Actinobacillus Pleuropneumoniae), Endotoxin and Dexamethasone on Enrofloxacin Pharmacokinetic Parameters in Swine

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LSU Historical Dissertations and Theses Graduate School

1998 The ffecE t of Pulmonary Infection (Actinobacillus Pleuropneumoniae), Endotoxin and Dexamethasone on Enrofloxacin Pharmacokinetic Parameters in Swine. Lynn O. Post Louisiana State University and Agricultural & Mechanical College

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Recommended Citation Post, Lynn O., "The Effect of Pulmonary Infection (Actinobacillus Pleuropneumoniae), Endotoxin and Dexamethasone on Enrofloxacin Pharmacokinetic Parameters in Swine." (1998). LSU Historical Dissertations and Theses. 6700. https://digitalcommons.lsu.edu/gradschool_disstheses/6700

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Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. THE EFFECT OF PULMONARY INFECTION {ACTINOBACILLUS PLEUROPNEUMONIAE) , ENDOTOXIN AND DEXAMETHASONE ON ENROFLOXACIN PHARMACOKINETIC PARAMETERS IN SWINE

A Dissertation

Submitted to the Graduate Faculty of the Louisiana State University and Agricultural and Mechanical College School of Veterinary Medicine in partial fulfillment of the requirements for the degree of

Doctor of Philosophy

The Interdepartmental Program in Veterinary Medical Sciences through the Department of Veterinary Physiology. Pharmacology and Toxicology

by Lynn O. Post B.S.. Cornell University. 1974 M.S.. Texas A&M University. 1982 D.V.M.. Texas A&M University. 1986 May 1998

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This microform edition is protected against unauthorized copying under Title 17, United States Code.

UMI 300 North Zeeb Road Ann Arbor, MI 48103

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. ACKNOWLEDGMENTS

This research would not have been possible without the cooperation between the

Food and Drug Administration. Center for Veterinary Medicine and Louisiana State

University. School of Veterinary Medicine. I am indebted to Dr. Michael J. Myers.

FDA/CVM and Dr. Charles R. Short. LSU. for serving as co-chairmen of my committee

and allowing me to complete my graduate research while employed at the FDA.

Carole Cope. Dottye Farrell, Sam Howard. Mark McDonald and Dr. Michael

Myers helped me through many long days, nights and weekends collecting and

processing blood and tissue samples. Dr. Michael Murtaugh. University of Minnesota,

generously donated the field strain of Actinobcicillus pleuropneumoniae. Dr. Ron

Snider. LSU. deserves special recognition for reviewing histopathology samples. Dr.

Joseph Kawalek, CVM. was very gracious in sharing his knowledge concerning

enrofloxacin metabolism in the pig. Dr. Richard Cullison. CVM. provided support for

the intravenous cannulation of the pigs. I appreciate the collaboration with Valarie

Reeves. CVM. on the HPLC method. Dr. Mary Bartholomew. CVM. was invaluable

for her practical statistical knowledge and advice. Drs. Marilyn Martinez and John

Baker. CVM, were largely responsible for overseeing the pharmacokinetic calculations

using WinNonLin.

I am sincerely grateful to committee members, Drs. Leonard Rappel. Steve

Nicholson and Ron Snider for their unselfish support and advice. This gratitude is

extended to Drs. Jack Caldwell and Woodrow Knight at FDA for their patience and

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. allowing me to conduct research in addition to my other duties in the Division of

Biometrics and Production Drugs.

I can not disregard the understanding of my wife. Marcia and my daughter.

Amber, for the numerous times that I missed family activities while working in the

laboratory. They make it all worthwhile.

iii

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. TABLE OF CONTENTS

ACKNOWLEDGMENTS...... ii

LIST OF TABLES...... vii

LIST OF FIGURES...... viii

ABSTRACT...... xi

INTRODUCTION...... 1 Factors Affecting Xenobiotic Disposition ...... 1 Infections and Drug Metabolism ...... 3 Cytokines and Metabolism ...... 5 Actinobacillus Pleuropneumoniae Infection in Swine ...... 8 Enrofloxacin ...... 14 Rationale ...... 17

CHAPTER I LITERATURE REVIEW ...... 21 Cytochrome P450 Inhibition ...... 21 History ...... 21 Cytokines as effectors in the acute-phase response ...... 22 Pro-inflammatory cytokines and infection ...... 23 Cytokines ...... 24 Tumor Necrosis Factor ...... 24 History ...... 24 Tumor necrosis factor and interleukin-1 in endotoxemia and sepsis ...... 27 Interleukin-1 ...... 30 History ...... 30 Interleukin-1 in inflammation and host defense ...... 31 Interleukin-6 ...... 33 History ...... 33 Acute-phase protein production ...... 34 Immune effects ...... 36 Hematopoiesis ...... 37 Anti-inflammatory mediators ...... 38 Xenobiotic Immunomodulation by Cytokines ...... 39 Cytokines in acute and chronic disease ...... 39 Glucocorticoids ...... 46 Efficacy of glucocorticoids in endotoxic shock ...... 49 Cytochrome P450 Subfamilies and Isozymes ...... 50 Rat...... 50 Swine ...... 53 Cytokine Mechanism of Action on Cytochrome P450 ...... 55 Interferons ...... 55

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Interleukin-1, interleukin-6 and tumor necrosis factor-a ...... 57 Endotoxin and infection effects on drug metabolism ...... 64 Endotoxin ...... 64 Infection ...... 69 .4 c lino bad 11 us Pleuropneumoniae...... 72 History ...... 72 Epidemiology ...... 74 Signs, pathology and lesions ...... 76 Virulence factors ...... 78 Capsule...... 78 Lipopolysaccharide ...... 80 Exotoxins ...... 84 Fluoroquinolones ...... 91 Biotransformation ...... 91 Flavin-containing monooxygenase ...... 95 Antimicrobial mechanism of action ...... 98 Pharmacokinetic characteristics...... 99 Enrofloxacin in veterinary medicine ...... 101

CHAPTER II INFECTION MODEL...... 105 Introduction ...... 105 Materials and Methods ...... 106 Animal management ...... 106 Experimental design ...... 107 Bacteria...... 109 Blood collection ...... 110 Pharmacokinetic analysis ...... 111 Plasma sample preparation ...... 111 Urine sample preparation ...... 112 Urine creatinine ...... 112 Recovery of the internal and external standards ...... 113 Aqueous standard curves for plasma ...... 114 Aqueous standard curves for urine ...... 115 High-performance liquid chromatography ...... 117 Interleukin-6 bioassay ...... 118 Interleukin-ip ELISA assay ...... 119 Health observations, necropsies and histopathology ...... 121 Statistics...... 122 Results...... 123 Animal observations and pathology ...... 123 Plasma interleukin-6 ...... 124 Plasma interleukin-1 ...... 124 Plasma enrofloxacin pharmacokinetic changes ...... 133 Area under the curve ...... 133

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Clearance, volume of distribution, mean residence time, half-life and elimination rate constant ...... 134 Urine enrofloxacin/creatinine and ciprofloxacin/creatinine ratios ...... 134 Discussion ...... 163

CHAPTER III ENDOTOXIN MODEL...... 173 Introduction ...... 173 Materials and Methods ...... 175 Animal management ...... 175 Experimental design ...... 175 Blood collection ...... 177 Pharmacokinetic analysis ...... 177 Urine and plasma sample preparation ...... 177 Urine creatinine ...... 177 Aqueous standard curves for urine and plasma ...... 178 High-performance liquid chromatography ...... 179 Interleukin-6 bioassay ...... 179 Plasma aspartate aminotransferase assay ...... 179 Health observations and necropsies ...... 180 Statistics...... 181 Results...... 181 Animal observations and pathology ...... 181 Plasma interleukin-6 ...... 182 Plasma enrofloxacin pharmacokinetic changes ...... 183 Area under the curve ...... 183 Clearance, volume of distribution, mean residence time, half-life and elimination rate constant ...... 184 Urine enrofloxacin/creatinine and ciprofloxacin/creatinine ratios ...... 184 Discussion ...... 219

SUMMARY AND CONCLUSIONS...... 227

REFERENCES...... 233

VITA...... 275

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. LIST OF TABLES

2.1. Experimental design for the Actinobacillus pleuropneumoniae infection study... 108

2.2. Lung/body weight (g/kg) ratios in control, dexamethasone. infected and infected- dexamethasone treatment groups ...... 127

2.3. Area under the curve for plasma interleukin-6 in pigs ...... 136

2.4. Area under the curve (mg/L x h) for plasma enrofloxacin in control, dexamethasone. infected and infected-dexamethasone treatment groups ...... 140

2.5. Plasma pharmacokinetic parameters of enrofloxacin in control, infected, dexamethasone and infected-dexamethasone treatment groups ...... 145

2.6. Total urine creatinine, enrofloxacin and ciprofloxacin in control and dexamethasone treatment groups for the infection study ...... 156

2.7. Urine enrofloxacin/creatinine and ciprofloxacin/creatinine ratios in control, infected, dexamethasone and infected-dexamethasone treatment groups ...... 157

3.1. Experimental design for the endotoxin study ...... 176

3.2. Lung/body weight (g/kg) ratios in control, dexamethasone. endotoxin and endotoxin-dexamethasone treatment groups. The lung weights were normalized with body weights...... 186

3.3. Plasma aspartate aminotransferase in control, endotoxin, dexamethasone and endotoxin-dexamethasone treatment groups ...... 189

.4. Area under the curve for plasma interleukin-6 in pigs in control, dexamethasone. endotoxin and endotoxin-dexamethasone treatment groups ...... 193

3.5. Area under the curve (mg/L x h) for plasma enrofloxacin in control, dexamethasone. endotoxin and endotoxin-dexamethasone treatment groups 197

3.6. Plasma pharmacokinetic parameters of enrofloxacin in control, endotoxin, dexamethasone and endotoxin-dexamethasone treatment groups ...... 202

3.7. Total urine creatinine, enrofloxacin and ciprofloxacin in control, endotoxin, dexamethasone and endotoxin-dexamethasone treatment groups ...... 212

3.8. Urine ciprofloxacin/creatinine and enrofloxacin/creatinine ratios in control, endotoxin, dexamethasone and endotoxin-dexamethasone treatment groups 214

vii

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. LIST OF FIGURES

1.1. The basic structure of the quinolones relates to the inhibition of methylxanthine metabolism. The N-C=N-C-N-C moiety is found in pipemidic acid and enoxacin. but not lomefloxacin or enrofloxacin ...... 93

1.2. The fluoroquinolone, enrofloxacin. and some of its metabolites ...... 94

2.1. Dorsal view of the trachea, heart and lungs of a control pig (non-infected. no- dexamethasone) ...... 125

2.2. Dorsal view of the trachea, heart and lung in an infected pig (no-dexamethasone). The gross lesion (5 x 6 cm) on the right caudal lung lobe (arrow) is described as necrotizing fibrinopurulent pneumonia that is consistent with Actinobacillus pleuropneumoniae infection ...... 126

2.3. Rank means±SD of lung/body weight ratios in control, infected, dexamethasone and infected-dexamethasone treatment groups ...... 128

2.4. Photomicrograph of a normal section of lung from a control pig with slight, chronic interstitial pneumonia ...... 130

2.5. Photomicrograph of a section of lung from an infected pig with necrotizing, fibrinopurulent pneumonia ...... 131

2.6. Photomicrograph of a section of lung from an infected pig with necrotizing, fibrinopurulent pneumonia with alveolar edema, bronchitis and bronchopneumonia ...... 132

2.7. Mean plasma concentration of interleukin-6 collected at 0, 1. 2. 3, 4. 6. 8. 10. 12. 24. 36. 48. 60. and 72 hours after infection or sham infection ...... 135

2.8. Rank means of area under the curve for plasma interleukin-6 ...... 137

2.9. Semilogrithmic plot of mean plasma concentrations of enrofloxacin measured at 0. 0.25. 0.5. 1. 2. 3, 4, 6. 8. 12. 24. 36. and 48 h after i.v. enrofloxacin (5 mg/kg BW) administration and 24 h after infection or sham infection ...... 139

2.10. Rank means±SD of area under the curve for plasma enrofloxacin in control, infected, dexamethasone and infected-dexamethasone treatment groups ...... 141

2.11. Means of area under the curve ratios for plasma enrofloxacin in control, infected, dexamethasone and infected-dexamethasone treatment groups ...... 143

viii

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 2.12. Rank means±SD of clearance for plasma enrofloxacin in control, infected. dexamethasone and infected-dexamethasone treatment groups ...... 146

2.13. Rank means±SD for volume of distribution of the elimination phase for plasma enrofloxacin in control, infected, dexamethasone and infected-dexamethasone treatment groups ...... 148

2.14. Rank means±SD o f volume of distribution at steady state for plasma enrofloxacin in control, infected, dexamethasone and infected-dexamethasone treatment groups...... 150

2.15. Rank means±SD of mean residence time (MRT) and half-life (t1/2) for plasma enrofloxacin in control, infected, dexamethasone and infected-dexamethasone treatment groups ...... 152

2.16. Chromatographs of standards (ciprofloxacin and enrofloxacin) and the internal standard (pipemidic acid); 0 h urine sample; and 48 h urine samples in a control pig ...... 154

2.17. Enrofloxacin and the major metabolite, ciprofloxacin, with four minor metabolites ...... 155

2.18. Rank means±SD of urine enrofloxacin/creatinine ratios in control, infected, dexamethasone and infected-dexamethasone treatment groups at 24. and 48 h after enrofloxacin administration ...... 158

2.19. Rank means±SD of urine ciprofloxacin/creatinine (pg/mg) ratios in control, infected, dexamethasone and infected-dexamethasone treatment groups at 24 and 48 h after enrofloxacin administration ...... 160

3.1. Rank means±SD of lung/body weight ratios in control, endotoxin, dexamethasone and endotoxin-dexamethasone treatment groups ...... 187

3.2. Rank means±SD of plasma aspartate aminotransferase in control, endotoxin, dexamethasone and endotoxin-dexamethasone treatment groups at 0. 24 and 72 h after endotoxin administration ...... 190

3.3. Mean plasma concentration of interleukin-6 measured at 0. 1, 3. 6 and 9 hours after endotoxin (2 pg/kg BW) administration at 0 h ...... 192

3.4. Rank means±SD of area under the curve for plasma interleukin-6 in control, endotoxin, dexamethasone and endotoxin-dexamethasone treatment groups ...... 194

3.5. Semilogrithmic plot of mean plasma concentrations of enrofloxacin measured at 0. 0.25. 0.5. 1. 2, 3. 4. 6. 8. 12. 24. 36. 48. 60. and 72 h after i.v. enrofloxacin (5 mg/kg BW) administration and 24 h after endotoxin or sham endotoxin ...... 196

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 3.6. Rank means±SD of area under the curve for plasma enrofloxacin in control, endotoxin, dexamethasone and endotoxin-dexamethasone treatment groups, comparing AUC(last) with AUC(inf) ...... 198

3.7. Means of area under the curve ratios for plasma enrofloxacin in control, endotoxin, dexamethasone and endotoxin-dexamethasone treatment groups ...... 200

3.8. Rank means±SD of clearance for plasma enrofloxacin in control, endotoxin, dexamethasone and endotoxin-dexamethasone treatment groups ...... 203

3.9. Rank means±SD of volume of distribution of the elimination phase for plasma enrofloxacin in control, endotoxin, dexamethasone and endotoxin-dexamethasone treatment groups ...... 205

3.10. Rank means±SD of volume of distribution at steady state for plasma enrofloxacin in control, endotoxin, dexamethasone and endotoxin-dexamethasone treatment groups ...... 207

3.11. Rank means±SD of mean residence time (MRT) and half-life (t 1/2) for plasma enrofloxacin in control, endotoxin, dexamethasone and endotoxin-dexamethasone treatment groups ...... 209

3.12. Chromatographs of standards (ciprofloxacin and enrofloxacin) and the internal standard (pipemidic acid); 0 h urine sample; and 72 h urine samples in an endotoxin treated pig ...... 211

3.13. Rank means±SD of urine enrofloxacin/creatinine ratios in control, endotoxin, dexamethasone and endotoxin-dexamethasone treatment groups at 24. 48 and 72 h after enrofloxacin administration ...... 215

3.14. Rank means±SD of urine ciprofloxacin/creatinine ratios in control, endotoxin, dexamethasone and endotoxin-dexamethasone treatment groups at 24. 48 and 72 h after enrofloxacin administration ...... 217

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. ABSTRACT

Efficacy studies are often done on diseased animals while pharmacokinetic

studies are done on healthy animals. Disease models are inherently difficult to

standardize because of the many variables associated with measuring pharmacokinetic

parameters in abnormal animals. Information derived from pharmacokinitic studies

with diseased animals could be useful to determine if therapeutic doses of a drug or

antimicrobial becomes less effective or toxic due to changes in distribution and

clearance.

