 6) C1 C2 C3 C4 C5 C6

0 282.15 258.64 275.27 280.51 257.87 277.81 272.04 ± 4.46

24 291.49 267.31 276.44 278.45 258.56 280.21 275.41 ± 4.62

48 271.75 271.22 282.16 274.56 263.27 282.38 274.22 ± 2.97

72 278.33 268.25 276.98 277.34 256.78 282.65 273.39 ± 3.83

96 286.13 267.56 283.65 282.55 261.18 284.97 277.67 ± 4.31

120 276.67 259.92 281.46 279.21 259.54 287.23 274.01 ± 4.73

Table 21: Effect of multiple dose intravenous administration of ketoprofen (3 mg.kg-1 of body weight, repeated for 5 days at 24 h intervals) on serum acid phosphatase.


Serum Acid Phosphatase (IU/L)


(n = 6) C1 C2 C3 C4 C5 C6

0 1.07 2.63 2.4 2.97 0.96 3.02 2.18 ± 0.38

24 1.27 2.83 2.54 2.84 1.37 2.94 2.30 ± 0.31

48 1.24 2.72 2.58 2.63 1.24 3.12 2.26 ± 0.33

72 1.41 2.78 2.72 2.42 1.19 2.88 2.23 ± 0.30

96 2.32 2.58 2.81 2.57 1.26 3.11 2.44 ± 0.26

120 2.06 2.71 2.65 2.63 1.35 3.13 2.42 ± 0.26

0

40

80

120

160

200

240

280

320

0 24 48 72 96 120

Treatment hour

AK

P (

IU/L

)


of body weight, repeated for 5 days at 24 h intervals) on serum alkaline phosphatase.

0.0

0.4

0.8

1.2

1.6

2.0

2.4

2.8

0 24 48 72 96 120

Treatment hour

AC

P (

IU/L

)

Figure 14: Effect of multiple dose intravenous administration of ketoprofen (3 mg.kg-1 of body weight, repeated for 5 days at 24 h intervals) on serum acid

phosphatase.

Table 22: Effect of multiple dose intravenous administration of ketoprofen (3 mg.kg-1 of body weight, repeated for 5 days at 24 h intervals) on serum aspartate

aminotransferase (AST/ SGOT).

Time of treatment

(hours)

AST/ SGOT (IU/L)


(n = 6) C1 C2 C3 C4 C5 C6

0 92.24 105.77 84.48 97.32 86.12 83.45 91.56 ± 3.56

24 86.23 102.62 83.67 96.43 87.83 81.78 89.76 ± 3.30

48 84.84 110.06 86.63 93.31 89.75 84.14 91.46 ± 3.97

72 83.59 104.28 85.21 94.62 92.02 85.35 90.85 ± 3.22

96 86.6 106.64 82.54 97.16 94.87 87.56 92.56 ± 3.59

120 84.61 108.37 86.19 95.72 95.55 87.24 92.95 ± 3.65


of body weight, repeated for 5 days at 24 h intervals) on serum alanine

aminotransferase (ALT/ SGPT).

Time of

treatment (hours)

ALT/ SGPT (IU/L)


(n = 6) C1 C2 C3 C4 C5 C6

0 28.56 34.19 31.45 33.78 27.64 28.51 30.69 ± 1.17

24 31.26 34.03 32.6 34.46 29.15 31.43 32.16 ± 0.80

48 29.36 34.15 32.88 32.47 26.57 32.52 31.33 ± 1.15

72 30.39 34.06 35.16 33.53 30.37 31.87 32.56 ± 0.82

96 32.82 36.54 34.91 35.28 29.78 31.08 33.40 ± 1.07

120 33.97 35.17 35.31 36.62 28.18 31.11 33.39 ± 1.29

0

10

20

30

40

50

60

70

80

90

100

0 24 48 72 96 120

Treatment hour

AS

T (

IU/L

)

Figure 15: Effect of multiple dose intravenous administration of ketoprofen (3 mg.kg-1 of body weight, repeated for 5 days at 24 h intervals) on serum aspartate aminotransferase (AST/ SGOT).

0

5

10

15

20

25

30

35

40

0 24 48 72 96 120

Treatment hour

AL

T (

IU/L

)


of body weight, repeated for 5 days at 24 h intervals) on serum alanine aminotransferase (ALT/ SGPT).

Table 24: Effect of multiple dose intravenous administration of ketoprofen (3 mg.kg-1 of body weight, repeated for 5 days at 24 h intervals) on serum lactate

dehydrogenase (LDH).

Time of

treatment (hours)

LDH (IU/L)


(n = 6) C1 C2 C3 C4 C5 C6

0 738.56 747.6 787.16 763.25 745.03 721.43 750.51 ± 9.19

24 758.52 751.16 774.72 757.63 728.33 744.36 752.45 ± 6.35

48 767.35 733.62 785.82 762.18 737.15 748.15 755.71 ± 8.11

72 754.39 744.52 783.34 778.14 708.25 731.37 750.00 ± 11.61

96 747.54 726.13 808.34 781.14 760.74 725.65 758.26 ± 13.23

120 762.52 755.21 817.57 775.42 730.45 736.52 762.95 ± 12.85

Table 25: Effect of multiple dose intravenous administration of ketoprofen (3 mg.kg-1 of body weight, repeated for 5 days at 24 h intervals) on total serum bilirubin.


Total Serum Bilirubin (mg/dl)


(n = 6) C1 C2 C3 C4 C5 C6

0 0.24 0.32 0.27 0.32 0.28 0.32 0.29 ± 0.01

24 0.26 0.26 0.24 0.26 0.23 0.38 0.27 ± 0.02

48 0.23 0.33 0.26 0.26 0.28 0.31 0.28 ± 0.01

72 0.28 0.35 0.26 0.34 0.27 0.36 0.31 ± 0.02

96 0.28 0.3 0.24 0.37 0.24 0.37 0.30 ± 0.02

120 0.25 0.29 0.28 0.33 0.26 0.35 0.29 ± 0.02

0

100

200

300

400

500

600

700

800

0 24 48 72 96 120

Treatment hour

LD

H (

IU/L

)

Figure 17: Effect of multiple dose intravenous administration of ketoprofen (3 mg.kg-1 of body weight, repeated for 5 days at 24 h intervals) on serum lactate dehydrogenase (LDH).

0.00

0.06

0.12

0.18

0.24

0.30

0.36

0 24 48 72 96 120

Treatment hour

To

tal

Bil

iru

bin

(m

g/d

l)


of body weight, repeated for 5 days at 24 h intervals) on total serum

bilirubin.

Table 26: Effect of multiple dose intravenous administration of ketoprofen (3 mg.kg-1 of body weight, repeated for 5 days at 24 h intervals) on serum creatinine.

Time of treatment

(hours)

Serum Creatinine (mg/dl)


(n = 6) C1 C2 C3 C4 C5 C6

0 1.27 1.13 1.19 1.24 1.14 1.21 1.20 ± 0.02

24 1.25 1.17 1.18 1.19 1.15 1.18 1.19 ± 0.01

48 1.24 1.15 1.16 1.2 1.15 1.23 1.19 ± 0.02

72 1.22 1.21 1.2 1.18 1.19 1.19 1.20 ± 0.01

96 1.19 1.18 1.15 1.17 1.14 1.28 1.19 ± 0.02

120 1.23 1.16 1.2 1.18 1.19 1.23 1.20 ± 0.01



Time of

treatment (hours)

BUN (mg/dl)


(n = 6) C1 C2 C3 C4 C5 C6

0 16.73 17.46 16.49 18.19 16.54 17.65 17.18 ± 0.28

24 16.87 17.21 18.61 17.64 15.32 17.68 17.22 ± 0.45

48 16.26 17.74 17.88 18.35 16.81 16.83 17.31 ± 0.33

72 16.77 17.39 18.03 17.55 16.38 17.43 17.26 ± 0.24

96 17.29 17.87 18.32 18.08 17.76 18.38 17.95 ± 0.17

120 17.22 17.11 18.17 18.16 17.54 19.12 17.89 ± 0.31

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

0 24 48 72 96 120

Treatment hour

Cre

ati

nin

e (

mg

/dl)

Figure 19: Effect of multiple dose intravenous administration of ketoprofen (3 mg.kg-1 of body weight, repeated for 5 days at 24 h intervals) on serum creatinine.

0

3

6

9

12

15

18

0 24 48 72 96 120

Treatment hour

BU

N (

mg

/dl)



Table 28: Effect of multiple dose intravenous administration of ketoprofen (3 mg.kg-1 of body weight, repeated for 5 days at 24 h intervals) on total serum protein.

Time of treatment

(hours)

Total Serum Protein (g/dl)


(n = 6) C1 C2 C3 C4 C5 C6

0 6.8 6.8 6.8 6.3 6.5 6.9 6.68 ± 0.09

24 6.8 6.8 6.8 6.2 6.6 7.1 6.72 ± 0.12

48 6.6 6.9 6.7 6.3 6.6 7.1 6.70 ± 0.11

72 7.0 6.9 6.7 6.3 6.5 7.1 6.75 ± 0.13

96 6.8 6.9 6.7 6.2 6.6 7.2 6.73 ± 0.14

120 6.7 6.9 6.8 6.4 6.6 7.0 6.73 ± 0.09

Table 29: Effect of multiple dose intravenous administration of ketoprofen (3 mg.kg-1 of body weight, repeated for 5 days at 24 h intervals) on serum albumin.

Time of

treatment (hours)

Serum Albumin (g/dl)


(n = 6) C1 C2 C3 C4 C5 C6

0 3.2 3.1 3.1 2.8 2.9 3.3 3.07 ± 0.08

24 3.1 3.2 3.0 2.7 3.0 3.3 3.05 ± 0.08

48 3.0 3.1 2.9 2.7 3.2 3.2 3.02 ± 0.08

72 3.1 3.3 3.1 2.7 3.1 3.4 3.12 ± 0.10

96 3.1 3.1 3.1 2.7 3.1 3.5 3.10 ± 0.10

120 3.2 3.0 3.2 2.8 3.1 3.4 3.12 ± 0.08

0

1

2

3

4

5

6

7

0 24 48 72 96 120

Treatment hour

To

tal

Pro

tein

(g

/dl)

Figure 21: Effect of multiple dose intravenous administration of ketoprofen (3 mg.kg-1 of body weight, repeated for 5 days at 24 h intervals) on total serum protein.