The objectives of this study were to: characterize a pulmonary infection model

in swine using Actinobacillus pleuropneumoniae; evaluate the pharmacokinetic changes

associated with the pulmonary infection model and dexamethasone after the infusion of

enrofloxacin and urine enrofloxacin and ciprofloxacin concentrations: and the

comparison endotoxin administration and dexamethasone to the pharmacokinetic

changes of the infection model.

In the infection study, infected pigs became ill within two hours of inoculation

and had microscopic lesions of necrotizing, fibrinopurulent pneumonia consistent with

Actinobacillus pleuropneumoniae infection; plasma interleukin-6 was slightly elevated

in the infected pigs and not the dexamethasone pigs: clearance of enrofloxacin was

increased in the dexamethasone treated pigs: volume of distribution of the elimination

phase and at steady state were decreased in the infection: and the urine

enrofloxacin/creatinine ratios were decreased in the dexamethasone treated pigs.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. In the endotoxin study, the endotoxin treated pigs exhibited toxic signs within

30 minutes; the plasma interleukin-6 concentrations were markedly elevated in the

endotoxin treated pigs; the dexamethasone treated pigs showed an increased clearance

of enrofloxacin; volumes of distribution was increased in the dexamethasone treated

pigs; the urine enrofloxacin/creatinine ratios were elevated in the endotoxin group

compared to control, dexamethasone and endotoxin-dexamethasone groups while the

ciprofloxacin/creatinine ratios in the endotoxin group was elevated and the

dexamethasone treated pigs had elevated urine ciprofloxacin/creatinine ratio at 24 h but

decreased at 48 and 72 h. These results show that the infection model was reliable,

infection had no effect on enrofloxacin distribution and clearance, dexamethasone

concurrent administration with enrofloxacin may reduce efficacy because of increased

clearance and the comparison of the endotoxin model compared to the infection model

gave incongruous results.

xii

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. INTRODUCTION

Factors Affecting Xenobiotic Disposition

It has been shown that the disposition of xenobiotic substances may be affected

by the disease process (Gibson & Skett. 1986; Monshouwer et al.. 1995b). Alterations

in absorption, distribution, metabolism and excretion depend on the microorganism,

compound and route of exposure of a drug or antimicrobial (Monshouwer et al., 1995a).

An alteration of one of these variables may have a profound impact on pharmacokinetic

parameters exhibited by a particular drug. All four processes can be affected during an

infection-induced acute-phase response. The acute-phase response occurs when

physiological homeostasis is upset by infection, inflammation, tissue injury, neoplastic

growth, or immunological disorders. The physiological response starts with a local

reaction at the site of injury and progress to a systemic reaction. The local reaction is

characterized by platelet aggregation, increase vascular permeability and activation and

accumulation of granulocytes and mononuclear cells. The systemic reaction is

characterized by fever, anorexia, increased white blood cell-counts. inhibition of gastric

function, increased release of endocrine hormones and alterations in plasma cation

concentrations. The term, acute-phase. refers to changes in several plasma proteins.

Delayed gastric emptying can reduce drug absorption from the duodenum and

jejunum. The rate of gastric emptying can be affected by anxiety, stress, food and

infections. In pigs, an experimentally induced acute-phase response decreased the

absorption rate of orally administered trimethoprim or oxytetracycline (Ladefoged.

1979; Pijpers et al.. 1991). Gastric secretion and gastrointestinal motility can be

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. affected during infection, which results in altered absorption rate (Groothius et al..

1978; Leenen & van Miert, 1969). The binding of drugs to plasma proteins can

influence drug disposition. Albumin and a l-acid glycoprotein (AGP) are the main drug

binding proteins. Inflammation and infection induce an acute-phase response which

decreases albumin and increases a r acid glycoprotein production (Eckersall et al.. 1996;

Heinrich et al., 1990; Kushner & Mackiewicz. 1993). Alpha,-acid glycoprotein is an

important binder of basic drugs such as propanolol. while albumin binds acidic drugs

like penicillin. Increased concentrations of a r acid glycoprotein and albumin can alter

the volume of distribution and drug half-life. The kidney is the most important organ

for the excretion of most drugs below 400 Daltons (Da) molecular weight and drugs

greater than 400 Da are excreted by the liver. Therefore, renal or hepatic dysfunction or

diminished renal or hepatic blood flow may affect the excretion rate of drugs

(Monshouwer et al.. 1995a). Metabolism can also be affected by either infection or

inflammation.

Domestic animals are especially at risk to infections, because of crowding and

poor housing conditions where outbreaks of disease are common (Fedorka-Cray et al..

1993a). The swine industry is an example of high density production. Diseases in

countries raising pigs were high and economic losses were large. Observations from

laboratory animals and humans provide supporting evidence that infection impairs drug

disposition, but information is lacking in domestic animals. Infection may induce

pharmacokinetic changes in domestic animals to the same extent as laboratory species

and humans. These alterations may have clinical consequences such as increased drug

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. toxicity or diminished drug efficacy: or decreased toxicity and increased efficacy.

Decreased half-life, decreased volume of distribution and increased clearance may be

manifested by decreased efficacy. On the other hand, increased half-life, increased

volume of distribution and decreased clearance may lead to drug toxicity. Alternatively,

increased half-life, increased volume of distribution and decreased clearance may signal

pharmacokinetic changes leading to excessive residue concentrations of veterinary

drugs and metabolites in food. The potential for a human food safety problem is likely

because levels of drugs residues may be increased in animal tissues due to decreased

metabolism of the parent compound.

Infections and Drug iMetabolism

The main site of drug metabolism is the liver, but lung. skin, kidney and small

intestine may also metabolize xenobiotic substances. Drug metabolism reactions are

classified into Phase I and Phase II reactions. Phase I reactions consist of oxidative and

reductive reactions that create or alter functional groups and Phase II reactions are

conjugations. Phase II reactions may depend upon the activities of Phase I enzymes.

Phase II reactions couple a drug or metabolite to an endogenous substrate such as

glucuronic acid, glutathione, sulphate, aceytl or glycine. Metabolism of most drugs is

accomplished by families I to IV of the microsomal cytochrome P450's (Nebert &

Gonzalez, 1987; Paine. 1991). Hydroxylation. dealkylation. dehalogenation.

epoxidation and deamination are typical oxidation reactions. In humans studies,

bacterial, viral and parasitic infections have been shown to impede drug metabolism by

suppressing cytochrome P450's (Armstrong & Renton. 1993a.b: Azri & Renton. 1987;

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Chang et al.. 1978: Descotes et al., 1985; Farquar et al.. 1983; Galtier et al.. 1986:

Glazier et al.. 1994: Kokwaro et al.. 1993; Kramer & McClain. 1981; Maffei et al..

1981: Renton. 1981.1986; Selgrade et al., 1984; Soykaef al.. 1976). A variety of

infections all showed an impairment of drug metabolizing activities, which

demonstrates that the source of infection does not necessarily dictate the expected

effect. Experimental animals exposed to either microbial infection or inflammatory

stimuli, endotoxin or turpentine, respond by manifesting physiological, hematological

and metabolic changes that characterize the acute-phase response (Ballmer et al., 1991;

van Miert. 1990).

Previous work has focused on Phase I metabolism, but only limited information

is available on the effects of infection on Phase II metabolism. Malarial infection

decreased the glucuronidation of 3'-azido-3' deoxythymidine and paracetamol in rat

liver microsomes (Ismail et al.. 1992). Ehrlichiaphagocytophilia or Trypanosoma

infection in goats depressed the glucuronidation of chloramphenicol and sulfadimidine

metabolites (Anika et al., 1986; van Gogh et al.. 1989). In Salmonella dublin infected

calves, it was observed that the elimination half-life of chloramphenicol was increased

(Groothuis & van Miert, 1987). Prolongation of the elimination half-life of paracetamol

in malaria-infected rats was also shown (Mansor et al.. 1991). Immunosupressed mice,

infected with Schistosoma mansoni, had prolonged pentobarbital induced sleep-times

(Tonelli et al.. 1995).

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Cytokines and Metabolism

The acute-phase response may be triggered by cytokines released from

macrophages-monocytes. Interleukins la and 1(5 (IL-1 a/p). tumor necrosis factors a

and P (TNF-a/p) and interferons a and P (INF-a/p) are cytokines that inhibit oxidative

and conjugative drug metabolism in laboratory animals (Craig et al., 1989b; Ghezzi et

al., 1986a.b; Morgan & Norman. 1990; Renton & Knickle. 1990). The interferon-

inducer (INF-a), tilorone. was shown to decrease the activity of several drug

metabolizing enzymes in the rat (Renton & Mannering. 1976b) such as Bordetella

pertusis. E. coli. lipopolysaccharide (LPS) and polyinosinic acid-polycytidylic acid

(poly IC). that induced INF decreased total cytochrome P450 (P450) and related enzyme

activities (Renton & Mannering. 1976a). Bordatellapertusis and LPS also induced

tumor necrosis factor-a (TNF-a). interleukin-l (IL-1) and interleukin-6 (IL-6). In

following years, many reports described the effects of cytokine inducers. polylC and

LPS effects on cytochrome P450 mediated drug metabolism (Cribb et al.. 1994;

Delaporte et al., 1993; Ghezziet al., 1986b; Morgan. 1989; Morgan & Norman. 1990;

Sakai et al.. 1992; Shedlofsky et al.. 1994; Stanley et al., 1988).

When it was proven that cytokines were involved in infections, cytochrome

P450 activity was decreased by lowering concentrations of mRNA and apoprotein to

suppress drug metabolism. Other studies were performed to directly expose laboratory

animals to cytokines. Tumor necrosis factor-a, IL-1. IL-6 or interferons depressed

cytochrome P450 dependent enzyme activities of P450IIC11 and P450I1C12 genes in

male and female rat livers, respectively (Ghezzi et al., 1986a.b.c; Shedlofsky et al.,

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 1987: Stanley et al.. 1991; Wright & Morgan. 1991). When administered in

combination to female rats. IL-1 and dexamethasone significantly depressed total P450

and P450IIC12 apoenzyme and mRNA (Wright & Morgan. 1991. Interleukin-la alone

significantly suppressed total P450 content. P450IIC11 apoenzyme and P450IIC12

mRNA. Interleukin-6 was not as potent, but it did potentiate the effects of

dexamethasone. In another investigation, the effects of IL-1 and IL-6 on P450IIC12

expression in rat hepatocyte cultures were examined (Morgan et al.. 1994). The effects

on expression of multiple cytochrome P450 gene products in male rat livers was also

examined in vitro. Interleukin-1 suppressed the expression of P450IIC12 mRNA and

protein in cultured hepatocytes. No consistent effect of IL-6 was observed. Maximal

suppression of P45II2C12 mRNA after 24 h of IL-1 treatment reached 12 and 32% of

control concentrations in two separate experiments. Cytochrome P450IIC12 protein

was suppressed to 28% of control concentrations as early as 12 h after IL-1 treatment.

Injection of IL-1. low doses of dexamethasone. or both, in male rats produced decreases

in total P450 and in P450IIIA2 and P450IIC11 mRNA and protein expression similar to

effects previously seen for P450IIC12 expression in females. Cytochrome P450IIEI

mRNA and protein was significantly suppressed only by the combination of IL-1 and

dexamethasone. Interleukin-6 treatment of male rats down-regulated the P450IIC11 and

P450IIE1 mRNAs at a dose of 4.5 pg/kg, which was lower than that required to induce

haptoglobin mRNA. a prototype acute phase gene product. Cytochrome P450IIC11

protein content of the microsomes was also decreased by IL-6 treatment, with a slower

time-course than for suppression of its mRNA. No significant effects of IL-6 treatment

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. were seen on P450IIIA2 mRNA or P450IIIA2/1. Therefore. IL-1 and IL-6 treatments.

in vivo. differentially affect subsets of P450 gene products in rat liver. The effects of

LPS. TNF-a and IL-1 on the mixed-fimction oxidase system, in vivo, may be due partly

to an induction of IL-6 in vivo (Chen et al.. 1992). The different sensitivities of the

enzymes to IL-6. but not to IL-1 or TNF-a may be due to the involvement of two

distinct mechanisms where ethylmorphine-N-demethylase and ethoxycoumarin-O-

deethylase were affected; ethoxyresorufin-O-deethylase was minimally affected and

pentoxyresorufm-O-deethylase was not affected.

The differential effects of IL-1 a and IL-P may be difficult to explain because

they both bind to the same biological receptor (Type I) and elicit the same biological

responses (Kalian et al.. 1986. 1990). For example, IL -la/p may coordinate the brain-

endocrine-immune responses to physiological and pharmacological stimuli by binding

to the same receptor (Takao et al.. 1992). The binding of radiolabeled IL-1R antagonist

(IL-ra) in homogenates of mouse hippocampus, spleen and testis was linear over a broad

range of membrane protein concentrations, saturable, reversible and of high affinity. In

competition studies. IL-lra. recombinant human IL-1 a. IL-ip and a weak IL-ip analog

inhibited radiolabeled IL-lra binding to mouse tissues in parallel with their biological

activities. The binding characteristics and distribution of IL-lra were comparable to

those of previously characterized Type I IL-1 receptors. The biological effects of IL-1 P

have been more extensively studied, because IL -la is not released into the systemic

circulation. Nonetheless, differential effects of IL-1 P on cytochrome P450 were

observed in arthritic rats (Ferrari et al.. 1993b). Interleukin-la may have similar

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. effects, but this has not been studied. Experimental arthritis and inflammation reduced

liver cytochrome P450-dependent monooxygenase activities with subsequent

impairment of drug metabolism. Arthritis was induced on day 0 by type II collagen and

a low dose (0.2 mg) of N-acetylmuramyl-L-alanyl-D-isoglutamine and human

recombinant IL-1 was administered subcutaneously at the daily dose of 0.02. 0.2 or 2.0

micrograms to each arthritic rat. from day 21 to 25 and on day 28. Ethoxyresorufin-O-

deethylation was depressed 6-fold in arthritic rat liver microsomes and the 2.0 pg

dosage of IL-1 potentiated this depression. Pentoxyresorufm-O-deethylation activity

was decreased by 50% in arthritic rats, following IL-1 treatment. Progesterone 60-

hydroxvlation and P450IIIA protein increased by 2-fold in both untreated arthritic rat

liver microsomes and after the low dose of IL-1. The two higher doses of IL-1

decreased this activity to baseline concentrations in the arthritic rats. Laurie acid

hydroxylation increased 2-fold in arthritic rats and was further potentiated by IL-1. This

study demonstrated that main liver drug-metabolizing isoenzymes during established

collagen-induced arthritis in rats were affected by systemic IL-1. Treatment with IL-1

mimicked or reversed the influence of the inflammatory process on these enzymes.

Actinobacillus Pleuropneumoniae Infection in Swine

This study tested a specific drug, enrofloxacin. in the presence of a pulmonary

infection with Actinobacillus pleuropneumoniae (APP). whereas previous studies have

employed non-therapeutic or classical substrates . such as antipyrine. instead of

pharmaceuticals to measure pharmacokinetic parameters in diseased animals

(Monshouwer et al.. 1995a). It is the intent of this study to characterize the disposition

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. and clearance of enrofloxacin in the infected animal with Actinobacillus

pleuropneumoniae using a field strain of serotype 1 that has a low virulence (Baarsch et.

al.. 1995: van Leengoed et. al.. 1987, 1989. 1990; Henselefn/.. 1996). Subsequently,

the morbidity rate was high, but the mortality rate was low.

Actinobacillus pleuropneumoniae can cause peracute. acute or chronic

respiratory disease in swine. The host's response to an acute infection includes a

moderate hyperthermia, lethargy and reduced feed and water consumption. The

infection occurs in many swine producing countries and causes great economic loss due

to high morbidity and mortality (Monshouwer. 1996). This study has employed a

domestic APP field strain. L91-2. belonging to serotype 1 which is common in the

United States (Baarsch et al.. 1995). The low virulence of the culture was produced by

serial in vitro passage on solid growth media. In the Netherlands serotype 9. strain

13261. was employed to perform pharmacokinetic studies. This strain was only passed

once in mouse lungs to decrease virulence (Monshouwer et al.. 1995a). In the present

study, only the attenuated field strain. L91-2, was inoculated into swine. High

morbidity, but low mortality occurred because of the reduced virulence of the field

strain, L91-2.

The acute and peracute forms of porcine Actinobacillus pleuropneumoniae

resemble septic shock in humans. Therefore, an Actinobacillus pleuropneumoniae

infection model may prove to be useful as a model for septic shock. Bacterial sepsis

produced by Actinobacillus pleuropneumoniae depends on a variety of virulence

factors: capsule, endotoxin, hemolysin and cytotoxins which play important roles in the

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. pathogenesis (Fedorka-Cray et al.. 1994). Other Gram-negative bacteria, such as

Escherichia coli. also produce endotoxin. Lipopolysaccharides have been shown to

induce TNF-a and other inflammatory cytokines which altered pharmacokinetic

parameters. Endotoxin down-regulated cytochrome P450 and decreased hepatic

microsomal enzyme activities. Furthermore, endotoxin stimulated transmembrane L-

arginine transport in pulmonary endothelial cells (Cendan et al.. 1995).

Lipopolysaccharide stimulation of plasmal membrane L-arginine transport was

mediated via an autocrine loop involving TNF-a and IL-1. The marked increase in

carrier mediated L-arginine transport activity produced by LPS. IL-1 and TNF-a may

represent an adaptive response by pulmonary endothelium to support arginine-

dependent biosynthetic pathways during sepsis. Lipopolysaccharide stimulation of

arginine transport was partially mediated through an autocrine mechanism that involves

IL-1 and TNF.

Definitive studies showing that cytokines affect P450 mediated reaction in food

and companion animals are lacking. Past research concentrated on viral or bacterial

infection and vaccinations to achieve changes in drug metabolism in domestic animals

(Beadle et al.. 1989; Short. 1986). For example, a soft tissue infection model was

created in horses by infecting subcutaneous tissue chambers with Streptococcus

zooepidemicus organisms (Beadle et al.. 1989). The horses were treated with cephapirin

which decreased systemic neutrophilia, pyrexia, anemia and chamber bacterial counts.

Cephapirin failed to eliminate the infection in the tissue chambers. Another

subcutaneous tissue chamber model was designed to study the interaction of Pasteurella

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. hemolytica. sulfadiazine/trimethoprim and bovine viral diarrhea virus in calves (Clarke

et al.. 1989b). Bacteria were not cleared from the tissue chambers, despite exceeding

the in vitro minimum bactericidal antimicrobial concentration in the chamber. The lack

of antimicrobial effectiveness was related to decreased pH and increased protein

concentrations in chamber fluids after inoculation. Infection with bovine viral diarrhea

virus had no effect on the Pasteurella hemolytica response to sulfadiazine/trimethoprim

antimicrobial therapy. Bovine viral diarrhea virus was thought to suppress host

responses to inflammation (Clarke et al.. 1989b).

Another tissue chamber study explored the effect of infection on the distribution

of sulfadiazine and trimethoprim in cattle (Clarke et al.. 1989a). Increased total serum

protein and albumin concentrations, decreased pH. distention of blood vessels and

erosion of surface capillaries were associated with marked increase in the penetration of

sulfadiazine and trimethoprim into infected tissue chambers compared to non-infected

chambers. A concurrent increase in the apparent volume o f distribution for sulfadiazine

and trimethoprim was associated with increased tissue penetration (Clarke et al..

1989a).

Naturally occurring diseases have provided much insight into the effects of

infection on pharmacokinetic parameters, but were difficult to control to insure

reproducibility between groups. Theophylline pharmacokinetic parameters were

unaltered in horses vaccinated with killed equine influenza vaccine (Short et al.. 1979).

This apparent contradiction to the previously mentioned studies may be explained by

vaccination failure with no measurable antibody titer or the failure to induce appropriate

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. cytokines that would cause pharmacokinetic changes to include down-regulation of

cytochrome P450. The APP infection model was developed as an experimental

respiratory disease model (Baarsch et al.. 1995: et al.. I995a.b). The Baarsch model

was standardized by optical density readings, while the Netherlands model was

standardized by plating serial dilutions in order to obtain the correct bacterial

concentration.

An in vitro study was conducted to determine the effect of APP infection on

Phase I and Phase II hepatic microsomal enzymes in castrated pigs (barrows)

(Monshouwer et al.. 1995b). Actinobacilluspleuropneumonicie infection produced

significant suppression of 33% or more of all oxidative activities after 24 h post

infection. All glucuonyltransferase activities were unaffected by APP infection after 24

and 40 hours. To further clarify the mechanism of oxidative suppression of enzyme

activities. mRNA analysis were performed by dot blot using a human cytochrome

P450IIIA4 cDNA probe (Monshouwer et al.. 1995b). Infection with APP seemed to

reduce P450iIIA activity associated with in vitro reduction of 6P-hydroxyIation of

testosterone, aniline hydroxylation. ethoxyresorufin O-deethylation and

pentoxyresorufin O-depentylation enzyme activities. While these results were

interesting, the suppression of model oxidative reactions may not be indicative of

effects with a therapeutic drug. Furthermore, the use of dot blots to measure total RNA

may not be as accurate as Northern blots. In another study, the effect of bacterial acute-

phase response model was investigated in vivo. Plasma clearances of antipvrine.

caffeine, paracetamol and indocyanine green were measured in healthy and APP

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. infected pigs (Monshouwer et al., 1995a). The most meaningful change was

suppression of oxidative hepatic biotransformation during the acute-phase response

elicited by APP. Again, the study predicted the fate of a model drug rather than verify

the fate of a pharmaceutical.

In one study, endotoxin was non-selective in down-regulating cytochrome P450

metabolism and depressed different forms of several P450's (Monshouwer et al..

1996a), while a separate study indicated that endotoxin was isoform specific (Dr.

Michael Myers, unpublished). Another report compared the endotoxin model to an APP

infection model in swine (Monshouwer et al.. I995a.b. 1996a). The lipopolvsaccharide

model showed a close similarity to the APP infection model based on the large decrease

of antipyrine plasma clearance. Total cytochrome P450 content and microsomal P450

enzyme activities were also decreased 24 h after LPS administration. In swine,

endotoxin decreased the activities of ethoxyresorufin O-deethyiase. caffeine 3-

demethylase and testosterone 6p-hydroxyIase which corresponded to rat P4501A1,

P450IA2 and P450IIIA. respectively. The decreased enzyme activities were

accompanied by a corresponding decrease in P450IA and P450IIIA apoprotein

concentrations. In rats and mice, all constitutive P450 isoforms were down-regulated by

LPS (Chen et al.. 1992: Ferrari et al., 1993a; Morgan. 1993). Western blots were

performed on P450IA and IIIA. The representative control and treated animals had

nearly equal band widths in the representative Western blots, which may be interpreted

that their was no difference between controls and treated pigs.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Growing swine (35 kg) and finishing swine (85 kg) received intravenous

endotoxin (2 or 20 (ig/kg) 24 h prior to harvesting the livers. Western blots of

constitutive P450 showed that endotoxin had no effect on the concentrations of P450IIE

or IIIA (Dr. Michael Myers, unpublished). The relative concentrations of P450IA2 and

IID6 were unaffected in finishing swine, but were reduced or absent in growing animals.

Endotoxin challenge completely eliminated detectable cytochrome P450IIA6 and IIB2

in growing and finishing swine. Endotoxin also eliminated cytochrome P450IA2 and

reduced the level of IID6 in growing swine. Growing swine had less ethoxyresorufin-

O-deethylase and coumarin-O-dealkylase than finishing swine and these changes were

independent of endotoxin dose. Therefore, it was concluded that: cytochrome P450

expression in swine was dependent upon the age of the animal: cytochrome P450's was

not universally down-regulated by endotoxin administration in swine; and. results from

growing animais may not adequately explain the metabolic fate of drugs in finishing

swine.

Enrofloxacin

Enrofloxacin. a fluoroquinolone antibiotic, was chosen to assess the influence of

infection on metabolism, for these primary reasons: First, it was very effective in

treating porcine pleuropneumonia caused by Actinobacillus pleuropneumoniae

(Gutierrez et al.. 1993). Forty-two antimicrobial agents were tested in 57 field strains of

Actinobacillus pleuropneumoniae. The in vitro susceptibility of 839 isolates of

Actinobacillus pleuropneumoniae showed that danofloxacin had high potency over

other less effective antimicrobials (Raemdonck et al.. 1994). Cephalosporins and

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. quinolones (ciprofloxacin, enrofloxacin and sparfloxacin) were the most active

antibiotics for treating porcine pleuropneumonia. Enrofloxacin fed in the diet (150

mg/kg) produced a marked control of Actinobacillus pleuropneumoniae infection with

reduction of the severity of thoracic lesions and a reduced prevalence of the organism in

the lung at necropsy (Smith et al.. 1991). In a Japanese study, enrofloxacin and

orbifloxacin were effective when administered subcutaneously. intramuscularly or

orally administered to swine for pneumonia due to Actinobacillus pleuropneumoniae

(Nakamura. 1995). In other countries, comparison o f minimum inhibitory

concentrations tested the effectiveness of 12 antimicrobial agents on swine pathogens,

including Actinobacillus pleuropneumoniae. in the United States. Canada and Denmark

(Salmon et al.. 1995). Enrofloxacin had similar minimum inhibitory concentrations

from one country to another, but variations in the MIC's of the remaining antimicrobial

agents were observed. Secondly, approval is being sought for the use of

fluoroquinolones in food animals (Hannan et al.. 1989: fCung et al.. I993a.b). Thirdly,

enrofloxacin undergoes N-dealkylation of the ethyl group on the piperazinvl ring, which

serves as useful marker to determine the effect of porcine Actinobacillus

pleuropneumoniae infection on metabolism. Presently, the most likely porcine

dealkylating P450 subfamily might be IIIA. Inhibitors of IIIA included tiamulin,

SKF525A, cimetidine and ketoconazole in pig livers (Witkamp et al.. 1994). When

tiamulin was added to the incubation medium, the N-demethylation rate of

ethylmorphine and the hydroxylation of testosterone at the 6p- and 1 la-positions were

strongly inhibited. The microsomal N-demethylation rate of erythromycin and the

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. hydroxvlation of testosterone at the 2P-position were inhibited to a lesser degree,

whereas the ethoxyresorufin-O-deethylation. aniline hydroxylation and testosterone

hydroxylations at the 15a- and 15p-positions were not affected by tiamulin. In the rat.

the IIIA subfamily was inducible by steroids (dexamethasone). macrolide antibiotics,

imidazole antifungals. clotrimazole and phenobarbital (Bachmann et al. 1992: Gonzalez.

1988; Watkins, 1990). Further support of oxidative metabolism by P450IIIA was

presented in a study using APP (Monshouwer et al.. 1995b) where the effect of APP

infection on both oxidative and conjugative microsomal enzyme activities was

investigated in castrated male commercial pigs. All oxidative enzyme activities were

suppressed by 33%. To further clarify the mechanism of the suppression of oxidative

enzyme activities, analysis of mRNA were conducted by dot blot analysis using a

human cytochrome P450IIIA4 cDNA probe. The reduction in cytochrome P450IIIA

activity, specific for 6a-hydroxyIation of testosterone, was postulated to be at a pre-

translational level, as measured by a decrease in the amount of mRNA. Flavin-

containing monooxygenases (FMO) are more likely than heme-protein P450s to be

involved in N-oxidation and N-dealkvlation of enrofloxacin. but absolute proof is

lacking in swine (Sorgel, 1989). Although, the specific isozyme for the dealkylation is

unknown, the biotransformation of enrofloxacin to ciprofloxacin may be decreased by

down-regulation of P450 due to APP infection or endotoxin in this study.

In the past, there has been widespread extra-label use of fluoroquinolones in

food animals and enrofloxacin, norfloxacin and ciprofloxacin were the ones most

frequently used in veterinary medicine. Fluoroquinolones have been used, in an extra-

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. label manner, by veterinarians to treat swine enteric infection, enzoonotic pneumonia,

septicemia, mastitis-metritis-agalactia and pleuropneumonia (Guitterrez et al.. 1993:

Kung et al.. 1993a.b). However, the extra-label use of fluoroquinolones in food animals

was banned in 1997 by the United States Food and Drug Administration. Therapeutic

use of approved fluoroquinolones in non-food animals was not affected by the ban.

Rationale

The hypothesis of this investigation is: infection with APP or endotoxin

administration may alter enrofloxacin pharmacokinetic parameters. Therefore, a

combination of one or more of the following five conclusions are possible: 1) In

domestic animals, pharmacokinetic studies have historically been conducted in healthy

animals, while efficacy studies are conducted in diseased animals. However, infected

animals may have altered pharmacokinetic parameters when compared to normal

animals. 2) Target animal safety problems could arise in drug development and

approval whereby therapeutic doses are actually toxic. 3) Conversely, increased rate of

clearance could indicate a need for more frequent dosing. Previously effective doses

may not be adequate to treat an infected animal. 4) Human food safety is an important

concern. Potential reductions in therapeutic drug clearance in food animals would result

in elevated residues concentrations even after a proper withdrawal period. Withdrawal

times are derived from non-infected animals. The purpose of the present study was to

determine if the pharmacokinetic parameters of a therapeutic drug (enrofloxacin) are

altered by APP infection and pleuropneumonia in swine. 5) Infection with APP does

not alter pharmacokinetic parameters.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. This study was designed to demonstrate that pharmacokinetic alterations can be

induced by a specific disease, porcine pleuropneumonia, coupled with a specific

antibiotic, enrofloxacin. in swine. Other investigators have approached this question,

but have not strongly connected a natural disease with a therapeutic drug to demonstrate

altered pharmacokinetic parameters.

Few studies have explored the possibility of altered pharmacokinetic parameters

in infected domestic animals. An Actinobacillus pleuropneumoniae infection model has

been used, but it is a different serotype (9) than the United States field serotype (1) used

in this study (Monshouwer et al., 1995a;b). The virulence may vary between the strains

and influence the experimental results. Variation in virulence may mean a variation in

bacterial toxins which may directly relate to the amount of P450 down-regulation and

the suppression of metabolism. The use of in vitro methods or classic substrates to

explore pharmacokinetic changes can not accurately predict real world pharmacokinetic

parameters without using a specific pharmaceutical, test species and microbial disease.

Dot blot analysis is a good screening tool, but can not compare to the specific molecular

weight controls of the protein and mRNA bands found in Western and Northern blots.

Large doses of endotoxin were administered in the endotoxin study (Monshouwer et al.,

1996a). However, a single dose of 20 pg/kg of endotoxin was nearly lethal to swine

while five doses of 17 pg/kg at 1 hour (hr) intervals was not lethal in separate study

(Monshouw'er et al.. 1995a; Myers et al., 1997). A single dose of 17 ug/kg LPS should

desensitize hepatic microsomal enzymes and create a maximum reduction of

metabolism (Dr. Michael Myers, unpublished). Furthermore, the results were related to

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. the weight of the pig; growing pigs respond differently to LPS than finishing pigs (Dr.