0.0

0.4

0.8

1.2

1.6

2.0

2.4

2.8

3.2

3.6

0 24 48 72 96 120

Treatment hour

Alb

um

in (

gm

/dl)


of body weight, repeated for 5 days at 24 h intervals) on serum albumin.

Table 30: Effect of multiple dose intravenous administration of ketoprofen (3 mg.kg-1 of body weight, repeated for 5 days at 24 h intervals) on blood glucose.

Time of treatment

(hours)

Blood Glucose (mg/dl)


(n = 6) C1 C2 C3 C4 C5 C6

0 62.34 67.65 60.98 64.97 63.88 65.55 64.23 ± 0.97

24 64.49 64.46 63.59 65.15 57.06 66.36 63.52 ± 1.35

48 58.46 69.51 64.64 63.65 62.48 67.14 64.31 ± 1.56

72 67.76 64.68 65.05 59.98 59.24 60.42 62.86 ± 1.41

96 63.81 62.78 66.48 63.47 63.64 68.37 64.76 ± 0.89

120 64.19 66.03 65.78 65.34 62.46 68.58 65.40 ± 0.83

0

10

20

30

40

50

60

70

0 24 48 72 96 120

Treatment hour

Glu

co

se (

mg

/dl)

Figure 23: Effect of multiple dose intravenous administration of ketoprofen (3 mg.kg-1 of body weight, repeated for 5 days at 24 h intervals) on blood glucose.

Discussion

CHAPTER V

DISCUSSION

Pharmacokinetic studies provide highly relevant information on the time

course of drugs and their concentration in the body and are mainly related to the

absorption, distribution, metabolism and elimination of therapeutic agent. It also

studies the relationship of these processes with regards to the intensity and duration of

characteristic effects of drugs. Thus, pharmacokinetic data of a drug provides an

important tool to achieve its effective dosage regimen.

Non-Steroidal Anti Inflammatory Drugs (NSAIDs) have been commonly used

to reduce pain and inflammation in different inflammatory and painful conditions. The

therapeutically effective concentration of NSAIDs are expressed as median effective

concentration (EC50) which must be achieved and maintained at the site of action in

animal body. Thus, a proper dosage regimen should be anticipated on the basis of

kinetic disposition of the drug in the animal body.

Ketoprofen is a promising and safer NSAID for treating various

musculoskeletal disorders including arthritis, other inflammatory and painful

conditions in animals including cattle. Considering this, it is prudent to investigate its

pharmacokinetics in domestic animal species like cattle in local environment, in

which it is to be employed clinically in Veterinary medicine. Therefore, present study

was conducted to determine pharmacokinetic parameters of ketoprofen in calves

following single dose intravenous and intramuscular administration at the dose rate of

3 mg.kg-1. Safety of repeated intravenous ketoprofen administration (3 mg.kg-1) for

five days was also evaluated.

5.1 Plasma levels and pharmacokinetics of single dose intravenous and intramuscular administration of ketoprofen in calves

5.1.1 Plasma levels

For a successful antiinflammatory therapy, the plasma concentration of an

antiinflammatory agent should not fall below the median effective concentration

(EC50) for COX-2 inhibition during the course of treatment. It is, therefore,

mandatory to measure the plasma drug concentrations at various time intervals after

the administration of drug. The plasma drug concentrations so determined are utilized

to compute various pharmacokinetic parameters, which are ultimately employed for

the determination of proper dosage regimen of a drug in a particular species for

suitable treatment.

In the present study, ketoprofen was given as a single dose at the rate of 3

mg.kg-1 via I.V. and I.M. routes in crossbred calves. Pharmacokinetic studies of

ketoprofen have been conducted following intravenous administration at the dose rate

of 1.5 mg.kg-1 (Landoni and Lees, 1995a), 3 mg.kg-1 (Landoni et al., 1995), 3.3

mg.kg-1 (De Graves et al., 1996) and 0.5 mg.kg-1 (Igarza et al., 2004) in cattle

including purebred calves, 3 mg.kg-1 (Landoni et al., 1999) in sheep, 2.2 mg.kg-1

(Musser et al., 1998) and 3 mg.kg-1 (Arifah et al., 2003; Pranvendra et al., 2005;

Pravin et al., 2005) in goats, 2.2 mg.kg-1 (Owens et al., 1995a; Sams et al., 1995;

Landoni and Lees, 1996) in horses, 2 mg.kg-1 (Lees et al., 2003) in cat and 2 mg.kg-1

(Alkatheeri et al., 1999; Alkatheeri et al., 2000) in camel. Pharmacokinetic studies of

ketoprofen have been carried out following intramuscular administration at a dose rate

of 3 mg.kg-1 (Pranvendra et al., 2005) in goat and 2 mg.kg-1 (Alkatheeri et al., 2000)

in camel. But, pharmacokinetic data following intramuscular administration of

ketoprofen in calves have not been reported.

In present study, following single dose I.V. administration of ketoprofen at the

dose rate 3 mg.kg-1 of body weight, peak plasma ketoprofen level of 31.63 ± 1.71

µg.ml-1 was obtained at 0.033 h and minimal level of 0.098 ± 0.004 µg.ml-1 was

obtained at 8 h. Ketoprofen concentration in plasma was not detectable after 8 h

following intravenous administration. Peak plasma ketoprofen level of 44 µg.ml-1 was

obtained in calves by Landoni et al. (1995) where as Pranvendra et al. (2005) reported

peak concentration of 18.64 μg.ml-1 obtained at 0.033 h, following I.V. administration

of ketoprofen in goats, which declined to 0.25 μg.ml-1 at 6 h. Pravin et al. (2005)

reported peak plasma concentrations of ketoprofen 9.87 ± 0.48 obtained at 0.042 h.

The drug was detectable up to 6 h in plasma.

Following single dose intramuscular administration in present study, the peak

(Cmax) and minimal detectable levels of 6.15 ± 0.24 and 0.090 ± 0.005 µg.ml-1 of

ketoprofen in calves were found at 0.5 (Tmax) and 18 h, respectively. Ketoprofen

concentration was not detectable in plasma samples beyond 18 h. Peak serum

concentration (12.2 g.ml-1) of ketoprofen at 1.5 h following intramuscular

administration at 2.0 mg.kg-1 dose rate was reported in camel by Alkatheeri et al.

(2005). It is evident from results that the maximum concentration (Cmax) of ketoprofen

in calves is lower than that of camel, however it was achieved faster in calves than

camels. The therapeutically effective concentration of ketoprofen for inhibition of

exudates PGE2 (EC50 for COX-2 inhibition), in calves with the value of 0.086 μg.ml-1

(Landoni et al., 1995), was maintained for up to 18 h. Thus, ketoprofen can be

administered intramuscularly for the effective treatment of arthritis, postoperative

analgesia and as antipyretic in calves.

5.1.2 Pharmacokinetics

Plasma drug concentrations measured at various time intervals in calves

following intravenous and intramuscular administration of racemic ketoprofen (rac-

KTP) in present study were employed for the calculation of absorption and

elimination half lives, apparent volume of distribution, volume of distribution at

steady state, total body clearance, mean residence time and bioavailability. The semi-

logarithmic plot of the plasma drug concentration as a function of time following

single dose intravenous and intramuscular administration of ketoprofen was analyzed

by non-compartmental technique. It was also used by Landoni and Lees (1995b),

Alkatheeri et al. (1999) and Castro et al. (2000) to describe the pharmacokinetic of

ketoprofen in horse, camel and cat, respectively.

Although I.V. is not the most likely route of administration of ketoprofen in

calves, I.V. pharmacokinetic study was performed to establish pharmacokinetic

variables, such as Vdarea, Vdss and ClB. The values of area under the plasma

concentration-time curves obtained after I.V. injection were employed for

determination of bioavailability of ketoprofen following I.M. administration.

The high value of distribution rate constant (α: 7.50 ± 0.86 h-1) and low value

of elimination rate constant (β: 0.45 ± 0.01 h-1) observed in the present study

following single dose intravenous administration of ketoprofen (KTP) indicate that

the drug is rapidly distributed and then relatively slowly eliminated. Similar values of

distribution rate constant for S(+) and R(-) KTP (6.40 and 4.97 h-1, respectively) and

elimination rate constant for S(+) and R(-) KTP (0.882 and 0.592 h-1, respectively)

were reported in horse (Landoni & Lees, 1996). Similar values of distribution rate

constant for S(+) and R(-) KTP (5.226 ± 0.488 and 9.227 ± 3.912 h-1, respectively)

and elimination rate constant for S(+) and R(-) KTP (0.848 ± 0.090 and 0.858 ± 0.111

h-1, respectively) were also reported in sheep (Landoni et al., 1999). However, in

calves faster distribution (33.72 ± 13.54 and 56.57 ± 18.45 h-1, respectively) and

elimination (2.13 ± 0.36 and 2.07 ± 0.34 h-1, respectively) for S(+) and R(-) KTP

were also reported (Landoni et al., 1995). In goats, lower value of distribution rate

constant for S(+) and R(-) KTP (3.90 ± 0.23 and 3.75 ± 0.30 h-1, respectively) but

similar elimination rate constant for S(+) and R(-) KTP (0.45 ± 0.11 and 0.42 ± 0.06

h-1, respectively) was observed (Arifah et al., 2003).

These values indicate that the rate of distribution and rate of elimination of

ketoprofen in calf is identical with horse and sheep and almost same as in goat.

However, differences in these parameters with that reported in calves (Landoni et al.,

1995) may be due to differences in environment, age, breed or pharmacokinetic data

analysis method. A previous report also described the differences in pharmacokinetic

behaviour of ketoprofen in cattle at different age and in different physiological

situations (Igarza et al., 2004).

The distribution half life (t1/2α) of ketoprofen following single dose

intravenous administration in the present study was 0.10 ± 0.01 h which is in close

agreement with distribution half life for S(+) and R(-) KTP (0.10 and 0.09 h,

respectively) reported in horse (Landoni and Lees, 1996), for S(+) and R(-) KTP (both

0.13) reported in sheep (Landoni et al., 1999), for rac-KTP (0.08 ± 0.05) reported in

goat (Musser et al., 1998), for rac-KTP (0.11 ± 0.04 h) in normal horses and (0.09 ±

0.06 h) in horses with acute synovitis (Owens et al., 1995a), and for S(+) and R(-)

KTP (0.18 ± 0.01 and 0.19 ± 0.01 h, respectively) reported in healthy goats (Arifah et

al., 2003). However, distribution half- life of ketoprofen following intravenous

administration was higher for S(+) and R(-) KTP (0.20 ± 0.03 and 0.13 ± 0.01 h,

respectively) reported in horses with acute synovitis in another study (Verde et al.,

2001).

The elimination half life (t1/2 β) of ketoprofen in present study, following single

dose intravenous administration in calves was calculated to be 1.55 ± 0.05 h. This

value is near to the elimination half life for S(+) and R(-) KTP (2.19 ± 0.42 and 1.30 ±

0.27 h, respectively) in calves following intravenous administration (Landoni & Lees,

1995a), for rac-KTP (1.63 h) in mare (Sams et al., 1995), for S(+) and R(-) KTP in

female (1.83 and 1.88 h, respectively) and male (2.33 and 2.11 h, respectively) camel

(Alkatheeri et al., 2000) and for rac-KTP (1.21 h) in goat (Pranvendra et al., 2005).

While shorter elimination half life of 0.42 ± 0.08 and 0.42 ± 0.09 h, respectively for

S(+) and R(-) enantiomers in calves (Landoni et al., 1995), 0.49 h for rac-KTP in

cattle (De Graves et al., 1996), 1.02 ± 0.47 and 0.63 ± 0.29 h, respectively for S(+)

and R(-) enantiomers in horse (Owens et al., 1995a), 0.86 ± 0.08 and 0.87 ± 0.10 h,

respectively for S(+) and R(-) enantiomers in sheep (Landoni et al., 1999) and 0.81 ±

0.02 h for rac-KTP in goat (Pravin et al., 2005) were reported. However, longer

elimination half life for S(+) and R(-) KTP (3.23 ± 1.56 and 1.63 ± 0.34 h,

respectively) in sheep (Landoni et al., 1999) and 4.19 h for rac-KTP in camel

(Alkatheeri et al., 1999) were also reported. Such variations in elimination half- life

might be attributable to interspecies and inter- individual variation.

The elimination half life of ketoprofen following intramuscular administration

in the present study was 3.40 ± 0.05 h. This value closely resembles to the elimination

half life of 3.28 h reported in camel (Alkatheeri et al., 1999) but higher than the value

of 0.46 h as reported in goat (Pranvendra et al., 2005). The longer elimination half- life

of ketoprofen following intramuscular administration observed in the present study

indicates that the drug being continuously absorbed during the elimination phase also.

The elimination half- life observed in the present study following intramuscular

administration is longer than intravenous administration of drugs. Longer half- life of

ketoprofen following intramuscular injection is advantageous for its use in cattle, as it

will require less frequent dosing.

To know the extent of penetration of drug in body tissue, knowledge of

volumes of distribution is necessary. The mean apparent volume of distribution

[Vd(area)] following single dose intravenous administration of ketoprofen (3 mg.kg-1) in

calves was 0.64 ± 0.03 L.kg-1. In goats, Similar value of Vd(area) of 0.67 ± 0.04 L.kg-1

(Pravin et al., 2005) and comparatively higher value of Vd(area) of 0.93 L.kg-1

(Pranvendra et al., 2005) were reported after intravenous administration of ketoprofen

at the dose rate of 3 mg.kg-1. Lower Vd(area) values of 0.28 ± 0.14 L.kg-1 in healthy

horses and 0.20 ± 0.08 L.kg-1 in horse with synovitis after intravenous administration

of ketoprofen at the dose rate of 2.2 mg.kg-1 is reported by Owens et al. (1995a).

Landoni et al. (1995) also reported lower value of Vd(area) for S(+) and R(-) KTP (0.22

± 0.06 and 0.20 ± 0.06 L.kg-1, respectively) in calves at the dose rate of 3 mg.kg-1.

The volume of distribution at steady state [Vd(ss)] following single dose

intravenous administration of ketoprofen (3 mg.kg-1) in calves was 0.35 ± 0.02 L.kg-1.

Igarza et al. (2004) reported similar value of Vd(ss) for S (+) KTP (0.28, 0.19 and 0.25

L.kg-1) and R(-) KTP (0.33, 0.25 and 0.30 L.kg-1) in cows with gestation, early

lactation and new born cattle, respectively. Similar values of Vd(ss) for S(+) (0.39 ±

0.07 L.kg-1) and R(-) KTP (0.29 ± 0.05 L.kg-1) were also observed in goat (Arifah et

al., 2003). However, lower Vd(ss) for rac-KTP were reported in healthy horses (0.14 ±

0.05 L.kg-1) and horses with induce synovitis (0.13 ± 0.03 L.kg-1) (Owens et al.,

1995a), for rac-KTP (0.23, 0.11 and 0.13 L.kg-1) in lactating goat, cattle and camel,

respectively (Musser et al., 1998; De Graves et al., 1996; Alkatheeri et al., 1999), for

S(+) KTP (0.26, 0.26 and 0.10 L.kg-1) and R(-) KTP (0.19, 0.17 and 0.14 L.kg-1) in

cattle, sheep and cat, respectively (Landoni and Lees, 1995a ; Landoni et al., 1999 ;

Lees et al., 2003) and for S(+) and R(-) KTP in female (0.10 and 0.09 L.kg-1,

respectively) and male (0.15 L.kg-1 for both enantiomer) camel (Alkatheeri et al.,

2000). The moderate volume of distribution following intravenous administration of

ketoprofen in cow calves in the present study is expected as, similar to most NSAIDs,

ketoprofen is also highly bound to plasma proteins.

The mean volume of distribution at steady state [Vd(ss)] after single

intramuscular administration of ketoprofen in calves at dose rate of 3 mg.kg-1 was

0.72 ± 0.02 L.kg-1. Distribution of ketoprofen following intramuscular administration

was larger in calves as compared to intravenous administration; favouring the

intramuscular use of the drug in calves to treat diseases associated with pain and

inflammation.

The area under plasma concentration time curve (AUC) is an important

parameter used to calculate clearance, volume of distribution and bioavailability of

drugs in pharmacokinetic studies. The area under curve following intravenous

administration in present study was calculated to be 10.42 ± 0.32 g.h.ml-1. Similar

AUC for S(+) KTP (9.94 ± 1.09 g.h.ml-1) and R(-) KTP (9.49 ± 0.84 g.h.ml-1) was

reported in calves by Landoni et al. (1995). Owens et al. (1995a) also reported similar

AUC for rac-KTP in healthy horses (12.06 ± 1.64 g.h.ml-1) and horses with synovitis

(10.10 ± 1.00 g.h.ml-1). However, lower AUC were reported for S(+) and R(-)

KTP (2.74 and 2.73 g.h.ml-1, respectively) in horses (Landoni and Lees, 1996), for

rac-KTP (3.05 ± 0.05 g.h.ml-1) in goat (Musser et al., 1998), for S(+) and R(-) KTP

(0.36 ± 0.05 and 0.20 ± 0.03 g.h.ml-1, respectively) in sheep (Landoni et al.,1999)

and for S(+)and R(-) KTP (5.15 ± 0.46 and 6.81 ± 0.66 g.h.ml-1, respectively) in

goat (Arifah et al., 2003). Following intramuscular administration of drug, relatively

higher value of area under curve (AUC) of 17.72 ± 0.39 g.h.ml-1 was reported in

present study, which is higher than the value of 3.97 g.h.ml-1 reported in goats by

Pranvendra et al. (2005). Alkatheeri et al. (1999) reported much higher value of AUC

up to 44 g.h.ml-1 in camel after intramuscular administration (2 mg.kg-1). The

difference in AUC may be due to interspecies variation or differences in dose or

different intramuscular bioavailability.

The total body clearance (ClB) is an important pharmacokinetic parameter that

gives sum of the clearance by each elimination organs. The total body clearance of

ketoprofen in present study following intravenous and intramuscular administration of

drug was 4.82 ± 0.16 and 2.83 ± 0.06 ml.min-1.kg-1, respectively. The value of total

body clearance following intravenous administration observed in the present study is

in agreement with the reported values of 5.50 ± 0.67 and 5.50 ± 0.50 ml.min-1.kg-1,

respectively for S(+) and R(-) KTP in calves (Landoni et al., 1995), 5.85 ± 0.83 and

4.77 ± 0.98 ml.min-1.kg-1 for S(+) KTP and 3.27 ± 0.50 and 3.87 ± 0.50 ml.min-1.kg-1

for R(-) KTP, respectively at the dose rate of 3 and 1.5 mg kg-1 in sheep (Landoni et

al., 1999) and 5.0 ± 0.05 and 3.83 ± 0.02 ml.min-1.kg-1, respectively for S(+) and R(-)

KTP at the dose rate 3 mg.kg-1 in goat (Arifah et al., 2003). However, lower ClB

values of 1.98 ± 0.20 and 2.51 ± 0.60 ml.min-1.kg-1, respectively for S(+) and R(-)

KTP in calves (Landoni and Lees, 1995a), 3.08 ± 0.38 and 3.66 ± 0.39 ml.min-1.kg-1,

respectively in healthy horses and horses with induce synovitis (Owens et al., 1995a)

and 2.8 ml.min-1.kg-1 in cattle (De Graves et al., 1996) were reported where as higher

ClB values of 9.53 and 12.33 ml.min-1.kg-1 in goats were reported by Pravin et al.

(2005) and Musser et al. (1998), respectively.