Michael Myers, unpublished). High doses of LPS should reduce some cytochrome

P450 activity to undetectable levels.

On one hand, cytochrome P450IIIA is an important constitutive enzyme in the

pig and total cytochrome P450 content was decreased in infected pigs (Monshouwer et

al.. 1995b). On the other hand. LPS down-regulation is selective for IIA6 and IIB2. but

there is no effect on IIE and IIIA (Dr. Michael Myers, unpublished). In the dot-blot.

two control and two infected animals were used to show that mRNA was reduced in the

treated pigs (Monshouwer et al.. 1995b). Measurement of protein by Western and

Northern blots would be more direct and reflect a more accurate picture of P450 status.

Unfortunately, there is no statistical analysis of variability between the control and

treated animals. The dot blot of total mRNA revealed visual differences in the size and

intensity of the blot between control and treated pigs. In a group of 23 normal pigs, the

amount of several cytochrome P450's varied by as much as 6 fold for IIIA (Dr. Michael

Myers, unpublished). The mean differences in enzyme activities were real, but within

normal variability with no statistical differences among the 23 control pigs (Dr. Michael

Myers, unpublished). The Western blot in the endotoxin study requires careful

interpretation, when the two control pigs were compared to the two LPS treated pigs

(Monshouwer et al., 1995b). Cytochrome P450IA and IIIA were decreased, but the

width of the protein bands in the photograph were almost identical. These studies with

dot blots fall short without specific P450 probes that target purified proteins (controls)

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. and use too few animals to allow for adequate quantitative and statistical analysis

pertaining to dot and Western blots.

Therefore, this study has four objectives to: I) to determine whether endotoxin

administration will serve as an acceptable model for Actionobacillus pleuropneumoniae

and other Gram-negative pathogens: 2) determine whether infection with APP alters the

disposition kinetics and urinary- metabolite profile of enrofloxacin: 3) determine whether

the potential inhibition of enrofloxacin metabolism is associated with specific

cytochrome P450 isozymes; and 4) compare the endotoxin exposure with the porcine

Actinobacillus pleuropneumoniae infection model.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. CHAPTER I

LITERATURE REVIEW

Cytochrome P450 Inhibition

History

Agents that inhibited reticuloendothelial system (RES) phagocytosis also

prolonged the sleep-times of sodium pentobarbital anesthesia (Samaras & Dietz. 1953).

Additional research by other investigators showed that increased RES phagocytosis

increased the barbiturate sleep-times during anesthesia (Di Carlo et al.. 1965; Wooles &

Borzelleca. 1966; Wooles & Munson. 1971). Sleep-times were used as an indirect

measure of hepatic microsomal activity and drug metabolism in vivo. This dilemma

was solved by demonstrating that prolonged barbiturate sleep-time was directly related

to the depression of the microsomes and was independent of activation of RES-

mediated phagocytosis (Wooles & Munson. 1971). Treated animals had abnormal

sleep-times lasting 10 days after administration of an RES inhibitor which meant that

the effect of the activating agents was long-lasting (Wooles & Munson. 1971).

Zymosan was one of the agents that enhanced phagocytic activity and depressed several

drug metabolizing enzymes; aminopyrine-N-demethvlase. /?-nitroanisole O-

demethylase and aniline hydroxylase in both male and female mice (Hojo et al.. 1976).

The effect on enzyme activities was also long-lasting (7 days) after exposure to

zymosan.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Cytokines as effectors in the acute-phase response

Infections are clinically manifested by stimulation of RES activity and other

components of the immune system such as pro-inflammatory cytokines (Myers and

Kavvalek. 1995: Whicher & Evans, 1990). The source of infection should be irrelevant

and infection should depress levels and activities of drug-metabolizing enzymes after

the release of cytokines in the acute-phase response (Myers and Kavvalek. 1995).

Therefore, infections are well-known inducers of the acute-phase response

(Monshouwer. 1996). The acute-phase response is characterized by an initial local

reaction at the site of injury and a subsequent systemic reaction (Kushner. 1982: van

Miert. 1995: Monshouwer et al., 1995a.b. 1996a). After tissue injury, infection,

inflammation, neoplastic proliferation, or immunological disorders, the local reaction

consists of processes such as platelet aggregation, increased vascular permeability and

activation and accumulation of granulocytes and mononuclear cells. The systemic

reaction consists of fever, anorexia, leukocytosis, inhibition of gastric emptying,

increased endocrine hormone release and alterations in plasma cation concentration. In

a classical sense, the acute-phase response infers fluctuations of plasma protein

concentrations at different rates and to different degrees (Gitlin & Colten. 1987).

Elevated acute-phase proteins include: serum amyloid A. fibrinogen, haptoglobin. C-

reactive protein and a,-acid glycoprotein (Heinrich et al., 1990; Kushner &

Mackiewicz. 1993). Albumin and transferrin plasma concentrations diminish during the

acute-phase response and are "negative" acute-phase proteins. Acute-phase proteins are

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. mostly synthesized in the liver and aid in the defense against the causes of tissue

damage and infection (Volanakis. 1993).

Activated immune cells secrete cytokines that have a complex role in the acute-

phase response (Monshouwer 1996). Cytokines are peptides or glycoproteins weighing

from 6 to 60 kiloDaltons (kDa) that act in a non-enzymatic. hormone-like fashion as

intercellular messengers (Richards et al.. 1995). Cytokines act on a variety of tissues

and a single cytokine can be released by multiple cell types (Richards et al.. 1995).

These proteins act in an autocrine or paracrine manner, but they may act systemically in

an endocrine manner. High affinity for receptors accounts for their potency

(Monshouwer et al.. 1996b). Over 80 cytokines are known to have a wide variety o f

functions, but mainly stimulate growth and differentiation. Every cell type expresses

multiple receptors and responds to many cytokines, which makes it impossible to assign

unique biological activities. Furthermore, a particular cytokine may have multiple

biological effects with other cytokines so that there is substantial redundancy in their

mode of actions (Paul. 1989).

Pro-inflammatory cytokines and infection

Acute-phase responses are readily induced by infection. Pro-inflammatory

cytokines, such as TNF-a. IL-1. IL-6 and the interferons have important roles in

response to infections by the host (Whicher & Evans. 1990a). In animals and humans.

TNF-a is one of the first cytokines to appear after release as an initial burst (Beutler et

al.. 1985). Generally, peak systemic concentrations were reached one to two hours after

induction. Interleukin-1 is released after TNF-a and before IL-6 (Fong et al.. 1989).

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Interleukin-6 is released as a secondary burst about one hour after TNF-a and it

gradually rises for several hours (Fong et al.. 1989; Schalaby et al.. 1989; Van Deventer

et al.. 1990).

The sequential release of TNF-a. IL.-1 and IL-6 is a common response to Gram-

negative bacteria, such as Escherichia. Actinobacillus and Pasteurella species. Species

differences account for many variations to the cytokine cascade, but variations were also

due to the character and timing of the stimulatory agent (Monshouwer. 1996). For

example, interferons are the main cytokine in the acute-phase response induced by viral

infection, because patients with confirmed viral infections have significantly increased

INF serum concentrations (Joklik. 1990). Endotoxins (lipopolysaccharide) or poly IC

are inflammatory stimuli that induce the cytokine cascade. Lipopolysaccharide induces

TNF-a. while poly IC is a potent IFN-a/p inducer.

Cytokines

Tumor Necrosis Factor

History

Tumor necrosis factor -a (TNF-a) can alter physiological and immunological

events as well as induce pathophysiological responses of several disease conditions

(Myers & Murtaugh. 1995). Tumor necrosis factor has a wide spectrum of physiologic

functions; mediates hemorrhagic necrosis in tumors, wasting in chronic disease,

stimulates release of platelet activating factor, activates hepatocytes. increases IL-6

synthesis and recruits immune and non-immune cells (Edwards et al.. 1994). Usually,

tumor necrosis factor is released from macrophages and monocytes as a proximal-

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. mediator before the release IL-1 and IL-6 in septic shock. Endotoxin increases serum

TNF-a concentrations by enhancing TNF-a gene transcription approximately 10-fold,

cellular TNF-a mRNA 100-fold and a 10.000-fold increase in the quantity of protein

secreted from activated monocytes.

Early investigators observed that Trypanosoma hrucei infection caused a marked

hypertriglyceridemia along with anorexia and wasting diathesis. At first, the condition

was attributed to deficiency of lipoprotein lipase (Kawakami & Cerami. 1981; Rouzer

& Cerami. 1980). Production of the active factor was induced by endotoxin in sensitive

mice, but was not induced in endotoxin resistant mice. Serum from sensitive mice that

had been treated with endotoxin induced the wasting syndrome in resistant mice

(Kawakami & Cerami. 1981). This factor was called cachectin. In orally fed animals,

infusion of cachectin/tumor necrosis factor-a caused weight loss and muscular wasting,

accompanied by anorexia (Matsui et al.. 1993). Despite muscle wasting, there were

gains in weight, protein and DNA contents of the viscera, but no significant metabolic

abnormalities. Oral feeding prevented loss of body weight, but cachectin-treated

animals had reduced nitrogen retention and carcass weight. By contrast, there were

gains in visceral protein concentrations, which in the liver was due to a marked

proliferation of biliary epithelium. In addition, cachectin-treated animals receiving feed

developed hyperglycemia, hyperosmolality, diuresis, dehydration, azotemia and

cholestasis. In the absence of the effects of anorexia, cachectin reduced nitrogen

retention and caused metabolic and multisystem dysfunction, comparable with the

effects of clinical sepsis.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. At the turn of the century, it was noted that some tumors underwent hemorrhagic

necrosis following streptococcal infection (Kawakami & Cerami. 1981). The necrosis

was caused by TNF-a and not a bacterial component. The serum factor inducing

hemorrhagic necrosis of transplantable tumors (TNF) and the macrophage hormone

associated with cachexia in cancer and certain infectious diseases were shown to be the

same protein, cachectin (Mullin & Snock. 1990). Because an association may exist

between TNF-a and the cachectic state, the effect of TNF-a on the permeability of

epithelial barriers was evaluated. Tumor necrosis factor was shown to affect the tight

junctional region between epithelial cells, lowered the transepithelial resistance and

potential difference and increased the flow of solute between cells and across the

epithelium. These effects were dose dependent, rapidly reversible and inhibited by a

monoclonal antibody to TNF-a. This suggested that the release of TNF-a at various

sites throughout the body produced a general breakdown in the barrier function of an

epithelial cell sheet (Mullin & Snock, 1990). Furthermore, the breakdown of the

epithelial barrier may not only explain cachexia in cancer and chronic infections, but it

may also explain hemorrhagic necrosis of some tumors.

A selective inhibitor of TNF-a synthesis is pentoxify lline (Strieter et al., 1988).

Pentoxifylline is a methylxanthine derivative that inhibits phosphodiesterases. The

subsequent increase in cAMP blocks accumulation of TNF-a mRNA by decreasing the

rate of transcription, but has no effect on transitional derepression (Han et al., 1990b:

Noel et al., 1990; Schonharting & Schade. 1989). Pentoxifylline has proven to be too

toxic for practical use in swine (Dr. Michael Myers, personal communication from Dr.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Michael Murtaugh). Other methylxanthines suppress TNF-a synthesis: theophylline,

theobromide. isobutyl methylxanthine (Strieter et al., 1988). These compounds were

nearly as effective, but molar concentrations were 2 to 5 times greater than

pentoxifylline. Prostaglandin E2. misprostel. ciprofloxacin and phenylcyclopentenone

were other agents that increase cyclic AMP, inhibit accumulation of TNF-a mRNA and

prevent TNF-a production in monocytes, macrophages and lymphocytes (Edwards et

al.. 1994: Kunkel et al., 1988: Mahatana et al., 1991). Inhibition of TNF-a may impact

other cytokines, as TNF-a induces the production and release of IL-l and IL-6.

Dexamethasone and other glucocorticoids inhibit TNF-a mRNA accumulation

(Beutler et al., 1986; Han et al.. 1990b; Luedke & Cerami. 1990). Dexamethasone may

alter TNF-a mRNA concentrations by inhibiting early transduction of proteins, but it

only inhibits TNF-a if present prior to endotoxin injection. Therefore, dexamethasone

is of limited benefit during septic shock.

Tumor necrosis factor and interleukin-1 in endotoxemia and sepsis

Tumor necrosis factor has immunological, hematopoeitic. cell differentiation,

reproduction, antiviral and endotoxic/septic shock effects (Myers & Murtaugh. 1995).

Tumor necrosis factor, in high doses, produces a variety of systemic endotoxic/septic

shock effects to include; hemodynamic, pulmonary, metabolic and pathological events

(Tracey. 1991). Chronic TNF-a exposure leads to cachexia that was characterized by

weight loss, protein and lipid depletion and anorexia. Chronic TNF-a administration

directly affects the hypothalamus to induce fever and the release of corticotropin-

releasing-factor (Martin & Tracey. 1991). Depletion of body fat. induction of insulin

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. resistance in adipocytes and decreased resting membrane potentials in myocytes are

direct effects (Goldblum et al.. 1989: Kiorpes et al.. 1990; Parrilo. 1993; Stephens et al..

1988a). Anabolic responses of the liver were indirect effects which reflected a cascade

of cytokine and hormone responses that persist for hours after the TNF-a blood

concentrations have dissipated (Nemerson. 1992). The effects of intravenous bolus

injections of TNF-a and IL-ip on hepatic mitochondrial energy metabolism were

investigated in rats (Shimahara et al.. 1995). The administration of TNF-a and IL-1 (3 in

rats induced a hypermetabolic state in hepatic mitochondrial energy metabolism, which

was a pattern similar to sepsis and presumably a compensatory reaction to the increased

energy consumption

Actinobacillus and Pasteurella species are Gram-negative pathogens that

produce respiratory bacterial infections in animals (Kiorpes et al.. 1990). Subsequently,

the lung becomes septic with increased vascular permeability, edema, injury' to

endothelial cells and death of the animal (Goldblum et al.. 1989; Stephens et al..

1988a). Mortality after sepsis was directly related to the induction of TNF-a and a

tissue factor which was an inducible cell surface glycoprotein (Henry & Moore. 1988;

Levi et al. 1993; Nemerson. 1992: Parrilo. 1993; Taylor et al. 1991). Tumor necrosis

factor may cause systemic hypotension and altered cell metabolism, while tissue factor

expression results in a local blood coagulation at the inflammatory site and

systemically. Coagulation activation occurs through the expression of tissue factor on

the cell membrane surface of circulating monocytes and tissue macrophages. Tumor

necrosis factor may shift the balance between procoagulant and anticoagulant activities

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. by initiating the tissue factor pathway of blood coagulation (Aderka, 1991). The most

severe consequence of the cascade was disseminated intravascular coagulopathy and a

rapid progression to death (Hoffman et al., 1990: Thomson et al.. 1974: Vestweber et

al.. 1983). Tumor necrosis factor mediation of lung injury was induced by LPS in

sheep, dogs, cattle, goats and pigs (Baarsch et al.. 1995; Egawa et al.. 1981: Eichacker

et al., 1991; Fukushimaet al., 1992: Gerros et al.. 1993; van Miert. 1990: van Miert et

al.. 1992; Redl et al., 1990; van Miert et al.. 1992). Psuedomonas aeruginosa and

Actinobacillus pleuropneumoniae have both been used in pigs to induce acute lung

injury in studies designed to measure pulmonary alveolar macrophage cytokines, effect

of pulmonary infection on pharmacokinetic parameters and pulmonary dysfunction and

metabolic acidosis associated with Gam-negative sepsis (Baarsch et al.. 1995:

Monshouwer et al.. 1995a.b: Windsor et al.. 1994).