The total body clearance following intramuscular administration obse rved in

the present study is higher than the reported value of 0.99 ml.min-1.kg-1 in camel

(Alkatheeri et al., 1999). However, it is lower than 14.43 ml.min-1.kg-1 observed in

goats (Pranvendra et al., 2005). Thus, there are marked interspecies and intraspecies

variations in clearance values of ketoprofen, which might be attributable to

differences in hepatic biotransformation process, renal excretion (elimination rates)

and volume of distribution of the drug at different ages and in different species. In the

present study, rapid clearance of ketoprofen was observed in calves following

intravenous route administration as compared to intramuscular route. Thus, drug is

supposed to persist for longer period in body following intramuscular administration.

The time required for an intact drug molecule to transit through body is termed

as mean residence time (MRT). The mean residence time observed following single

dose intravenous administration in present study was 1.20 ± 0.06 h which is similar

for S(+) KTP (0.79 ± 0.11 and 1.90 ± 0.72 h) and R(-) KTP (0.95 ± 0.13 and 1.47 ±

0.20 h) in sheep at the dose rate of 3 and 1.5 mg kg-1, respectively (Landoni et al.,

1999), for S(+) and R(-) KTP (0.99 and 1.25 h, respectively) in cattle with early

lactation (Igarza et al., 2004) and for rac-KTP (0.95 ± 0.02 h) in goat (Pravin et al.,

2005). While higher MRT values of 2.67 ± 0.79 and 1.69 ± 0.25 h for S(+) for R(-)

KTP, respectively in calves (Landoni et al., 1995) and 2.08 and 2.13 h for S(+) for R(-

) KTP, respectively in female camel (Alkatheeri et al., 2000) were reported. Lower

MRT value was reported for rac-KTP in healthy horses and horses with induce

synovitis (0.76 ± 0.25 and 0.62 ± 0.13 h, respectively) (Owens et al., 1995a). These

values indicate that ketoprofen remains for a shorter span of time in cattle, sheep, and

goat following intravenous administration of drug correlating with its relatively faster

elimination from the body.

The mean residence time calculated following single dose intramuscular

administration in present study was 4.22 ± 0.07 h. Similar MRT value of 3.77 h was

reported in camel after intramuscular administration at the dose rate of 2 mg.kg-1. The

MRT data indicates that ketoprofen remains for a longer time after intramuscular

administration than intravenous administration in calves.

5.1.3 Systemic Bioavailability

The value of the absolute bioavailability of ketoprofen in calves following

intramuscular administration at the dose rate of 3 mg.kg-1 was 77.31 ± 2.23 percent.

Bioavailability of ketoprofen obtained in the present study is similar to 80.15 percent

reported in goat by Pranvendra et al. (2005) and 71-96 percent in human by Jamali

and Brocks (1990). However, in camel and Japanese quail birds it is reported to be

121.1 and 56 percent, respectively (AlKatheeri et al., 1999; Graham et al., 2005).

There is also wide variation reported in oral bioavailability of ketoprofen in different

animal species like 5.75 to 54.17 percent [S(+) ketoprofen] and 2.67 to 50.50 percent

[R(-) ketoprofen] for different oral formulation in horses (Landoni and Lees, 1995),

90 percent in dogs (Schmitt and Guentert, 1990), 112 percent [S(+) ketoprofen] and

85 percent [R(-) ketoprofen] in cats (Lees et al., 2003) and 24 percent in Japanese

quail (Graham et al., 2005). Good bioavailability (77.31 percent) of ketoprofen and

maintenance of therapeutic concentration up to 18 h following intramuscular injection

suggests that ketoprofen is most suitable for intramuscular administration for the

treatment of painful and inflammatory conditions in cattle.

5.2 Dosage regimen of ketoprofen in crossbred calves

Pharmacokinetic behaviour of ketoprofen varies in different domestic animal

species including cattle. Therefore, the therapeutic dosage regimens of this drug are

needed to be established in cattle by assessing kinetic parameters. The ultimate

objective of a satisfactory dosage regimen of antiinflammatory, analgesic and anti-

pyretic agents are to maintain the plasma or serum drug level above minimum

effective concentration [EC50] during the treatment. Ketoprofen inhibits both COX-1

and COX-2 with the IC50 ratio (serum TxB2 : exudates PGE2) of 1.37 in cattle.

However, therapeutic effects of NSAIDs are due to inhibition of COX-2 enzyme.

Inhibitory activity of ketoprofen on inflammatory exudate prostaglandin E2 (PGE2) is

considered as measure of COX-2 inhibition. For racemic ketoprofen, the mean EC50

value for inhibition of exudate PGE2 in cattle was 0.086 μg.ml-1 (Landoni et al.,

1995). In the present study, following intramuscular administration, ketoprofen

concentrations in calf plasma were greater than 0.086 g.ml-1 up to 18 h (Table 11).

Thus, COX-2 inhibitory EC50 concentrations persisted up to 18 h in calves after I.M.

injection of ketoprofen at 3 mg.kg-1 body weight. Therefore, ketoprofen given at the

dose rate of 3 mg.kg-1 body weight and repeated at 18 h would be satisfactory dosage

regimen in calves to achieve its therapeutic goals.

5.3 Safety Studies

NSAIDs are routinely employed to treat inflammatory conditions and to

alleviate pain and fever in animals. Prolonged therapy or high dosage regimen

implemented during the treatment may exhibit the toxic effect, if any. Considering

this fact, safety study of a drug is indeed necessary to notify any alteration in the

physiological or biochemical parameters of the body fluid and tissue along with

therapeutic effects. If alterations are very minor, the drug is relatively safe and if

severe, then adverse effects of such drug will observed. As ketoprofen is likely to be

used repeatedly for 3 to 5 days in many cases, its safety assessment is necessary by

evaluating haematological and biochemical parameters.

Reported adverse effect of ketoprofen includes gastrointestinal ulcers,

hepatopathy and nephropathy, but these harmful effects were only observed after long

term administrations (25 to 90 days) of ketoprofen or when given repeatedly at high

dose rates (Collins et al., 1998; Jerussi et al., 1998; Cabre et al., 1998b; Narita et al.,

2005; Luna et al., 2007).

In the present study none of the calf exhibited clinical symptoms of adverse

reaction or toxicity. The value of Hb, PCV, TLC, and DLC estimated during

treatment hours did not differ significantly from corresponding control values. The

mean values of serum alkaline phosphatase, acid phosphatase, aspartate

aminotransferase, alanine aminotransferase, lactate dehydrogenase, total serum

bilirubin, serum creatinine, blood urea nitrogen, total serum prote in, serum albumin

and blood glucose during treatment period were also not differ significantly from their

respective control values.

It indicates that repeated administration of ketoprofen within therapeutic

dosage regimen in calves was well tolerated. In the present study of safety evaluation,

all the haematological and blood biological parameters were found within normal

range. This suggests that long term administration at recommended dosage of

ketoprofen is safe for use in cattle.

Summary and

Conclusions

CHAPTER VI

SUMMARY AND CONCLUSIONS

Detailed pharmacokinetics of ketoprofen in crossbred calves was investigated

by non-compartmental analysis in the present study, following a single dose

intravenous and intramuscular administration. Safety of repeated intravenous

administration of ketoprofen in calves was evaluated by monitoring haematological

and blood biochemical profiles. Plasma ketoprofen concentration was determined by

HPLC assay.

Following single dose intravenous administration of ketoprofen at the dose

rate of 3.0 mg.kg-1 of body weight in calves, various pharmacokinetic parameters like

distribution half- life (t1/2) (0.10 ± 0.01 h), elimination half- life (t1/2) (1.55 ± 0.05 h),

apparent volume of distribution [Vd(area)] (0.64 ± 0.03 L.kg-1), volume of distribution

of drug at steady state [Vd(ss)] (0.35 ± 0.02 L.kg-1), total body clearance (ClB) (4.82 ±

0.16 ml.min-1.kg-1), area under plasma drug concentration-time curve (AUC) (10.42 ±

0.32 µg.h.ml-1), area under first moment of curve (AUMC) (12.37 ± 0.38 µg.h2.ml-1),

and mean residence time (MRT) (1.20 ± 0.06 h) were determined.

Following single dose intramuscular administration of ketoprofen at the dose

rate of 3.0 mg.kg-1 of body weight in calves, peak plasma concentration (Cmax) of 6.15

0.24 g.ml-1 was attained at 0.50 h (Tmax). Based on the plasma ketoprofen

concentrations, various pharmacokinetic parameters like elimination half- life (t1/2)

(3.40 ± 0.05 h), volume of distribution of drug at steady state [Vd(ss)] (0.72 ± 0.02

L.kg-1), total body clearance (ClB) (2.83 ± 0.06 ml.min-1.kg-1), area under plasma drug

concentration-time curve (AUC) (17.72 ± 0.39 µg.h.ml-1), area under first moment of

curve (AUMC) (74.93 ± 2.02 µg.h2.ml-1), mean residence time (MRT) (4.22 ± 0.07

h), and mean absorption time (MAT) (3.02 ± 0.10 h) were determined. The

intramuscular bioavailability of ketoprofen in calves was 77.31 ± 2.23 per cent.

Longer elimination half- life, extensive volume of distribution at steady state

and slower total body clearance of ketoprofen following intramuscular administration

as compared to intravenous administration makes it more suitable for intramuscular

use in calves. Furthermore, therapeutically effective concentration (EC50 for COX-2

inhibition) of ketoprofen in calves ( 0.086 g.ml-1) was maintained in plasma up to 8

and 18 hours following intravenous and intramuscular administration, respectively.

Plasma concentration profile also support intramuscular use of ketoprofen in calves

for convenient approach. Therefore, based on observed plasma drug concentration

profile following intramuscular administration, optimal dosage regimen of ketoprofen

would be 3.0 mg.kg-1 repeated at an interval of 18 h.

Safety of repeated intravenous administration of ketoprofen (3.0 mg.kg-1 at 24

h interval for 5 days) was evaluated by means of various haematological (Hb, PCV,

TLC and DLC) and blood biochemical parameters (AKP, ACP, AST, ALT, LDH,

Total Bilirubin, Serum Creatinine, BUN, Total Serum Protein, Serum Albumin and

Blood glucose). All the parameters were found to fluctuate within normal range

during treatment hours and the mean values were not significantly different from

corresponding control hour (0 h) values. No clinical symptoms of adverse reaction or

toxicity were observed throughout this period. Results suggest that repeated use of

ketoprofen at twenty-four hours for five days is safe in calves.

In conclusion, therapeutically effective concentration of ketoprofen was

maintained up to 18 h after intramuscular administration. Observed plasma ketoprofen

concentrations and calculated pharmacokinetic parameters shows suitability of the

drug for intramuscular use in the treatment of diseases associated with pain and

inflammation of calves. Therefore, ketoprofen given intramuscularly at the dose rate

of 3 mg.kg-1 body weight and repeated at 18 h would be satisfactory dosage regimen

in calves to achieve its therapeutic goals. The systemic bioavailability of ketoprofen

following intramuscular administration in the calves was 77.31 per cent. Ketoprofen

(3 mg.kg-1) was found safe, following repeated intravenous administration for five

days at twenty-four hour interval, based on evaluation of the hematological and

biochemical parameters.

References

REFERENCES

Abas, A. and Meffin, P.J. (1987). Enantioselective disposition of 2-arylpropionic acid

non-steroidal antiinflammatory drugs. IV. Ketoprofen disposition. J.

Pharmacol. Exp. Ther., 240(2): 637-641.

Aberg, G.; Ciofalo, V.B.; Pendleton, R.G.; Ray, G. and Weddle, D. (1995). Inversion

of R(-) to S(+) ketoprofen in eight animal species. Chirality, 7(5): 383-387.

Alkatheeri, N.A.; Wasfi, I.A. and Lambert, M. (1999). Pharmacokinetics and

metabolism of ketoprofen after intravenous and intramuscular administration

in camels. J. Vet. Pharmacol. Ther., 22(2): 127-135.

Alkatheeri, N.A.; Wasfi, I.A.; Lambert, M.; Saeed, A. and Khan, I.A. (2000).

Pharmacokinetics of ketoprofen enantiomers after intravenous administration

of racemate in camels: effect of gender. J. Vet. Pharmacol. Ther., 23(3): 137-

143.

Altman, R.D.; Honig, S.; Levin, J.M. and Lightfoot, R.W. (1988). Ketoprofen versus

indomethacin in patients with acute gouty arthritis: a multicenter, double blind

comparative study. J. Rheumatol., 15: 1422-1426.

Anfossi, P.; Villa, R.; Montesissa, C. and Carli, S. (1997). Intramuscular

bioavailability of ketoprofen lysine salt in horses. Vet. Q., 19(2): 65-68.

Arifah, A.K.; Landoni, M.F.; Frean, S.P. and Lees, P. (2001). Pharmacodynamics and

pharmacokinetics of ketoprofen enantiomers in the sheep. Am. J. Vet. Res.,

62(1): 77-86.

Arifah, A.K. and Lees, P. (2002). Pharmacodynamics and pharmacokinetics of

phenylbutazone in calves. J. Vet. Pharmacol. Ther., 25(4): 299-309.

Arifah, A.K.; Landoni, M.F. and Lees, P. (2003). Pharmacodynamic, chiral

pharmacokinetic and PK-PD modeling of ketoprofen in the goat. J. Vet.

Pharmacol. Ther., 26(2): 139-150.

Arrioja, D.A. (2002). Anafen Injection (large animal) and Anafen Injection and

Tablets (small animal) (Merial-Canada). Compendium of veterinary products,

CD ed. Port Huron, MI: North American Compendiums.

Baggot, J.D. (1977). In: Principles of Drug Disposition in Domestic Animals: The

Basis of Veterinary Clinical Pharmacology. 1 st Ed., W.B. Saunders Co.,

Philadelphia, U.S.A, pp. 144-189.

Baggot, J.D. (1995). Pharmacokinetics: Disposition and fate of drugs in the body. In:

Veterinary Pharmacology and Therapeutics (Edi. Adams, H.R.). 7 th Ed., Lowa

State University Press. Ames, pp. 18-52.

Boothe, D.M. (2001). The analgesic, antipyretic, antiinflammatory drugs. In:

Veterinary Pharmacology and Therapeutics (Edi. Adams, H.R.). 8 th Ed., Lowa

State University Press. Ames, pp. 433-451.

Burr, J.L.; Baynes, R.E.; Smith,G. and Riviere, J.E. (2006). Pharmacokinetics of

flunixin meglumine in swine after intravenous dosing. J. Vet. Pharmacol.

Ther., 29(5): 437-440.

Cabre, F.; Fernandez, M.F.; Calvo, L.; Ferrer, X.; Garcia, M.L. and Mauleon, D.

(1998a).

Analgesic, antiinflammatory, and antipyretic effects of S(+) ketoprofen in

vivo. J. Clin. Pharmacol., 38: S3-10.

Cabre, F.; Fernandez, M.F.; Zapatero, M.I.; Arano, A.; Garcia, M.L. and Mauleon D.

(1998b). Intestinal ulcerogenic effect of S(+) ketoprofen in the rat. J. Clin.

Pharmacol., 38: 27S-32S.

Caldwell, J.; Hutt, A.J. and Fournel-Gigleux, S. (1988). The metabolic chiral

inversion and disposition of the 2-arylpropionic acids and their biological

consequences. Biochemical pharmacol., 37: 105-114.

Castro, E.; Soraci, A.; Fogel, F. and Tapia, O. (2000). Chiral inversion of R(–)

fenoprofen and ketoprofen enantiomers in cats. J. Vet. Pharmacol. Ther., 23:

265-271.

Chan, K.K.H. and Gibaldi, M. (1982). Estimation of statistical moments and steady

state volume of distribution for a drug given a by intravenous infusion. J.

Pharmacokinet. Biopharm., 10: 551-558.

Chandrasekharan, N.V., Hu Dai, K., Roos, L.T., Evanson, N.K., Tomsik, J., Elton,

T.S. and Simmons, D.L. (2002). COX-3, a cyclooxygenase-1 variant inhibited

by acetaminophen and other analgesic/antipyretic drugs: cloning, structure and

expression. Proceedings of the National Academy of Sciences, 99: 13926–

13931.

Collins, A.J.; Davis, J. and Dixon, A.S.J. (1998). A prospective endoscopic study of

the effect of orudis and oruvail on the upper gastrointestinal tract, in patients

with osteoarthritis. Br. J. Rheumatol., 27:106-109.

Corveleyn, S.; Henrist, D.; Van der Weken, G.; Baeyens, W. and Remon, J.P. (1996).

Bioavailability of ketoprofen in horses after rectal administration. J. Vet.

Pharmacol. Ther., 19(5): 359-363.

Cutler, D.J. (1978). Theory of the mean absorption time, an adjunct to conventional

bioavailability studies. J. Pharm. Pharmacol., 30: 476-478.

Davis, J.L.; Papich, M.G.; Morton, A.J.; Gayle, J.; Blikslager, A.T. and Campbell,

N.B. (2007). Pharmacokinetics of etodolac in the horse following oral and

intravenous administration. J. Vet. Pharmacol. Ther., 30(1): 43-48.

De Graves, F.J.; Riddell, M.G. and Schumacher, J. (1996). Ketoprofen concentrations

in plasma and milk after intravenous administration in dairy cattle. Am. J. Vet.

Res., 57(7): 1031-1033.

De Graves, F.J.; Ravis, W.R.; Johansen, D.; Campbell, J.D. and Duran, S.H. (1998).

Stereospecific pharmacokinetics of free and protein-bound ketoprofen in

serum and synovial fluid of horses after intravenous and intramuscular

administration. Am. J. Vet. Res., 59(6): 739-743.

De Ruiter, J. (2002). Non-Steroidal Antiinflammatory Drugs (NSAIDs). In: Principles

of Drug Action: 2. (http://www.duc.auburn.edu/~deruija/nsaids_2002.pdf.

:accessed and downloaded from website on 31-01-2008).

Delbeke, F.T.; Baert, K. and De Backer, P. (1998). Disposition of human drug

preparations in the horse. VI. Tiaprofenic acid. J. Chromatography B, 701(1-

2): 207-214.

Deneuche, A.J.; Dufayet, C.; Goby, L.; Fayolle, P. and Desbois, C. (2004). Analgesic

comparison of meloxicam or ketoprofen for orthopedic surgery in dogs. Vet.

Surgery, 33(6): 650-660.

Dolan, S.; Kelly, J.G.; Huan, M. and Nolan, A.M. (2003). Transient upregulation of

spinal cyclooxygenase-2 and neuronal nitric oxide synthase following surgical

inflammation. Anesthesiology, 98: 170-180.

http://www.ncbi.nlm.nih.gov/sites/entrez?Db=PubMed&Cmd=Search&Term=%22De%20Graves%20FJ%22%5BAuthor%5D&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVCitation

http://www.ncbi.nlm.nih.gov/sites/entrez?Db=PubMed&Cmd=Search&Term=%22Johansen%20D%22%5BAuthor%5D&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVCitation

http://www.ncbi.nlm.nih.gov/sites/entrez?Db=PubMed&Cmd=Search&Term=%22Campbell%20JD%22%5BAuthor%5D&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVCitation

Dollery, C. (1991). Therapeutic Drugs: Volume 2, Churchill Livingstone, London,

UK, pp: 25-27.

Donnelly, M.T.; Richardson, P.; Hawkey, C.J.; Courtauld, E. and Stack, W.A. (2000).

Dose-dependent effects of ketoprofen on the human gastric mucosa in

comparison with ibuprofen. Aliment. Pharmacol. Ther., 14(5): 543-549.

Dow, S.W.; Rosychuk, R.A.; McChesney, A.E. and Curtis, C.R. (1990). Effects of

flunixin and flunixin plus prednisone on the gastrointestinal tract of dogs. Am.

J. Vet. Res., 51(7): 1131-1138.

Earley, B. and Crowe, M.A. (2002). Effects of ketoprofen alone or in combination

with local anesthesia during the castration of bull calves on plasma cortisol,

immunological, and inflammatory responses. J. Ani. Sci., 80(4): 1044-1052.