Previous studies demonstrated that the administration of the cytokines IL-1 and

TNF to rats in a fasting state can produce many and. if not most, of the metabolic

alterations found in patients with sepsis and injury (Ling et al.. 1995). Consequently,

another study was designed to define the metabolic effects of IL-l and TNF-a during a

fed state by continuous intravenous feeding for 20 hours and to characterize the unique

effects of IL-1 among other cytokines by employing the IL-1 receptor antagonist. IL-ra

(5 mg/kg). The effects were also explored during the endotoxemic condition induced by

infusion of 200 fig/kg of endotoxin in rats. The results showed that during feeding IL-1

was responsible for the increase of glucose flux and plasma insulin, the development of

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. insulin resistance and plasma zinc depression. In other words, IL-1 initiated conditions

that mimicked sepsis and injury that were similar to events in the fasting state.

The changes in energy expenditure have a more complex mechanism. The

results suggested that certain host responses to cytokines or endotoxin, related to protein

metabolism, differ between the fed and fasting states. These data may have clinical

relevance for the insulin-resistant state that develops during severe infection, because

IL-1 receptor antagonists may be used in conjunction with nutritional therapy to offer

additional advantages in the treatment of these drastic metabolic disorders.

Cytokines can be produced within the nervous system by various cell types,

including astrocytes, which secrete them in response to pathological processes such as

viral infections (Yu et al.. 1995). Astrocytes were known to play an important role in

the homeostasis of the nervous system by contributing to the regulation of local energy

metabolism. Tumor necrosis factor-a and IL-la markedly stimulate glucose uptake and

phosphorylation in primary cultures of neonatal murine astrocytes. The effects of TNF-

a and IL-la on glucose uptake and phosphorylation may be mediated by the

phospholipase A2 signal transduction pathway. These results demonstrated that TNF-a

and IL-la can fundamentally perturb the energy metabolism of astrocytes, possibly

impairing their ability to provide adequate energy substrates for neurons.

Interleukin-1

History

Three different investigators identified IL-I. The first described a pyrogenic

factor (endogenous pyrogen) that was produced by activated peritoneal exudate cells

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. (Heyman & Beeson. 1949). The second investigator described a factor that was

synthesized from activated mononuclear phagocytes and acted as a co-stimulator of

thymocyte proliferation, termed lymphocyte activating factor (Gery & Waksman. 1972:

Gery et al.. 1971). The third investigator described a soluble mediator, leukocyte

endogenous mediator, that regulated the acute-phase response (Kampschmidt. 1984).

The three biological factors were identified and the identical factors were given the

name, "interleukin-1” (Dinarello. 1984; Kampschmidt. 1984). Additional work

revealed that IL-1 was produced by every nucleated cell type if not most cells had IL-1

receptors on their surface (Dower & Urdal, 1987). Monocytes and macrophages were

stimulated to produce IL-l by lipopolysaccharide. interferon-y (INF-y). muranyl

dipeptide, microorganisms, poly IC and complement 5a.

Interleukin-1 in inflammation and host defense

Interleukin-1 is a key promoter of inflammation. There are two forms of

interleukin-1: IL -la and IL-1 p. In humans, there was a difference in the time frame that

the two forms are produced; the a form was produced early and then the cell switched

to producing the p form (Dr. Michael Myers, personal communication). Even though

there was only a 16 to 20% homology between the two forms, they both bound to the

same Type I receptor (Bomsztyk et al., 1989; Horuk et al., 1989). There was also

binding to Type II receptors, but without initiating biological activity. Hence. IL-1 type

II receptors are also termed decoy receptors since they have no biological effects (Dr.

Michael Myers, personal communication). Adherence of leukocytes to endothelial cells

and the degradation of cartilage have been examined in detail. In bovine and porcine

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. domestic species, IL-1 and other cytokines activate endothelial cells to express surface

molecules that implement the adhesion and extravasation of IL-1 activated neutrophils

(Canning & Baker. 1990; Goodman et al., 1991; Johnston et al., 1992; Modat et al.,

1990; Sample & Czuprynski. 1991; Zimmerman et al.. 1992). Interleukin-1 stimulates

collengenase production and proteoglycan degradation by cartilage explants in vitro

(Buideetal.. 1992; Conquer et al.. 1992; Mauviel et al.. 1988; Mori et al.. 1989). The

influence of IL-l on cartilage degradation may be an important part of the inflammatory

process in arthritic joints (Lederer & Czuprynski. 1995). Lyme disease patients and

rheumatoid arthritis patients have increased production of IL-1. which further supports

the role of IL-l as a biological mediator in the pathology of arthritis (Beck et al.. 1989;

Smith et al.. 1989).

Most of the literature on IL-1 comes from studies on mice and human cells.

Veterinary researchers must extrapolate from these studies to a particular species

(Lederer & Czuprynski. 1995). There was limited literature available on the biological

effects of IL-1 in cattle, pigs and chickens (Lederer & Czuprynski. 1995). Interleukin-

la and IL-1 (3 are natural porcine isoforms (Huether et al.. 1993; Maliszewski et al.,

1990). These bind with similar high affinity to porcine chondrocytes and synovial

fibroblasts (Bird & Saklatvala. 1986,1987). Up-regulation of IL-(3 mRNA was observed

for LPS. phorbol myristate acetate and TNF-a (Huether et al.. 1993). Dexamethasone

and IL-4 down-regulated mRNA transcription (Huether et al.. 1993). Interferon alone

did not induce release of IL-1 activity in porcine monocytes, but IL-1 was increased by

co-stimulation with LPS (Cllarieys et al.. 1990).

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Porcine and human IL-1 have been shown to modulate a host of biological

responses by porcine cells in vitro. Human IL-1 inhibited proteogylcan synthesis

without stimulating its degradation by porcine cartilage explants (Nietfeld et al.. 1990).

Porcine IL-la decreased collagen synthesis by human rheumatoid synovial cells

(Mauviel et al.. 1988). Interleukin-1 inhibited progesterone secretion and increased the

growth of high density cultures of granulosa cells from small or medium sized ovarian

follicles, but had no effect on mature granulosa cells (Fukuoka et al., 1989). Little is

published on the effects of IL-1 on porcine leukocytes. It is known that human IL-la

induced natural killer (NK) cell lysis of tumor cell targets (Knoblock & Canning. 1992).

Interleukin-6

History

Interleukin-6 is a pleiotropic molecule with notable purposes in inflammation,

hemopoiesis and lymphocyte activation and differentiation (Richards el al.. 1995).

Rodent models of inflammation have provided in vivo information on the function of

IL-6. but little data was available in most food-producing animals. Most of the

investigators have reported work completed in human, rodent and swine systems.

The first observations of IL-6 effects occurred in 1921 when inflamed patients

yielded blood samples with high erythrocyte sedimentation rates. Erythrocyte

sedimentation rates are still used today as non-specific markers of inflammation. The

increased erythrocyte sedimentation rate w'as due to elevation of serum liver proteins.

Some of these proteins were acute-phase proteins such as fibrinogen. Interleukin-6 w as

largely responsible for the induction of these proteins from the liver. Early studies that

*% -» j j

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. sought to isolate and identify IL-1 were obscured by the probable contamination of

samples with IL-6 and the induction of IL-6 in vivo by crude preparations of IL-1. It

was later determined that IL-I induced fever and the production of liver acute-phase

proteins in rat models of inflammation (Kampschmidt & Upchurch. 1974:

Kampschmidt et al., 1982). When monocyte or macrophage products were examined, it

was shown that IL-1 and TNF-a were not fully active (Gauldie et al., 1987b; Koj et al..

1984; Perlmutter et al., 1986). A distinct cytokine . termed hepatocyte stimulation

factor (HSF). was identified which induced a full spectrum of hepatic acute-phase

responses (Bauer et al.. 1984; Baumann et al.. 1987a.b: Darlington et al.. 1986:

Goldman & Liu. 1987: Koj et al.. 1984; Ritchie & Fuller. 1983; Woloski & Fuller.

1985). The IL-6 molecule was purified and cloned and was subsequently proven to be a

potent molecule for regulating hepatocyte responses (Gauldie et al.. 1987a: Hirano et

al.. 1986).

Acute-phase protein production

It was suggested that mediators responsible for serum acute-phase proteins were

released by leukocytes at sites of inflammation (Koj, 1974). These mediators acted

systemically on lymphocytes. The primary role of IL-6 in vivo was to initiate the

hepatic inflammatory response. Interleukin-6 serum assays of animals undergoing an

inflammatory response revealed a peak concentration of IL-6 at 6-12 h before peak

concentrations of serum acute-phase proteins (Gauldie et al.. 1992; Jablons et al., 1989;

Nijsten el al.. 1987; Schreiber. 1987; Schreiber et al.. 1989). The 6 to 12 h time lag

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. reflected the time required to synthesize and release cytokines into the systemic

circulation, interact with hepatic receptors and stimulate hepatic synthesis and secretion.

Treatment with a single low dose (80 to 800 ng) of IL-1 24 h before a lethal

bacterial challenge of granulocytopenic and normal mice enhanced non-specific

resistance (Vogels et al.. 1993). Interleukin-1 induced secretion of acute-phase proteins,

liver proteins w hich possess several detoxifying effects. The role of these proteins in

the IL-1-induced protection was investigated. Inhibition of liver protein synthesis with

D-galactosamine completely inhibited the IL-1-induced synthesis of acute-phase

proteins. D-galactosamine pretreatment abolished the protective effect of IL-1 on

survival completely in neutropenic mice infected with Pseudomonas aeruginosa or

partially in non-neutropenic mice infected with Klebsiella pneumoniae. Pretreatment

with IL-6. a cytokine induced by IL-1. did not reproduce the protection offered after IL-

1 pretreatment, nor did it enhance or deteriorate the IL-1-enhanced resistance against

infection. A protective effect of IL-1 via effects on glucose homeostasis during the

acute-phase response was investigated by comparing plasma glucose concentrations in

IL-1-treated mice and control mice before and during infection. Although glucose

concentrations in IL-1-pretreated mice were somewhat higher in the later stages of

infection, no significant differences from concentrations in control mice were present

and the glucose concentrations in control and treated animals never fell to hypoglycemic

values. Interleukin-1-induced non-specific resistance was mediated neither by the

induction of IL-6 nor by the effects of IL-1 on glucose homeostasis. Acute-phase

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. proteins generated after IL-1 pretreatment seem to play a critical role in the IL-l-

induced protection to infection.

Immune effects

Interleukin-6 is a multifunctional cytokine whose circulating concentrations are.

under physiological conditions, below detection, but whose production was rapidly and

strongly induced by several pathological and inflammatory stimuli (Poli et al.. 1994).

Interleukin-6 has been implicated in a number of cell functions connected to immunity

and hematopoiesis. Cells of the immune system that were affected by IL-6 were: B

lymphocytes, helper T lymphocytes, cytokine T lymphocytes and NK cells (van Snick.

1990). In vitro. B lymphocytes require IL-6 for late stage differentiation of B cells

(Muraguchi et al.. 1988). Interleukin-6 augmented secondary antibody responses in

vivo (Takatsuki et al.. 1988). Interleukin-6 played a major role in the development of

antigen specific humoral responses, but there were no supporting experiments in food-

producing animals. Interleukin 6 may also synergize with IL-1 to regulate the initial T-

cell activation (van Snick. 1990). Lastly. IL-6 may increase the lysing activity of

natural killer cells (Luger et al.. 1989).

Interleukin-6 deficient mice were unable to mount a normal inflammatory

response to localized tissue damage generated by turpentine injection (Fattori et al..

1994). The induction of acute-phase proteins was dramatically reduced, mice did not

lose body weight and only suffered from mild anorexia and hypoglycemia. In contrast,

when systemic inflammation was elicited through the injection of bacterial

lipopolysaccharide. these parameters were altered to the same extent both in wild-type

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. and IL-6-deficient mice, demonstrating that under these conditions IL-6 function was

dispensable. Moreover, LPS-treated IL-6-deficient mice produce three times more

TNF-a than wild-type controls, suggesting that increased TNF-a production might be

one of the compensatory mechanisms through which a normal response to LPS was

achieved in the absence of IL-6. Different patterns of cytokines were involved in

systemic and localized tissue damage which identified IL-6 as an essential mediator of

the inflammatory response to localized inflammation. Interleukin-6 may regulate

(locally) bone turnover and are essential for the bone loss caused by estrogen deficiency

in mice (Poli et al.. 1994).

Changes in the parameters of specific and non-specific immunity after challenge

of the immunized animals with Marburg virus were characterized (Ignat'ev et al.. 1994).

Mediators of the immune response. TNF-a and rNF. were shown to play different roles

in the development of the disease and "protection" of animals. The survival rate of the

immunized animals after the challenge with Marburg virus was determined by the

intensity and level of the immune response after challenge rather than by the

concentrations of post-vaccination immunity at the time of the challenge.

Hematopoiesis

Interleukin-6 interacted with IL-3 to enhance stem cell differentiation (Wong &

Clark. 1988). Strong inducing effects on megakaryocyte colony formation were

impressive (Akira et al.. 1990; Kishimoto. 1989). Interleukin-6 and IL-11 may have a

use in supporting cancer patients, but high concentrations of IL-6 cause pathological

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. increases in proliferation of hematopoietic myeloid cell lineage on multiple myeloma

and Castleman's disease (Akira et al., 1990; Zhang et al.. 1990).

Anti-inflammatorv mediators

The initiation of the acute-phase response by IL-6 has been well characterized

(Bauman & Gauldie. 1990; Gauldie, 1991; Koj. 1985; Richards & Gauldie. 1995;

Travis & Salvesen, 1983). On the other hand. IL-6 has been shown to inhibit the release

of pro-inflammatory cytokines IL-1 and TNF-a from monocytes in vitro (Aderka et al..

1989; Mancilla et al., 1990). In vivo. LPS induced inflammation in the rat lung was

reduced by IL-6 (Guo et al., 1992; Ulichet a l, 1991). Interleukin-6 also inhibited joint

inflammation in a collagen-induced arthritis model (Ikuta et al.. 1991). Tissue

inhibition of metalloproteinases-1 was enhanced by IL-6. The balance of

metalloproteinases and tissue inhibitors of metalloproteinases-1 help govern the net

catabolism of connective tissue matrices in normal physiological processes and in

pathological conditions involving joints and lungs (Lotz & Gueme. 1991).

Other systemic functions of IL-6 in inflammation include actions in the brain.

Interleukin-6 was shown to enhance ACTH production in vitro and to increase ACTH

in serum of rats (Perlstein et al., 1993; Woloski et al.. 1985). These ACTH effects

probably cause increases in glucocorticoids in serum after inflammatory stimuli.

Increased glucocorticoids may modulate numerous functions such as decreased cytokine

production, suppression of lymphocyte responses and enhancement of regulation of

various acute-phase protein genes (Richards et al., 1995). In addition. IL-6 has also

been shown to increase body temperature (Akira et al.. I990a.b). Thus, IL-6 has a

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. major role in several aspects of the systemic inflammatory response. Analysis of these

activities in mice, rats and rabbits and correlation with IL-6 levels in humans, suggest

that similar activities of IL-6 may occur in pigs, cattle and poultry in vivo. However,

this has not been determined.

Xenobiotic Immunomodulation by Cytokines

Cytokines in acute and chronic disease

Interleukin-1. IL-6 and TNF-a act synergisticallv to induce fever, initiate acute-

phase protein synthesis, activate neutrophil and macrophage antimicrobial activity,

decrease serum iron and zinc concentrations and induce tissue catabolism (Dinarello.

1986; Hardie & Kruse-Elliot. 1990; Le & Vileck. 1987; Whicher & Evans. 1990b).