Elmas, M.; Uney, K.; Karabacak, A. and Yazar, E. (2005). Pharmacokinetics of

flunixin-meglumine following intravenous administration in angora rabbits.

Bull. Vet. Inst. pulawy., 49(2): 85-88.

Evans, A.M. (1992). Enantioselective pharmacodynamics and pharmacokinetics of

chiral non-steroidal antiinflammatory drugs. Eur. J. Clin. Pharmacol., 42:

237-256.

Faulkner, P.M. and Weary, D.M. (2000). Reducing pain after dehorning in dairy

calves. J. Dairy Sci., 83(9): 2037-2041.

Figueras, A.; Capella, D.; Castel, J.M. and Laorte, J.R. (1994). Spontaneous reporting

of adverse drug reactions to NSAID. A report from the Spanish system of

pharmacovigilance, including an early analysis of topical and enteric coated

formulations. Eur. J. Clin. Pharmacol., 47: 297-303.

Foster, M.L. and Jamali, F. (1988). Stereoselective pharmacokinetic of ketoprofen in

the rat. Drug metab. Dispos., 16(4): 623-626.

Francischi, J.N.; Chaves, C.T.; Moura, A.C.L.; Lima, A.S.; Rocha, O.A.; Ferreira-

Alves, D.L. and Bakhle, Y.S. (2002). Selective inhibitors of cyclooxygenase-2

(COX-2) induce hypoalgesia in a rat paw model of inflammation. Br. J.

Pharmacol., 137: 837-844.

Fumikazu, Y.; Yoko, H. and Takeshi, H. (2003). Experimental studies on

phototoxicity, phoallergenicity and prolonged photosensitivity due to

nonsteroidal antiinflammatory drugs (NSAIDs). Japanese J. Dermatol.,

113(4): 405-411.

http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Search&itool=pubmed_AbstractPlus&term=%22Donnelly+MT%22%5BAuthor%5D

http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Search&itool=pubmed_AbstractPlus&term=%22Richardson+P%22%5BAuthor%5D

http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Search&itool=pubmed_AbstractPlus&term=%22Hawkey+CJ%22%5BAuthor%5D

http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Search&itool=pubmed_AbstractPlus&term=%22Courtauld+E%22%5BAuthor%5D

http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Search&itool=pubmed_AbstractPlus&term=%22Stack+WA%22%5BAuthor%5D

Gabriel, S.E.; Jaakkimainen, L. and Bombardier, C. (1991). Risk for serious

gastrointestinal complications related to use of non-steroidal anti-

inflammatory drugs: A meta-analysis. Ann. Intern. Med., 328:1313-1316.

Geisslinger, G.; Mangel, S.; Wissel, K. and Brune, K., (1995). Pharmacokinetic of

ketoprofen enantiomers after different doses of recemate. Br. J. Clin.

Pharmacol., 40: 73-75.

Ghezzi, P.; Melillo, G.; Meazza, C.; Sacco, S.; Pellegrini, L.; Asti, C.; Porzio, S.;

Marullo, A.; Sabbatini, V.; Caselli, G. and Bertini, R. (1998). Differential

contribution of R(-) and S(+) isomers in ketoprofen antiinflammatory

activity: Role of cytokine modulation. J. Pharmacol. Exp. Ther., 287: 969-

974.

Gibaldi, M.; Nagashima, R. and Levy, G. (1969). Relationship between drug

concentration in plasma or serum and amount of drug in the body. J. Pharm.

Sci., 58: 193-197.

Gibaldi, M. and Perrier, D. (1982). In: Pharmacokinetics, 2nd Ed., Marcel Dekker Inc.,

New York.

Gilroy, D.W.; Colville-Nash, P.R.; Willis, D.; Chivers, J.; Paul-Clark, M.J. and

Willoughby, D.A. (1999). Inducible cyclooxygenase may have anti-

inflammatory properties. Nature Med., 6: 698-701.

Glew, A.; Aviad, A.D.; Keister, D.M. and Meo, N.J. (1996). Use of ketoprofen as an

antipyretic in cats. Canadian Vet. J., 37(4): 222-225.

Graham, J.E.; Kollias-baker, C.; Craigmill, A.L.; Thomasy, S.M. and Tell, L.A.

(2005) Pharmacokinetics of ketoprofen in Japanese quail (Coturnix japonica).

J. Vet. Pharmacol. Ther., 28(4): 399-402.

Green, G.A. (2001). Understanding NSAIDs: from aspirin to COX-2. Clin.

Cornerstone Sports Med., 3: 50-59.

Gregoricka, M.J.; Sutherland, S.F.; Dedrickson, B.J. and Busch, K.R. (1990).

Assessment of the intramuscular administration of ketoprofen. Equine Pract.,

12(7): 15-22.

Hart, F.D. and Huskisson, E.C. (1984). Nonsteroidal anti inflammatory drugs - current

status and rationale therapeutic use. Drugs, 27: 232-235.

Hawkey, C.J. (1999). COX-2 inhibitors. Lancet, 353(9149): 307-314.

http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=Search&Term=%22Hawkey%20CJ%22%5BAuthor%5D&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVAbstractPlusDrugs1

Horii, Y.; Ikenaga, M.; Shimoda, M. and Kokue, E. (2004). Pharmacokinetics of

flunixin in the cat: enterohepatic circulation and active transport mechanism in

the liver. J. Vet. Pharmacol. Ther., 27(2): 65-69.

Igarza, L.; Soraci, A.; Auza, N. and Zeballos, H. (2002). Chiral inversion of R(-)

ketoprofen: Influence of age and differing physiological status in dairy cattle.

Vet. Res. Commun., 26: 29-37.

Igarza, L.; Soraci, A.; Auza, N. and Zeballos, H. (2004). Some pharmacokinetic

parameters of R(-) and S(+) ketoprofen: The influence of age and differing

physiological status in dairy cattle. Vet. Res. Commun., 28: 81-87.

Jamali, F. and Brocks, D.R. (1990). Clinical pharmacokinetic of ketoprofen and its

enantiomers. Clin. Pharmacokinet., 19(3): 197-217.

Jamali, F.; Lovalin, R. and Aberg, G. (1997). Bi-directional chiral inversion of

ketoprofen in CD-1 mice. Chirality, 9(1): 29-31.

Jaussaud, P.; Bellon, C.; Besse, S.; Courtot, D. and Delatour, P. (1993).

Enantioselective pharmacokinetics of ketoprofen in horse. J. Vet. Pharmacol.

Ther., 16(3): 373-376.

Jelic-Ivanovic, Z.; Spasic, S.; Majkic-Singh, N. and Todorovic, P. (1985). Effects of

some antiinflammatory drugs on 12 blood constituents: protocol for the study

of in vivo effects of drugs. Clin. Chem., 31: 1141-1143.

Jerussi, T.P.; Caubet, J.F.; McCray, J.E. and Handley, D.A. (1998). Clinical

endoscopic evaluation of the gastroduodenal tolerance to (R)- ketoprofen, (R)-

flurbiprofen, racemic ketoprofen, and paracetamol: a randomized, single-

blind, placebo-controlled trial. J. Clin. Pharmacol., 38: 19-24.

Johnston, S.A. and Fox, S.M. (1997). Mechanisms of action of antiinflammatory

medications used for the treatment of osteoarthritis. J. Am. Vet. Med. Assoc.,

210(10): 1486-1492.

Jusko, W.J. and Gibaldi, M. (1972). Effects of changes in elimination on various

parameters of the two compartment open model. J. Pharm. Sci., 61(8): 1270-

1272.

Kadir, A.; Ali, B.H.; Al Hadrami, G.; Bashir, A.K.; Landoni, M.F. and Lees P.

(1997). Phenylbutazone pharmacokinetics and bioavailability in the

dromedary camel (Camelus dromedarius). J. Vet. Pharmacol. Ther., 20(1):

54-60.

http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Search&term=%22Horii+Y%22%5BAuthor%5D

http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Search&term=%22Ikenaga+M%22%5BAuthor%5D

http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Search&term=%22Shimoda+M%22%5BAuthor%5D

http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Search&term=%22Kokue+E%22%5BAuthor%5D

http://www.clinchem.org/

Kalpravidh, M.; Lumb, W.V.; Wright, M. and Heath, R.B. (1984). Effects of

butorphanol, flunixin, levorphanol, morphine, and xylazine in ponies. Am. J.

Vet. Res., 45(2): 217-223.

Kantor, T.G. (1986). Ketoprofen: a review of its pharmacologic and clinical

properties. Pharmacotherapy, 6: 93-103.

Klasco, R.K. (2003). In: USP DI: Drug information for the healthcare professional.

Volume I. Greenwood Village, Thomson Micromedex, Inc.

Landoni, M.F. and Lees, P. (1995a). Pharmacokinetic and pharmacodynamics of

ketoprofen enantiomers in calves. Chirality, 7(8): 586-597.

Landoni, M.F. and Lees, P. (1995b). Influence of formulation on pharmacokinetic and

bioavailability of racemic ketoprofen in horse. J. Vet. Pharmacol. Ther., 18:

446-450.

Landoni, M.F. and Lees, P. (1996). Pharmacokinetics and pharmacodynamics of

ketoprofen enantiomers in the horse. J. Vet. Pharmacol. Ther., 19: 466-476.

Landoni, M.F.; Cunningham, F.M. and Lees, P. (1995). Pharmacokinetics and

pharmacodynamics of ketoprofen in calves applying PK/PD modeling. J. Vet.

Pharmacol. Ther., 18: 315-324.

Landoni, M.F.; Comas, W.; Mucci, N.; Anglarilli, G.; Bidal, D. and Lees, P. (1999).

Enantiospecific pharmacokinetics and pharmacodynamics of ketoprofen in

sheep. J. Vet. Pharmacol. Ther., 22(6): 349-359.

Lees, P.; May, S.A. and White, D. (1990). Pharmacokinetics and dosage regimens of

antiinflammatory drugs. Ann. Rech. Vet., 21(1): 735-738.

Lees, P.; Aliabadi, F.S. and Landoni, M.F. (2002). Pharmacodynamics and

enantioselective pharmacokinetics of racemic carprofen in the horse. J. Vet.