These were beneficial events in the formation of an effective inflammatory and immune

response to invading microorganisms and in tumor surveillance. The inflammatory

reaction was beneficial for humans when its effects were limited to the pathogens

(Hakim. 1993). The insufficiency o f a component of the inflammatory reaction such as

the production of reactive oxygen species (ROS). which was seen in chronic

granulomatous disease, leads to severe and recurrent bacterial infections. In other

situations, inflammatory reactions were deleterious because they were directed against

normal tissues instead, or in addition to. pathogens.

In some instances, the behavior of the phagocytes was modified, because they

have been primed by inflammatory molecules such as TNF, LPS. interleukins or

interferons. Priming often led to a decreased speed of locomotion of the leukocytes

with an increased susceptibility to their stimuli. The combination of these effects leads

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. to a premature release by the phagocytes of their killing and degradative factors.

Production of ROS such as that seen during irradiation, drug metabolism, or ischemia

followed by reperfusion induced inflammatory reactions with a secondary amplification

of ROS production. Acute ROS production can also lead to thrombosis, whereas

chronic ROS production induced a chronic inflammatory reaction of the endothelium

with atherosclerosis as a possible consequence. Reactive oxygen species may either

positively or negatively control the activity of inflammatory molecules. The

multiplicity of the cross reactions between ROS and inflammation suggests a search for

new therapeutic drugs that disconnect these two events.

In contrast, pro-inflammatory cytokines also have adverse effects in both acute

and chronic diseases (Dinarello. 1986: Hardie & Kruse-Elliot. 1990: Lahdevirta et al..

1988: Le & Vileck. 1987; Morris & Moore. 1991; Sprouse et al.. 1987; Whicher &

Evans. 1990b). Tumor necrosis factor and IL-1 were involved in the induction of shock

associated with Gram-negative sepsis, trauma and bums (Dinarello. 1991; Eichacker et

al.. 1991: Hardie & Kruse-Elliot. 1990: Tracey & Cerami. 1989; Whicher & Evans.

1990b). Pathogenic sequellae associated with IL-1 and TNF-a release included

hypotension, depressed myocardial function, vascular leakage, microvascular

thrombosis, tissue neutrophilic infiltration and necrosis and multiple organ failure.

Chronic production of pro-inflammatory cytokines caused cachexia found in

inflammatory and neoplastic disorders (Bistrian et al.. 1992; Cotran. 1987; Grunfeld &

Feingold. 1992: Le & Vileck. 1987; Tracy & Cerami. 1992). The cachexia was a result

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. of increased oxidation of amino acids, muscle proteolysis and accelerated synthesis of

acute-phase proteins in the liver.

Tumor necrosis factor and IL-1 were also involved in tissue injury associated

with inflammation through interaction with neutrophils and endothelial cells. (Cotran.

1987; Le & Vileck, 1987; Ward. 1991 a.b). Both cytokines sustain the mobilization of

mature neutrophils from bone marrow and activate the neutrophils. Exposure of

endothelial cells to TNF-a or IL-1 induced expression of leukocyte adhesion molecules

and contact between endothelial cells and neutrophils exposes endothelial cells to toxic

oxygen radicals and proteases. Tumor necrosis factor and IL-1 enhanced the

susceptibility of endothelial cells to injury by increasing concentrations of xanthine

oxidase and iron inducing the generation of oxygen radicals and promoted conversion

into intracellular hydroxyl radicals. Tumor necrosis factor and IL-1 increased the

amount and surface expression of tissue factor by endothelial cells. Tissue factor

expression promoted thrombus formation at the site of inflammation and may play a

role in disseminated intravascular coagulopathies during shock and sepsis.

Acute inflammation is a natural response to tissue injury during sepsis.

Excessive tissue destruction, shock, multiple organ failure and death are deleterious

effects caused by pro-inflammatory cytokines. Therapeutic regimes to limit

inflammatory responses may be life saving if accompanied by an antibiotic.

Approaches to limit inflammation include: inducing neutropenia, blocking neutrophil

migration from the blood, antioxidants, iron chelators, antiproteases, inhibitors or

antagonists of IL-1 and TNF-a and inhibitors of IL-1 and TNF-a production, inhibitors

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. of cyclooygenase isozymes (COX-1 and COX-2) (Cotran. 1987; Landoni & Lees. 1996;

Le & Vileck, 1987; Ward, 191a.b). Therapeutics, such as ibuprofen. which prevent

effector cells from reaching the site of inflammation or which inhibit synthesis of pro-

inflammatory cytokines have the most promise for prevention of tissue injury

(Eichackeret al.. 1992; Mullen et al., 1993; Nacy et al.. 1990; Stephenset al.. 1988b).

Pro-inflammatory cytokines regulate local inflammatory reactions, but may also

gain access to the circulation and evoke systemic effects known as the acute-phase

response (van Miert, 1995). A study was performed to improve the understanding of

the pathophysiology of pro-inflammatory cytokines in ruminants using TNF-a. IL-la/p

and INF-a/y (van Miert. 1995). The following aspects were perceived from this study:

a) cytokine therapy given before or just after microbial challenge induces in vivo

antimicrobial activity, cytokines potentiate in vivo antimicrobial activity of antibiotics:

b) cytokines may act as biological response modifiers for enhancing specific immunity

to vaccines and c) cytokines may affect drug absorption, disposition and metabolite

formation in disease states.

Cytokine therapy in conjunction with antibiotics was further explored in a study

using Staphylococcus aureus (Sanchez et al.. 1994a.b). A bovine blood

polymorphonuclear neutrophil system was used to determine the ability of cytokines,

alone or in combination with various antibiotics, to enhance the intracellular killing of

Staphylococcus aureus sequestered within these phagocytes. Pretreatment of blood

polymorphonuclear neutrophils with human recombinant TNF-a. bovine macrophage

supernatant TNF-a. or human recombinant IL-ip enhanced the killing of S. aureus by

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. the phagocytes compared with that of untreated polymorphonuclear neutrophils. This

enhancement was dose-dependent and required 30 minutes of contact between the

cytokine and the blood polymorphonuclear neutrophils. However, mammary gland

polymorphonuclear neutrophils, macrophages and blood monocytes showed no

enhanced killing activity following pretreatment with TNF. Pretreatment of

polymorphonuclear neutrophils with TNF-a and subsequent exposure to various

antibiotics after infection with S. aureus enhanced intracellular bacterial killing by

certain antibiotics, including paldimycin. rifampin and ciprofloxacin, but not antibiotics

that do not normally kill intracellular S. aureus. Cytokine therapy may be a useful

adjunct to antibiotic therapy in the treatment of bovine staphylococcal infection and

mastitis.

Tumor necrosis factor plays a central role in the initiation of inflammation and

inhibitors of TNF-a production would be a logical choice as inhibitors of inflammation

(Cavaillon & Cavaillon. 1990). Production of TNF-a can be blocked at two

pharmacological sites. The first site, blocked by drugs such as dexamethasone and

cyclosporin A. halted pre-translational derepression without significantly inhibiting

transcription (Han et al.. 1990a: Trinchieri. 1991: Weinberg et al., 1992). Blocking at

the second site for compounds that increased cAMP. such as pentoxifylline,

prostaglandin E2. forskolin and dibutyrl cAMP. prevented translation of TNF-a (Han et

al.. 1990a: Trinchieri. 1991). Increased concentrations of cyclic AMP blocked

accumulation of TNF-a mRNA. as cyclic AMP caused a decrease in the rate of

transcription (Han et al., 1990a; Noel et al.. 1990). It had no effect on post-

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. transcriptional control of mRNA. The mechanism for this effect was not clearly

defined, but may involve the retention of a necessary transcription factor (NF-k P) in the

cytoplasm due to the phosphorylation of an associated inhibitor protein (I-k P).

Blocking TNF-a mRNA by increasing cAMP concentrations may require more than a

therapeutic dose of pentoxifylline. A pharmacological dose of pentoxifylline had no

protective effects on the acute-phase response reactions induced by a pyrogenic dose of

Escherichia coli LPS (van Duin et al., 1995). However, both dexamethasone which

blocks arachadonic acid release and cyclosporin A have been reported to prevent IL-1

production (Cavaillon & Cavaillon. 1990).

Chlorpromazine (CPZ) inhibited TNF-a production and protected against

endotoxic shock in mice (Menzozzi et al.. 1994). The effect of pretreatment with CPZ

(4 mgdcg intraperitoneally 30 min before endotoxin) was compared with dexamethasone

(3 mg/kg) after induction of endotoxin-induced cytokines (IL-la. IL6. IL-10 and TNF)

in the serum of mice. The inhibitory effect of CPZ and dexamethasone on serum and

spleen-associated TNF-a release was studied. Induction of IL-1 and IL-6 was inhibited

by dexamethasone but not by CPZ. Dexamethasone did not affect IL-10. while CPZ

potentiated its induction. Chlorpromazine also inhibited spleen-associated TNF-a

induction in LPS-treated mice, suggesting an effect on the synthesis of TNF.

Chlorpromazine inhibited TNF-a induction by Gram-positive bacteria (heat-killed

Staphylococcus epidermidis) and by anti-CD3 monoclonal antibodies. Intraperitoneal

administration of CPZ also inhibited the induction of brain-associated TNF-a induced

by intra-cerebroventricular injection of LPS. Therefore. CPZ was a more specific

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. inhibitor of TNF-a production than dexamethasone. In particular. CPZ increased the

induction of IL-10. which is a "protective" cytokine known to inhibit LPS toxicity and

TNF-a production. Chlorpromazine inhibited TNF-a production in vivo, irrespective of

the TNF-a stimulus used to induce TNF. Finally, dexamethasone can potentiate TNF-a

production when administered 24 h before LPS which is called "rebound'’. CPZ did not

induce the "rebound” effect of dexamethasone when administered 24 h before LPS but

it did inhibit TNF-a production after 24 h . Chlorpromazine has potent

immunomodulatory effects in vivo: it induces humoral autoimmunity in up to 50% of

patients, inhibits delayed-type hypersensitivity reactions and suppresses lethal immune

hyperactivation in animal models of septic shock (Tarazona et al.. 1995). In an in vivo

model of acute superantigen-driven immune activation, CPZ independently down-

regulated the production of various T cell-derived cytokines (IL-2. IFN-y. IL-4. TNF-a

and GM-CSF) and up-regulated the secretion of IL-10. The mechanism by which CPZ

and related drugs enhance humoral autoimmune reactions, block cellular immune

responses and prevent lethal septic shock in vivo may explain the mechanism of action

of CPZ in endotoxic shock.

The effect of different inhibitors of cytochrome P450 on TNF-a production can

repress TNF-a production (Fantuzzi et al.. 1993). Metyrapone and SKF525A (100 and

50 mg/kg, i.p.. respectively) suppressed serum TNF-a induced by co-treatment with

LPS. (2.5 micrograms/mouse). Inhibition was independent of endogenous

corticosteroids since it was also observed in adrenalectomized mice. In vitro production

of TNF-a by endotoxin-stimulated human monocytes was also inhibited by metyrapone

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. and SKF525A. Since lipoxygenase (LO) inhibitors also block TNF-a production and

metyrapone was reported to inhibit LO. it was suggested that inhibition by metyrapone

and SKF525A might be due to inhibition of either LO or a cytochrome P450 implicated

in the oxidation of endogenous substrates involved in the inflammatory response.

Glucocorticoids

Dexamethasone and other glucocorticoids inhibited TNF-a mRNA

accumulation (Beutler et al.. 1986; Han et al.. 1990b; Luedke et al., 1990).

Dexamethasone was believed to alter TNF-a mRNA levels by inhibiting early

transduction proteins. This may explain why dexamethasone inhibited TNF-a

production only if present prior to administration of endotoxin. It also explains why

dexamethasone was of limited benefit during septic shock. Thalidomide also

destabilized TNF-a mRNA and reduced its half-life from approximately 30 to 17 min.

The effect of thalidomide was selective for TNF-a (Moreira et al., 1993). Its

mechanism of action was distinct from those of the glucocorticoids and pentoxifylline,

thus accounting for the synergistic effects of these drugs.

Glucocorticoids acts both at the transcriptional and post-transcriptional level to

inhibit TNF-a synthesis (Han et al.. 1990a; Trinchieri. 1991). Endotoxin induced TNF-

a gene expression was associated with an increase in NF-kB nuclear factors binding kB

motifs. Tumor necrosis factor action was mediated through receptor signaling

pathways, as microinjection of TNF-a into the cell has no effect (Niitsu et al.. 1985).

Tumor necrosis factor rapidly induced production of diacylglycerol (DAG) from

membrane phospholipids as a result of activation of a phosphatidylcholine-specific

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. phospholipase C (PC-PLC) following binding to the type I receptor. Diacylglycerol

formation did not result in changes in cellular Ca~+ or inositol trisphosphate (Shutze et

al.. 1991). Diacylglycerol produced by PC-PLC resulted in the activation of two

additional signal pathways, protein kinase C (PK.C) and acidic sphingomyelinase which

breaks down cellular sphingomyelin to produce ceramide (Schutze et al.. 1992). Protein

kinase C triggered the activation of the proto-oncogenes c-myc. c-fos and c-jun.

The sphingomyelin/ceramide pathway functions to activate NF-kB. Activation

of NF-kB resulted when the inhibitory subunit I-kB was released from the cytoplasmic

complex containing the p50 and p65 (rel A) subunits. This dimer was then translocated

to the nucleus. Ceramide produced from neutral sphingomyelinase also may activate

NF-kB (Yang et al.. 1993). Neutral sphingomyelin (SM) was directly activated by the

TNF-a receptor (Kim et al.. 1991). Neutral SM broke down membrane sphingomyelin

to ceramide. which activated a membrane-bound. 97 kDa-protein kinase. This activated

kinase then phosphorvlated substrates containing the motif X-Ser/Thr-Pro-X which was

the consensus sequence for the action of mitogen-activated protein (MAP) kinases.

Tumor necrosis factor binding resulted in the phosphory lation of 42- and 44-kDa MAP

kinases (Vietor et al.. 1993: Raines et al.. 1993). Tumor necrosis factor activation of

the MAP kinases are responsible for production of arachidonic acid through the

activation of both a secreted phospholipase A2 (sPLA2) and a cellular phospholipase

A2 (cPLA2) (Lin et al.. 1993: Hoek et al.. 1993). Arachidonic acid metabolites play a

role in the induction of c -fos and cell cytotoxicity (Jones et al.. 1993: Chang et al..

1992; Reid et al.. 1991).

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Glucocorticoids strongly inhibited translational derepression of TNF-a (Han et

al.. 1990a). A highly conserved region in the 3* untranslated mRNA region may be

responsible for post-translational control. The 3' untranslated region contains the

(UUAU)n sequence common to many cytokine mRNAs. which may regulate messenger

RNA stability (Jongeneel, 1992). Stimuli that induced NF-kB translocation to the

nucleus and binding to the promoter region were potent inducers of TNF. Endotoxin

was a strong inducer of NF-kB in macrophages and phorbol mvristate acetate (PMA).

the classic inducer of protein kinase C activity and TNF-a itself, also elicited the

appearance of NF-kB in the nucleus. Various transactivator viral proteins, such as the

Taxi gene of HTLV-I. activated the TNF-a promoter through the NF-kB enhancer

element (Albrecht et al.. 1992). These observations showed that the activation of NF-

k B was a major route by which TNF-a expression was induced in target cells. Since

NF-kB was a universal transcription factor and was activated by numerous conditions,

including bacterial products, viruses and their products, cytokines, mitogens and

physical and oxidative stresses, it was not surprising that expression of TNF-a has been

implicated in many physiological and pathophysiological processes (Baeuerle &

Henkel, 1994).

Another mechanism of TNF-a suppression may be through lipocortin induced

inhibition of phospholipase A2 (Goulding & Guyre. 1992). Many of the anti

inflammatory effects of glucocorticoids were dependent on lipocortin 1 synthesis.

Lipocortin 1 is a calcium binding protein in the annexin family. Lipocortin inhibited

phopholipase A2 which resulted in decreased production of platelet activating factor.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. leukotrienes and prostaglandins (Fradin et al., 1988). The binding of lipocortin to

neutrophils and monocytes reduced chemotaxis and attenuated the release of oxygen

radicals and eicosanoids (Goulding & Guyre. 1992).