Pharmacol. Ther., 25(6): 433–448.

Lees, P.; Taylor, P.M.; Landoni, F.M.; Arifah, A.K. and Waters, C. (2003).

Ketoprofen in the cat: pharmacodynamics and chiral pharmacokinetics. Vet. J.,

165 (1): 21-35.

Lees, P.; Landoni, M.F.; Giraudel, J. and Toutain, P.L. (2004). Pharmacodynamics

and pharmacokinetics of nonsteroidal antiinflammatory drugs in species of

veterinary interest. J. Vet. Pharmacol. Ther., 27: 479–490.

Lemke, K.A.; Runyon, C.L. and Horney, B.S. (2000). Effect of pre-operative

administration of ketoprofen on post-operative pain in dogs after elective

ovariohysterectomy. Vet. Anaes. Analg., 27(2): 97-111.

Lemke, K.A.; Runyon, C.L. and Horney, B.S. (2002). Effects of pre-operative

administration of ketoprofen on whole blood platelet aggregation, buccal

mucosal bleeding time, and haematologic indices in dogs undergoing elective

ovariohysterectomy. J. Am. Vet. Med. Assoc., 220(12): 1818-1822.

Liversidge, G.G. (1981). Ketoprofen. In: Analytical Profiles of Drug Substances:

Volume 10. (Ed. Florey, K.), Academic Press, London, UK, pp. 443-471.

Lockwood, P.W.; Johnson, J.C. and Katz, T.L. (2003). Clinical efficacy o f flunixin,

carprofen and ketoprofen as adjuncts to the antibacterial treatment of bovine

respiratory disease. Vet. Record, 152(13): 392-394.

Lombardino, J.G. and Wiseman, E.H. (1982). Piroxicam and other antiinflammatory

oxicams. Medicinal Res. Rev., 2(2): 127-152.

Longo, F.; Autefage, A.; Bayle, R.; Gool, F. and Van-Gool, F. (1990). Efficacy of

ketoprofen in musculoskeletal diseases of horses. Bulletin-Mensuel-de-la-

Societe-Veterinaire-Pratique-de-France. 74(6): 349-364.

Luna, S.P.; Basilio, A.C.; Steagall, P.V.; Machado, L.P.; Moutinho, F.Q.; Takahira,

R.K. and Brandao, C.V. (2007). Evaluation of adverse effects of long-term

oral administration of carprofen, etodolac, flunixin meglumine, ketoprofen,

and meloxicam in dogs. Am. J. Vet. Res., 68(3): 258-264.

MacAllister, C.G.; Morgan, S.J.; Borne, A.T. and Pollet, R.A. (1993). Comparison of

adverse effects of phenylbutazone, flunixin meglumine, and ketoprofen in

horses. J. Am. Vet. Med. Assoc., 202(1):71-77.

Mauleon, D.; Artigas, R.; Garcia, M.L. and Carganico, G. (1996). Preclinical and

clinical development of dexketoprofen. Drugs, 52: 24-46.

Mengle-Gaw, L.J. and Schwartz, B.D. (2002). Cyclooxygenase-2 inhibitors: promise

or peril? Mediators of Inflammation, 11: 275–286.

Mercer, H.D.; Baggot, J.D. and Sams, R.A. (1977). Application of pharmacokinetic

methods to the drug residue profile. J. Toxicol. Environ. Health., 2(4): 787-

801.

http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Search&itool=pubmed_AbstractPlus&term=%22Lemke+KA%22%5BAuthor%5D

http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Search&itool=pubmed_AbstractPlus&term=%22Runyon+CL%22%5BAuthor%5D

http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Search&itool=pubmed_AbstractPlus&term=%22Horney+BS%22%5BAuthor%5D

http://www3.interscience.wiley.com/journal/112226614/home

http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Search&itool=pubmed_AbstractPlus&term=%22Luna+SP%22%5BAuthor%5D

http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Search&itool=pubmed_AbstractPlus&term=%22Basilio+AC%22%5BAuthor%5D

http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Search&itool=pubmed_AbstractPlus&term=%22Steagall+PV%22%5BAuthor%5D

http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Search&itool=pubmed_AbstractPlus&term=%22Machado+LP%22%5BAuthor%5D

http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Search&itool=pubmed_AbstractPlus&term=%22Moutinho+FQ%22%5BAuthor%5D

http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Search&itool=pubmed_AbstractPlus&term=%22Takahira+RK%22%5BAuthor%5D



http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Search&itool=pubmed_AbstractPlus&term=%22Brandao+CV%22%5BAuthor%5D

http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Search&itool=pubmed_AbstractPlus&term=%22MacAllister+CG%22%5BAuthor%5D

http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Search&itool=pubmed_AbstractPlus&term=%22Morgan+SJ%22%5BAuthor%5D

http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Search&itool=pubmed_AbstractPlus&term=%22Borne+AT%22%5BAuthor%5D

http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Search&itool=pubmed_AbstractPlus&term=%22Pollet+RA%22%5BAuthor%5D

Milligan, B.J.; Duffield, T. and Lissemore, K. (2004). The utility of ketoprofen for

alleviating pain following dehorning in young dairy calves. Canadian Vet. J.,

45: 140-143.

Montoya, L.; Ambrose, L.; Kreil, V.; Bonafine, R.; Albarellos, G.; Hallu, R. and

Soraci A. (2004). A pharmacokinetic comparison of meloxicam and

ketoprofen following oral administration to helthy dogs. Vet. Res. Commun.,

28(5): 415-428.

Musser, J.M.; Anderson, K.L. and Tyczkowska, K.L. (1998). Pharmacokinetic

parameters and milk concentrations of ketoprofen after administration as a

single intravenous bolus dose to lactating goats. J. Vet. Pharmacol. Ther.,

21(5): 358-363.

Naidoo, V.; Wolter, K.; Cromarty, A.D.; Bartels, P.; Bekker, L.; McGaw, L.; Taggart,

M.A.; Cuthbert, R. and Swan, G.E. (2008). The pharmacokinetics of

meloxicam in vultures. J. Vet. Pharmacol. Ther., 31(2): 128-134.

Narita, T.; Tomizawa, N.; Sato, R.; Goryo, M. and Hara, S. (2005). Effects of long-

term oral administration of ketoprofen in clinically healthy beagle dogs. J. Vet.

Med. Sci., 67 (9): 847-853.

Nazifi, S.; Rezakhani, A. and Maharloo, F.G. (2002). Comparative study on the

effects of flunixin meglumine and ketoprofen on haematological and some

biochemical parameters of cattle. J. Faculty Vet. Med., University of Tehran,

57(2): 95-99.

Notari, R.E. (1973). Pharmacokinetics and molecular modification: Implication in

drug design and evaluation. J. Pharm. Sci., 62(6): 865-881.

Owens, J.G.; Kamerling, S.G. and Barker, S.A. (1995a). Pharmacokinetics of

ketoprofen in healthy horses and horses with acute synovitis. J. Vet.

Pharmacol. Ther., 18(3): 187-195.

Owens, J.G.; Kamerling, S.G.; Stanton, S.R. and Keowen, M.L. (1995b). Effects of

ketoprofen and phenylbutazone on chronic pain and lameness in the horse.

Equine Vet. J., 27: 296-300.

Pasloske, K.; Renaud, R.; Burger, J. and Conlon, P. (1999). Pharmacokinetics of

ketorolac after intravenous and oral single dose administration in dogs. J. Vet.

Pharmacol. Ther., 22(5): 314-319.

http://www.ncbi.nlm.nih.gov/sites/entrez?Db=PubMed&Cmd=Search&Term=%22Musser%20JM%22%5BAuthor%5D&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVCitation

http://www.ncbi.nlm.nih.gov/sites/entrez?Db=PubMed&Cmd=Search&Term=%22Anderson%20KL%22%5BAuthor%5D&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVCitation

http://www.ncbi.nlm.nih.gov/sites/entrez?Db=PubMed&Cmd=Search&Term=%22Tyczkowska%20KL%22%5BAuthor%5D&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVCitation

http://lib.bioinfo.pl/auth:Narita,T

http://lib.bioinfo.pl/auth:Tomizawa,N

http://lib.bioinfo.pl/auth:Sato,R

http://lib.bioinfo.pl/auth:Goryo,M

http://lib.bioinfo.pl/auth:Hara,S



http://www.ncbi.nlm.nih.gov/sites/entrez?Db=PubMed&Cmd=Search&Term=%22Owens%20JG%22%5BAuthor%5D&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVCitation

http://www.ncbi.nlm.nih.gov/sites/entrez?Db=PubMed&Cmd=Search&Term=%22Kamerling%20SG%22%5BAuthor%5D&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVCitation

http://www.ncbi.nlm.nih.gov/sites/entrez?Db=PubMed&Cmd=Search&Term=%22Barker%20SA%22%5BAuthor%5D&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVCitation

Pibarot, P.; Dupuis, J.; Grisneaux, E.; Cuvelliez, S.; Plante, J.; Beauregard, G.;

Bonneau, N.H.; Bouffard, J. and Blais, D. (1997). Comparison of ketoprofen,

oxymorphone hydrochloride, and butorphanol in the treatment of

postoperative pain in dogs. J. Am. Vet. Med. Assoc., 211(4): 438-444.

Picco, E.J.; Diaz David, D.C.; Encinas, T.; Rubio, M.R. and Boggio, J.C. (2004).

Pharmacokinetics of sodium meclofenamate in pre-ruminant cattle. Arq. Bras.

Med. Vet. Zootec., 56(6): 695-700.

Pranvendra, K.; Ahmed, A.H.; Singh, K.P.; Hore, S.K.; Rahal, A. and Ahuja, V.

(2005). Bioavailability and pharmacokinetics of ketoprofen in indigenous

goats. Paper presented at national seminar on “Veterinary Pharmacology in

post WTO era and fifth annual conference of ISVPT” held at Madras

Veterinary College, Chennai, pp. 37.

Pravin, K.; Jayachandran, C.; Sinha, S.; Nirbhay, K. and Thakur B. S. (2005).