Efficacy of glucocorticoids in endotoxic shock

Synthetic glucocorticoids, such as dexamethasone and non-steriodal anti

inflammatory drugs, such as phenylbutazone, are often used as immunosuppressors in

veterinary practice (Bertini et al.. 1989; Edwards et al.. 1994). The scope o f treatment

extends from mild allergic reactions to septic shock. Glucocorticoids inhibit TNF-a

mRNA transcription and prevent translation when corticosteroids were administered

prior to LPS administration. A lag time is needed for blocking mRNA transcription and

translation. Cytokines. TNF-a in particular, may contribute to inflammation through

eicosanoid activation. Arachidonic acid metabolites seem to function as secondary

messengers to escalate the inflammatory processes. Pretreatment with the

cyclooxygenase inhibitor, ibuprofen. and other non-steriodal anti-inflammatory agents

prior to endotoxin exposure may dampen cytokine-mediated prostaglandin responses.

At the same time, negative feedback inhibition of cytokine production by prostaglandins

is also blocked. The specific actions of these therapeutic steroids and non-steroids on

cytokine mediated alteration in cytochrome P450 metabolism of antibiotics was

discovered in the present decade. Efficacy of the veterinary immunomodulators in

domestic animals has often been assumed, because of extrapolation from human and rat

data (Myers & Kawalek. 1995).

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Dexamethasone attenuation of experimental endotoxin-induced acute respiratory

failure in pigs has been evaluated (Olson et al., 1988). Dexamethasone (5 mg/kg) was

given intravenously 18 and 1 hour before administration of endotoxin. The

hemodynamic alterations attributed to endotoxin infusion were attenuated and

leukotriene B4 was blocked (Weiss. 1995). These preliminary data suggested that

modulation of inflammatory cytokines may be beneficial in attenuating acute infectious

pneumonia and septic and endotoxic shock. Perhaps combinations of drugs, such as a

phosphodiesterase inhibitor and a cvclooxygenase and/or lipoxygenase inhibitor may

prove to be effective.

Cytochrome P450 Subfamilies and Isozymes

Rat

The following cytochrome P450s subfamilies have been identified in the rat: IA.

11 A. IIB, IIC. IID. HE. IIG. Ill A. IVA. IVB. IVF. VII. XIA. XIB. XVII. XIX and XXVII

(Nelson et al., 1993). Analysis of interspecies differences of P450 primary sequence

showed a strong evolutionary conservation of the mammalian IA genes (Wrighton &

Stevens. 1992). In the rat. IA1 was constitutively expressed at low levels in the liver

and in extrahepatic tissues, such as the lung (Ryan & Levin. 1990). After treatment

with polycyclic aromatic hydrocarbons (PAH), such as 3-methylcholanthrene. IA1

concentrations were increased by induction. The second gene of the P450IA subfamily

vvas IA2. which plays a major role in the rat with the bioactivation of a multitude of

carcinogens and mutagens (Guengerich. 1988: Nebert et al.. 1987).

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. The P450IIA subfamily has been extensively studied in the rat. This subfamily

had three members (Gonzalez. 1988; Nebert et al.. 1991). The IIA1 isozyme was the

most extensively studied and was originally referred to as testosterone 7a-hydroxylase

(Ryan et al.. 1979). The second rat form was IIA2. Although it was highly related to

IA1 by amino acid sequence, it did not demonstrate the same degree of specificity with

respect to testosterone hydroxylation (Arlotto et al.. 1989). Furthermore. IIA1 and IIA2

were not expressed similarly in the untreated rat and were not induced by PAH (Arlotto

et al.. 1989: Ryan & Levin. 1990; Ryan et al.. 1979). The third rat IIA subfamily

member . IIA3 was expressed exclusively in the lung, where the concentrations of

mRNA encoding IIA3 were induced by 3-methylcholanthrene (Kimura et al.. 1989).

The P450I1B6 subfamily were the major forms of P450 induced by

phenobarbital and the most widely studied in humans (Adesnik & Atchison. 1985: Ryan

& Levin. 1990). Phenobarbital induced IIB1 and IIB2 in the rat. where only IIB2 was

expressed in untreated rats (Adesnik & Atchison. 1985: Ryan & Levin. 1990). The

substrate specificity of these two forms overlap, but the activity of IIB1 is greater than

IIB2 for the majority of substrates (Guengerich et al.. 1982; Ryan & Levin. 1990).

Most of the P450IIC subfamily was composed o f a large number of forms that

were historically referred to as the constitutive P450s (Ryan & Levin. 1990). This

designation includes most of the rat forms that were expressed in a gender-specific

manner. As of 1992. seven rat IIC genes had been identified.

The HE subfamily is a very important metabolic system in regards to toxicology.

In the rat. the HE subfamily contains only one member. IIE1 (Nerbert et al.. 1991).

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. This P450 was unusual, because it was induced by many common small organic

molecules (Wrighton & Stevens, 1992). Furthermore. IIE2 often catalyzed the

conversion of numerous low molecular weight agents to metabolites that were more

chemically reactive than the parent compound. Therefore. IIE2 was first isolated as the

low Km N-nitrosodimethylamine N-demethylase. which was induced after treatment

with pvrazole, acetone, isoniazid, ketone, isopropanol and ethanol (Patten et al., 1986;

Ryan et al., 1985). Rat IIE1 was also induced by physiological status, such as fasting,

diabetes and obesity that result in accumulation of acetone or ketones. It has been

demonstrated that rat HE 1 was responsible for the metabolic activation of a large

number of halogenated toxins to include: carbon tetrachloride, enflurane. alcohols. N-

nitrosodimethylamine. aniline, benzene, acetone and acetominophen.

The final P450 subfamily involved in hepatic drug metabolism is the IIIA

subfamily. The various members of this subfamily have been widely studied, because

they were shown to be responsible for the metabolism of clinically and toxicologically

important agents. The IIIA subfamily is inducible by steroids, macrolide antibiotics,

imidazole antifungals. clotrimazole and phenobarbital (Bachmann et al. 1992; Gonzalez.

1988: Watkins. 1990). Two genes were identified in the rat. IIIA 1 and IIIA2 (Hardwick

et al.. 1983; Gonzalez et al., 1986). However, it is important to know that various

purification procedures and immunochemical characterizations indicate that three or

four proteins may be present in the P450III family (Halpert. 1988: Hostetler et al., 1987:

Graves et al.. 1987). It was found that rat III Al was induced by glucocorticoids, such

as dexamethasone and was not expressed in untreated adult rats (Gonzalez et al.. 1986;

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Wright & Paine. 1994) On the other hand. IIIA2 was not induced by glucocorticoids

and was expressed only in adult male rats. Finally, both IIIA 1 and IIIA2 were induced

by phenobarbital (Gonzalez et al., 1986). These forms of rat P450 were specifically

responsible for erythromycin N-demethylation. steroid 6p-hydroxylation and the

formation of a stable metabolic intermediate complex with triacetyloleandomycin

(Wrighton et al.. 1985; Sonderfan et al., 1987). The stable metabolic intermediate

complex with triacetyloleandomycin inhibited IIIA1 and IIIA2. Hepatocytes from

female rats lack P450IIIA2 (Fau et al., 1994). Hepatotoxicity due to flutamide. an

androgen, was lower in female rats than in male rats. Flutamide led to the covalent

binding of reactive electrophilic metabolites in male rat hepatocyte proteins. The

female rat did not produce reactive electrophilic metabolites which resulted in lower

covalent binding to proteins. In a like manner, male rats had reduced covalent binding

and lactate dehydrogenase release from hepatocytes after piperonyl butoxide. a P450

inhibitor, administration. The toxicity in male rats was increased by P-naphthoflavone

which induces P450IA. It was concluded that flutamide was toxic to rat hepatocytes as

a result of the P450-mediated (IIIA and IA) formation of electrophilic metabolites. To

date, much less is known about the regulation of expression or substrate specificity of

other rat IIIA subfamily members, as compared to humans.

Swine

The following cytochrome P450 families and subfamilies have been identified in

the pig: XIA1. XVII and XXIA1 (Nelson et al., 1993). Compared to the rat. swine

P450 subfamilies and isozymes have not been extensively studied (Axen et al., 1994).

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Therefore, there is very little published information concerning substrates, inducers and

inhibitors. A small number of studies have appeared in the literature and seem to deal

primarily with the P450IIIA subfamily. One of these studies investigated the

simultaneous use of the antibiotic, tiamulin, with certain ionophoric antibiotics

(monensin. salinomycin) which may give rise to a toxic interaction in pigs and poultry

(Witkamp et al.. 1994). The effects of tiamulin on hepatic cytochrome P450 activities

in vitro were tested using pig liver microsomes. When tiamulin was added to the

incubation medium, the N-demethvlation rate of ethylmorphine and the hydroxylation

of testosterone at the 60- and 1 la-positions was strongly inhibited. Tiamulin inhibited

these activities more than SKF525A or cimetidine. but less than ketoconazole. The

microsomal N-demethylation rate of erythromycin and the hydroxylation of testosterone

at the 20-position were inhibited to a lesser degree, whereas the ethoxyresorufin-O-

deethylation. aniline hydroxylation and testosterone hydroxylations at the 15a- and

150-positions were not affected by tiamulin. Complexation by tiamulin of cytochrome

P450 resulting in a loss of CO-binding capacity was not demonstrated. Thus, it was

suggested that a selective inhibition of cytochrome P450 enzymes was present in pigs,

probably those belonging to the P450IIIA subfamily. The mechanism of this interaction

was unclear. However, interactions between tiamulin and those veterinary drugs or

endogenous compounds which undergo oxidative metabolism by P450 enzymes must

be considered.

A second study involving P450IIIA was conducted (Monshouwer et al.. 1995b).

The effect of APP infection on both Phase I (oxidative) and Phase II (conjugative)

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. microsomal enzyme activities was investigated in castrated male conventional pigs.

After 24 h. Actinobacillus pleuropneumoniae infection resulted in a significant

suppression of 33% or more of all oxidative enzyme activities determined. After 40 h .

the activities were still suppressed, but did not differ from the results after 24 h . On the

contrary, all glucuronosyltransferase activities measured were not affected by APP

infection after both 24 and 40 h . To further clarify the mechanism of the suppression of

oxidative enzyme activities, analysis of mRNA were conducted by dot-blot analysis

using a human cytochrome P450IIIA4 cDNA probe. The results indicated that APP

infection suppressed oxidative enzyme activities. The reduction in cytochrome

P450IIIA activity, specific for 6a-hydroxylation of testosterone, was postulated to be at

a pre-translational level, as measured by a decrease in the amount of mRNA.

In another study, a cytochrome P450XXVII catalyzing la-hydroxylation of 25-

hydroxyvitamin D3 was purified from pig liver mitochondria (Axen et al.. 1994). It

also catalyzed 27-hydroxylation of 25-hydroxy vitamin D3 and 25-hydroxyIation of

vitamin D3. The ratio between the la-. 27- and 25-hydroxylase activities remained

essentially constant during the purification. This was an example of a constitutive P450

subfamily in porcine liver.

Cytokine Mechanism of Action on Cytochrome P450

Interferons

Interferon induction was thought to be the most likely suppresser of P450.

because all agents that induce INF. such as the antiviral drug tilorone. depressed mixed-

function oxidase (Leeson et al.. 1976: Renton & Mannering. 1976b). The down

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. regulating effect of 12 different INF inducing agents on P450 content and reactions

supported this hypothesis. Tilorone, Mengo RNA virus. Escherichia coli

lipopolysaccharide. B. pertussis vaccine, hepatic RNA, poly IC and statolon. from a

fungal mycophage. were tested. In addition to the interferons, these agents induced the

production and release of several other cytokines: IL-1, IL-6 and TNF-a.

Testing of purified and hybrid interferons in mice showed that there was a

relationship between INF concentrations and changes in P450 activity and

concentrations (Ghezzi et al., 1986a.b). Interferons administered in vivo inhibited

microsomal metabolism, in vitro, of benzo(a)pyrene. hexobarbital, 7-ethoxycoumarin.

benzamphetamine and zoxazolamine (Ghezzi et al.. 1986a.b.c). Murine in vivo and

human in vitro studies showed increased hexobarbital sleep-times and decreased

metabolism of antipyrine. p-nitroanisole. theophylline. 16P hydroxylation of

androstenedione and acetominophen (Craig et al.. 1989a; Franklin & Finkle. 1986;

Moochhala & Lee. 1991; Moore et al.. 1983;Okuno. 1990; Smith et al.. 1983; Taylor et

al.. 1985).

Interferon influenced constitutive P450 expression both at the steady state

mRNA and P450 apo-protein biosynthesis level (Craig et al.. 1990). Messenger RNA

concentrations were depressed 6-8 h after exposure to INF and reached maximum

suppression at 24 h post-treatment. Cytochrome P450 apo-protein did not diminish

until 8-12 h post-treatment. Reduced P450 mRNA mirrored decreased concentrations

of hepatic P450 protein (Craig et al.. 1990; Morgan, 1989.1991; Stanley et al.. 1991).

In a more recent rat study, the response of constitutive enzymes activities to down-

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. regulation of INF was not influenced by any of the induction protocol using

polyinsonoic acid-polycytidylic acid (Anari et al., 1995). The interferon inducers

depressed the constitutive and induced activities of ethoxyresorufin O-deethylase.

benzyl-oxyresorufin O-dealkylase and p-nitrophenol hydroxyalse at all concentrations

of induction. Cytochrome P450 induction did not invariably confer resistance to

interferon-medicated down-regulation of the enzymes. Furthermore, the mechanism of

induction did not determine the response to INF. It was concluded that species and

duration or level of induction were the major influences on the observed response of

P450 enzymes to INF down-regulation.

Interleukin-1, interleukin-6 and tumor necrosis factor-a

Interleukin-1. IL-6 and TNF-a are cytokines produced during an inflammatory

response, including endotoxin challenge (Bertini et al.. 1988.1989: Myers & Kawalek.

1995). Interferons. TNF-a and IL-6 all down-regulated P450 by affecting hepatic P450

concentrations and activity. Interferons are potent blockers of P450 synthesis, but

definitive proof is lacking that interferons are the primary agents: they may even

enhance degradation of P450s. Heat-stable LPS induced serum factor could also

depress P450 after heat-inactivation of all interferons (Egawa et al.. 1981). It was

decided that the heat-stable factor was not interferon which led to the discovery of other

blockers of P450 synthesis. Dextran sulfate induced production of a factor that

decreased hepatic P450 concentrations and inhibited in vitro aminopvrine and benzo-

(a)-pyrene metabolism (Peterson & Renton, 1986). The serum samples from the

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. animals contained no detectable INF. The serum factor could have been a combination

of IL-1. IL-6 and TNF-a (Myers & Kawalek, 1995).

More recent research suggests that IL-6 may have an inhibitory effect on

cytochrome P450IIIA and may alter drug metabolism (Liao et al., 1996). Cyclosporine

is a drug used to prevent rejection of transplanted organs and is metabolized by

P450IIIA. Thus, the inhibitory effect of IL-6 may alter cyclosporine concentrations

which, in turn, may increase its adverse effects, such as nephrotoxicity. Interleukin-6

was administered in vivo to rats, resulting in decreased steady state concentrations of

mRNA and apoprotein for P450 IIC12. microsomal ethylmorphine-N-demethvlase and

ethoxycoumarin-O-deethylase activities (Williams et al., 1991; Wright & Morgan.

1991).

IL-1 also depressed P450 by inhibiting gene expression (Myers & Murtaugh.

1995). Ethyoxycoumarin-O-deethylase activity decreased in microsomes from mice

treated in vivo with IL-1. but did not affect activity when added to microsomes from

untreated animals. In a study to compare cytokines, cultured rat hepatocytes were used

to measure the relative activities of human IL-6, IL-1 a and -P and INF-y (Clark et al..