Disposition kinetics of ketoprofen in goats after intravenous administration.

Paper presented at national seminar on “Veter inary Pharmacology in post

WTO era and fifth annual conference of ISVPT” held at Madras Veterinary

College, Chennai, pp. 33.

Pyorala, S.; Laurila, T.; Lehtonen, S.; Leppa, S. and Kaartinen, L. (1999). Local tissue

damage in cows after intramuscular administration of preparations containing

phenylbutazone, flunixin, ketoprofen and metamizole. Acta Veterinaria

Scandinavica, 40(2):145-150.

Rao, G.S.; Malik, J.K.; Siddaraju, V.B. and Shankaramurthy, N.C. (2007).

Pharmacokinetics and bioavailability of nimesulide in goats. J. Vet.

Pharmacol. Ther., 30(2): 157-162.

Riegelman, S.; Loo, J. and Rowland, M. (1968a). Shortcoming in Pharmacokinetic

analysis by conceiving the body to exhibit properties of a single compartment.

J. Pharm. Sci., 57(1):117-123.

Riegelman, S.; Loo, J. and Rowland, M. (1968b). Concept of volume of distribution

and possible errors in evaluation of this parameter. J. Pharm. Sci., 57(1): 128-

133.

Riviere, J.E. (1999). In: Comparative Pharmacokinetics: Principles, Techniques, and

Applications. Lowa State University Press. Ames.

Roberts, L.J. and Morrow, J.D. (2001). Chemical classification of analgesic,

antipyretic and non steroidal antiinflammatory drugs. In: Goodman and

Gillman‟s The Pharmacological Basis of Therapeutics (Eds. Hardman, J.G.

and Limbird, L.E.), 10th Ed., McGraw-Hill Companies, Inc., USA, pp. 691.

Rowland, M.; Benet, L.J. and Grahum, G.C. (1973). Clearance concept in

pharmacokinetics. J. Pharmacokinet. Biopharm., 1(2): 123-136.

Sams, R. (1978). Pharmacokinetics and metabolic considerations as they apply to

clinical pharmacology. In: Proceedings of the second Equine Pharmacology

Symposium (Eds. Powers, J.D. and Powers, T.E.). Am. Assoc. Equine Pract.,

Colorado.

Sams, R.; Gerken, D.F. and Ashcraft, S.M. (1995). Pharmacokinetic of ketoprofen

after multiple intravenous doses to mares. J. Vet. Pharmacol. Ther., 18(2):

108-116.

Santos, Y.; Ballesteros, C.; Ros, J.M.; Lazaro, R.; Rodriguez, C. and Encinas, T.

(2001). Chiral pharmacokinetics of ketorolac in sheep after intravenous and

intramuscular administration of the racemate. J. Vet. Pharmacol. Ther., 24(6):

443-446.

Schmitt, M. and Guentert, T.W. (1990). Biopharmaceutical evaluation of ketoprofen

following intravenous, oral and rectal administration in dogs. J. Pharm. Sci.,

79(7): 614-616.

Shargel, L. and Yu, A.B.C. (1993). In: Applied Biopharmaceutics and

Pharmacokinetics, 4th Ed., Appleton and Lange, Stamford.

Shpigel, N.Y.; Longo, F.; Saran, A.; Ziv, G. and Trenti, F. (1994). Ketoprofen

efficacy as adjunctive therapy in the treatment of field cases of clinical

mastitis in dairy cows. Proceedings 18th World Buiatrics Congress: 26th

Congress of the Italian Association of Buiatrics, Bologna, Italy, 1: 595-598.

Smith, C.J.; Zhang, Y.; Koboldt, C.M.; Muhammad, J.; Zweifel, B.S.; Shaffer, A.;

Talley, J.J.; Masferrer, J.L.; Seibert, K. and Isakson, P.C. (1998).

Pharmacological analysis of cyclooxygenase-1 in inflammation. Proceedings

of the National Academy of Sciences, 95: 13313–13318.

Smith, J.B. and Willis, A.L. (1971). Aspirin selectively inhibits prostaglandin

production in human platelets. Nature (New Biology), 231: 235–237.

Solankar, A.K. and Jagtap, A.G. (2005). Chronobiological and

chronopharmacological studies of ketoprofen and its solid dispersion form

using adjuvant arthritis model in rats. Indian J. Exp. Biol., 43(1): 46-52.

Soraci, A.; Tapia, O.; Castro, E. and Sanmartin, M.F. (1996). Asimetria molecular:

importancia de su conocimiento. Acta Bioquimica Clinica Latinoamericana, 3:

151-163.

Sridevi, S. and Diwan, P.V.R. (2002). Optimized transdermal delivery of ketoprofen

using pH and hydroxypropyl-β-cyclodextrin as coenhancers. Eur. J. Pharm.

Biopharm., 54: 151-154.

Steinmeyer, J. (2000). Pharmacological basis for the therapy of pain and inflammation

with nonsteroidal antiinflammatory drugs. Arthritis Res., 2: 379-385.

Sweetman, S.C. (2002). In: Martindale: The Complete Drug Reference, 33rd Ed., The

Pharmaceutical Press, London, UK, pp: 47-48.

The British Pharmacopoeia. (2002). The Pharmaceutical Press, London, UK, pp.

1003-1004.

Thompson, L. (2006). Anti- inflammatory Agents. In: Merck Veterinary Manual. (Edi.

Kahn, C.M.). Merck & Co., Inc., Whitehouse Station, NJ, USA.

USP Veterinary Pharmaceutical Information Monographs (2004). Anti-

inflammatories. J. Vet. Pharmacol. Ther., 27 (supplement 1): 75-84.

Vane, J.R. (1971). Inhibition of prostaglandin synthesis as a mechanism of action for

aspirin- like drugs. Nature (New Biology), 231: 232–235.

Vane, J.R. and Botting R. M. (1987). Inflammation and the mechanism of action of

antiinflammatory drugs. F.A.S.E.B. J., 1: 89-96.

Vane, J.R. and Botting, R.M. (1998). Anti- inflammatory drugs and their mechanism

of action. Inflammation Res., 47: 78-87.

Vane, J.R.; Bakhle, Y.S. and Botting, R.M. (1998). Cyclooxygenase 1 and 2. Annu.

Rev. Pharmacol. Toxicol., 38: 97-120.

Vavra, I. (1987). Ketoprofen. In: Nonsteroidal antiinflammatory drugs: Mechanisms

and clinical use (Eds. Lewis, A.J. and Furst D.E.). New York: Marcel Dekker,

pp: 419-438.

http://lib.bioinfo.pl/auth:Solankar,AK

http://lib.bioinfo.pl/auth:Jagtap,AG





http://www.merckvetmanual.com/mvm/htm/present/mvm_mercklink.htm

Veng-Pedersen, P. (1991). Stochastic interpretation of linear pharmacokinetics: A

linear system analysis approach. J. Pharm. Sci., 80: 621-631.

Verde, C.R.; Simpson, M.I.; Frigoli, A. and Landoni, M.F. (2001). Enantiospecific

pharmacokinetics of ketoprofen in plasma and synovial fluid of horses with

acute synovitis. J. Vet. Pharmacol. Ther., 24(3): 179-185.

Vermeulen, B. and Remon, J.P. (2001). The oral bioavailability of ibuprofen

enantiomers in broiler chickens. J. Vet. Pharmacol. Ther., 24(2): 105 -109.

Vinagre, B.; Rodriguez, C.; San Andres, M.I.; Boggio, J.C; San Andres, M.D.;

Encinas, T. and Vinagre, E. (1998). Pharmacokinetics of indomethacin in

sheep after intravenous and intramuscular administration. J. Vet. Pharmacol.

Ther., 21(4): 309-314.

Wagner, J.G. (1967). Method of estimating relative absorption of a drug in a series of

clinical studies in which blood levels are measured after single and/or multiple

doses. J. Pharm. Sci., 56: 652-653.

Wagner, J.G. (1968). Pharmacokinetics. Ann. Rev. Pharm., 8(3): 67-94.

Wallace, J.L.; Bak, A.; McKnight, W.; Asfaha, S.; Sharkey, K.A. and Mac-Naughton,

W.K. (1998). Cyclooxygenase 1 contributes to inflammatory responses in rats

and mice: implications for gastrointestinal toxicity. Gastroenterology, 115:

101–109.

Wanwimolruk, S.; Wanwimolruk, S.Z. and Zoest, A.R. (1991). Sensitive HPLC assay

for ketoprofen in human plasma and its application to pharmacokinetic study.

J. Liq. Chromatography, 14(20): 3685-3694.

Wilcke, J.R.; Crisman, M.V.; Scarratt, W.K. and Sams, R.A. (1998).

Pharmacokinetics of ketoprofen in healthy foals less than twenty-four hours

old. Am. J. Vet. Res., 59(3): 290-292.

Willoughby, D.A.; Moore, A.R. and Colville-Nash, P.R. (2000). COX-1, COX-2 and

COX-3 and the future treatment of chronic inflammatory disease. The Lancet,

355: 646–648.

Yamaoka, K.; Nakagawa, T. and Uno, T. (1978). Statistical moments in

pharmacokinetics. J. Pharmacokinet. Biopharm., 6: 79-98.

http://www.ncbi.nlm.nih.gov/sites/entrez?Db=PubMed&Cmd=Search&Term=%22Wilcke%20JR%22%5BAuthor%5D&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVCitation

http://www.ncbi.nlm.nih.gov/sites/entrez?Db=PubMed&Cmd=Search&Term=%22Crisman%20MV%22%5BAuthor%5D&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVCitation

http://www.ncbi.nlm.nih.gov/sites/entrez?Db=PubMed&Cmd=Search&Term=%22Scarratt%20WK%22%5BAuthor%5D&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVCitation

http://www.ncbi.nlm.nih.gov/sites/entrez?Db=PubMed&Cmd=Search&Term=%22Sams%20RA%22%5BAuthor%5D&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVCitation

Documents

STUDIES ON PHARMACOKINETICS, BIOAVAILABILITY AND …...Veterinary College, Anand and Director of Student’s Welfare, Anand Agricultural University, Anand, for providing me constant