1995. 1996). These cytokines were used to inhibit phenobarbital and/or 3-

methylcholanthrene induction of cytochrome P450IIB1. P450IIB2. P450IA1 and

P450IA2. All cytokines produced a concentration-dependent inhibition of

phenobarbital induction of benzyloxyresorufin O-dealkylase activity. All cytokines

were less effective in inhibiting the 3-methylchoIanthrene induction of ethoxyresorufin

O-dealkylase. These results suggested that inducible isoforms of P450 differ in their

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. susceptibility to regulation by cytokines. Cytokines possess differential activity to

inhibit the induction of P450 isoforms, where IL-1 P and IL-6 were the most effective.

The mechanism for the inhibition of drug-metabolizing enzymes by IL-1 p was

investigated (Ferrari et al.. 1993a). Interleukin-1 treatment on various drug-

metabolizing enzymes in male and female rats induced both cytochrome P450IIIA1

activity and protein in females. In males. IL-1 repressed P450IIIA2 activity without

decreasing the protein. Cytochrome P450IA1 activity was impaired in males, but was

retained after dexamethasone pretreatment. Interleukin-1 did not change P450IIB1/2

activity and protein, but counteracted their induction by dexamethasone. Uridine

diphospho-glucuronosyltransferase. P450IA2 (bilirubin), activity and its induction by

dexamethasone were not affected by IL-1 treatment. Both P450IIC11 and epoxide

hydrolase activities were repressed by IL-1 treatment, as well as dexamethasone

treatment. The incongruity between the variations of the activities and the protein of

P450IIIA2 suggested a post-translational regulation. Interleukin-1 treatment mimics

glucocorticoid effects for P450IIC11. IIIA 1 and for microsomal epoxide hydrolase, but

not for P450IA1 and IIB1/2. Cytochrome P450IA1 was impaired in males, but its

activity was restored after dexamethasone pretreatment. Both P450IIC11 and epoxide

hydrolase activities were repressed by IL-1 treatment, but were not altered by

dexamethasone.

In a study using rat hepatocytes in primary culture. IL-1P strongly inhibited

basal ethoxyresorufin O-deethylase and pentoxyresorufin O-deethylase activities and

fully blocked their induction by pentobarbital in a dose-dependent relationship while IL-

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 6 only slightly antagonized pentobarbital-induced pentoxyresorufin activity (Abdel-

Razzak et al., 1995). This demonstrated that IL-1 (3 can suppress basal P450 activities,

as well as pentobarbital-inducible expression of five cytochrome P450 mRNA's in

cultured hepatocytes. A previous experiment provided the first demonstration that

various cytokines act directly on human hepatocytes to affect expression of major P450

genes and that a wide range of responses can be observed among the enzymes for a

given cytokine, suggesting that different regulatory mechanisms may be involved

(Abdel-Razzak et al., 1993).

Interleukin-1 triggered the down-regulation of several hepatic cytochrome P450

gene products, but the cellular signaling pathways involved are not known (Chen et al..

1995). The role of sphingomyelin hydrolysis to ceramide in the suppression of

P450IIC11. a major constitutive form of cytochrome P450. by IL-1 was examined.

Treatment of rat hepatocytes cultured on matrigel with IL-la caused a rapid turnover of

sphingomyelin and an increase in cellular ceramide. with no change in cellular

phosphatidylcholine. The ceramide was composed mainly of a D-erythro-sphingosine

backbone, suggesting that it was derived from sphingolipid hydrolysis rather than from

increased de novo synthesis. Treatment of the cells with either N-acetyl-D-erythro-

sphingosine (C2-ceramide) or bacterial sphingomyelinase suppressed the expression of

P450IIC11 and induced the expression of the interleukin-1-responsive a,-acid

glycoprotein mRNA. In contrast, the acute-phase gene p-fibrinogen. which was

induced by IL-6 but not by IL-1. did not respond to C2-ceramide. N-Acetyl-D-erythro-

sphinganine mimicked the effect of C2-ceramide on P450IIC11. but not on a,-acid

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. glycoprotein expression. These results were consistent with a role for ceramide or a

related sphingolipid in mediating the down-regulation of P450IIC11. the induction of

a r acid glycoprotein and perhaps other cellular effects of IL-1 in hepatocytes.

Interleukin-1 is known to repress a number of hepatic drug-metabolizing

enzymes in rats and humans (Parmentier et al.. 1993). The effect of interleukin-ip on

lauric acid 12-hydroxylase (IVA family) was studied in cultured fetal rat hepatocytes

after clofibric acid induction. Dexamethasone was used as an agent promoting

differentiation and long-term maintenance of active hepatocytes. Dexamethasone and

clofibric acid in combination allowed maximal (13.5-fold) induction of IVA 1. Lauric

acid 12-hydroxylase activity was found to increase with time in culture. Interleukin-ip

adversely affected P450IVA clofibric acid-induced activity, totally eliminating the

effect of induction at doses exceeding 5 ng/ml. This repression was dose-dependent.

The mechanism by which interleukin-ip prevents the development of cytochrome

P450IVA activity was unclear.

The cytokine. TN F-a . is a primary inflammatory' mediator after liver injury.

Several cytokines impair the regulation of cytochrome P450 genes in liver, but the

specificity of these effects remains unclear (Nadin et al.. 1995). Tumor necrosis factor-

a depressed ethoxycoumarin-O-deethylase activity and the steady state concentrations

of mRNA and apo-proteins for P450IIC12 and IID6 (Bertini et al.. 1988; Chen et al.,

1992; Wright & Morgan, 1991). Microsomal ethylmorphine-N-demethylase and

ethoxycoumarin-O-deethylase (P450 IIC12) were inhibited by exogenous IL-6 in rats.

Messenger RNA steady state concentrations were depressed along with decreased apo-

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. protein (Williams et al, 1991; Wright & Morgan. 1991). The effects o f recombinant

murine TNF-a on the expression of specific constitutive P450s in male rat liver was

investigated (Nadin et al.. 1995). Tumor necrosis factor-a down-regulated P450IIC11

and IIIA2 in male rat liver at a pre-translational level, whereas two other constitutive

P450s. IIA1 and IIC6, seem refractory to TNF-a. Thus, impaired P450 regulation by

TNF-a resembled the combined effects of autologous interferons on IIIA2 and

interleukins on IIC11.

Inhibition of cytochrome P450 was not limited to hepatic microsomes but can

occur in other tissues, such as Leydig cells (Li et al.. 1995). Testosterone biosynthesis

in Leydig cells depended on the action of 17 a-hydroxylase/C 17-20 Ivase cytochrome

P450 (P450cl7). which was encoded by the Cypl7 gene. Tumor necrosis factor-a

inhibits cAMP-stimulated testosterone production in mouse Leydig cells. The

inhibition of testosterone production was parallel to the inhibition of P450cl7

messenger RNA and protein concentrations. Tumor necrosis factor-a stimulated PK.C

a-translocation was further demonstrated by indirect immunofluorescence assay.

Phorbol myristic acid, a known activator of protein kinase C and TNF-a a had a similar

inhibitory effect on P450cl7 expression, testosterone production and P450XVII

activity.

Dexamethasone is a well known inhibitor of TNF-a production when given

shortly before lipopolysaccharide (Fantuzzi et al., 1995). However, dexamethasone

may potentiate TNF-a production when administered 24-48 h before LPS. This may be

due to induction of P450IIIA. which was known to produce nitric oxide (NO). This

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. suggests that P450IIlA-generated NO might be involved in TNF-a induction. Nitric

oxide is becoming more recognized as a signaling molecule in many organs, although

its role in the liver remains to be fully elucidated (Milboume & Bygrave. 1995). Liver

cells produced NO in response to a variety of stimuli including infection with

Corynebacterium parvum. LPS and cytokines. Within the liver. NO modulated some

fundamental intracellular functions such as protein synthesis, mitochondrial electron

transport and components of the citric acid cycle. Drug metabolism and blood storage

may be other intracellular roles of nitric oxide. Additionally. NO acts to protect the

liver from immunological damage in models of hepatic inflammation. Inflammatory

stimulation of the liver led to the induction of nitric oxide (NO) biosynthesis (Stadler et

al.. 1994). The interference of NO with specific P450-dependent metabolic pathways

was investigated, because NO binds to the catalytic heme moiety of cytochromes

P450IA. The results indicated that inhibition of NO biosynthesis in patients suffering

from systemic inflammatory response syndromes may help to restore biotransformation

capacity of the liver.

Although nitric oxide synthetase (NOS) is a cytochrome P450-like hemoprotein

with additional sequence homology to cytochrome P450 reductase, the role of the

cytochrome P450 system in cytokine-mediated NO synthesis is unknown (Kuo et al..

1995). Inhibition of P450IIIA activity was associated with a concentration-dependent

decrease in cytokine-mediated NO production. Concentrations of NOS mRNA and

protein were not altered. The NOS enzyme assay was notable for stable concentrations

of intermediate. N-OH-L-arginine and decreased production of the final end product. L-

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. citrulline. Cytochrome P450IIIA isoform may play a post-translational role in cytokine-

mediated NO synthesis in this model of isolated rat hepatocytes in primary culture. Rat

liver microsomes catalyzed the oxidative denitration of N omega-hydroxy-L-arginine

(NOHA) by hydrogenated nicotinamide adenine dinuleotide phosphate (NADPH) and

0 2 with formation of citrulline and nitrogen oxides like NO and N 02- (Boucher et al..

1992). Classical inhibitors of NO-synthetases. like N omega-methyi-and N omega-

nitro-arginine. failed to inhibit the microsomal oxidation of NOHA to citrulline and

N 02-. On the contrary, classical inhibitors of hepatic cytochromes P450 like CO,

miconazole, dihydroergotamine and troleandomycin. strongly inhibited this

monooxygenase reaction. Oxygenation of NOHA by NADPH and 0 2 with formation of

citrulline and NO can be efficiently catalyzed by cytochromes P450, including the IIIA

subfamily.

Endotoxin and infection effects on drug metabolism

Endotoxin

Active infection with bacterial proliferation was not necessary for changes in

drug biotransformation. Heat-killed or formalin-fixed Corynebacterium parvum and

bacillus Calmette-Guerin, an immunomodulator. depresses hepatic P450 drug

metabolism (Farquar et al.. 1976: Soyka et al.. 1976). Endotoxin from other Gram-

negative and positive bacterial cell walls are potent inhibitors of hepatic mixed-function

oxidase (Azri & Renton, 1987; Gorodischer et al., 1976; Lavicky et al.. 1988; Sasaki et

al., 1984; Sonawane et al., 1982; Stanley et al., 1988; Williams & Szentivanyi. 1983:

Yoshida et al., 1982). Escherichia coli. Bordatella pertussis, diphtheria toxoid and

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. tetanus toxoid all contain cell wall endotoxins that alter P450 activity (Ashner et al.,

1992.1993; Fantuzzi et al., I994a.b). Pertussis toxin was a necessary component for the

sustained effects of diphtheria-tetanus-pertussis vaccine on hepatic drug metabolism

which suggested a role for INF-y in this process (Ashner et al.. 1993). Bacterial

endotoxin such as Escherichia coli or B. pertussis endotoxin decreased P450 activities

in mice (Fantuzzi et al., 1994b; Sasaki et al.. 1984). Endotoxin treated rats experienced

a dramatic decrease in steady state concentrations of P450IIC12 mRNA and apo-protein

(Morgan. 1989). The mechanism for these effects was thought to be suppression of

cytochrome P450 activity and concentrations by TNF. interleukins, interferons and

other cytokines (Myers & Kawalek. 1995). The common theme that emerges from the

preceding studies was that infections, whether viral, bacterial, or parasitic, all depressed

P450-mediated metabolism. The next question was how were these vastly different

agents all affected by the mixed-function oxidase system? In a series of papers, it was

shown that both poly IC and tilorone decreased the levels of enzymatically-active P450.

cytochrome b5 and 5-aminoIevulinic acid synthetase (ALA-S) (El Azhary &

Mannering, 1979). Conversely, heme oxygenase activity increased following treatment

with poly IC. 6-Aminolevulinic acid synthetase was the rate-limiting enzyme

controlling heme biosynthesis, whereas heme oxygenase degrades heme (Tephley et al.,

1973; Maines. 1988). Administration of interferon inducers such as poly IC and

tilorone seemed to simultaneously decrease heme synthesis and increase heme

degradation.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Functional P450 consists of both the P450 apoprotein and the heme moiety with

independent synthesis of both components. Either component could be the target of the

interferon-inducing agents. The turnover rate of the heme moiety had a biphasic

pattern, indicating that different P450 molecules have different turnover rates (Levin &

Kuntzman. 1969). Poly IC and tilorone increased the turnover rate of short-lived P450

molecules, but did not affect the long-lived variants (El Azhary et al., 1980). However,

a transient increase in heme ievels was also noted. This suggested, but did not prove,

that the ultimate target was the synthesis of the P450 apoprotein.

Xanthine oxidase had been proposed as being another mechanism for the

depression of P450 levels and was observed after animals were treated with interferon

or interferon inducers (Myers & Kawalek. 1995). Added support for this hypothesis

came from studies showing an inverse relationship between production of interferon and

depression of P450 protein levels and P450-mediated enzymatic activity (Singh et al..

1982). Strains of mice that produced high levels of interferon had low levels of P450

following challenge with Newcastle disease virus, whereas mouse strains that produced

lower levels of interferon exhibited a small effect on P450 concentrations. The

differences in P450 levels between these two strains of mice correlated to

ethylmorphine-/V-demethylase activity and high interferon-producing strains exhibiting

the lowest levels of enzymatic activity. The induction of xanthine oxidase paralleled the

levels of interferon produced (Deloria et al., 1983). High interferon-producing mice had

levels of xanthine oxidase that were higher than those found in mice producing lower

levels of interferon. The inhibiting effect on P450 levels and its monooxygenase

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. activity was greatest in high interferon-producing mice (Deloria et al.. 1983).

Formation of lipid peroxides (Ali et al., 1985) has also been proposed as one possible

mechanism to explain the decrease in the P450 levels following interferon induction.

However, inhibiting lipid peroxidation failed to prevent poly 1C induced P450 loss

(Mannering et al.. 1989).

Additional studies revealed that incubation with free radical scavengers

superoxide dismutase and iV-acetylcysteine protected the P450 enzymes from the effects

of added xanthine oxidase (Ghezzi et al.. 1985). In vivo treatment with allopurinol. an

xanthine oxidase inhibitor, or iV-acetylcysteine canceled the effect of poly IC on both

P450 and xanthine oxidase. However, while allopurinol protected against the effects of

poly IC. it also decreased the basal concentrations of P450 and xanthine oxidase.

Although there were effects of xanthine oxidase on P450 levels, there was no significant

effect on P450-mediated ethoxycoumarin O-deethvlase activity (Ghezzi et al.. 1985).

Chickens cannot form xanthine oxidase and it was shown that polv IC treatment of

chickens did not affect P450 levels (Moochhala & Renton. 1991). Nonetheless,

brooder-raised chickens may have very low levels of P450-mediated enzymatic activity

when compared with pen-raised birds (Dr. Joseph Kaw'alek. unpublished). Using

animals with such low activity would minimize any changes in P450 concentrations that

may have been affected in these chickens. In addition, they could not show a protective

role for the free radical scavenger iV-acetylcysteine, or that added xanthine oxidase

affected the in vitro activity of liver microsomes.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. The above results were subsequently contradicted using mice, rats and hamsters

(Reiners et al., 1990; Cribb & Renton. 1993). While no one group showed a role for

xanthine oxidase, an earlier study had been designed to feed diets containing tungstate

to inactivate xanthine oxidase in mice (Mannering et al., 1988). In the tungstate study,

a role for either xanthine oxidase or lipid peroxides in the poly IC mediated decrease in

hepatic P450 was not discovered. Therefore, it was concluded that induction of

xanthine oxidase and reduction in P450 content were coincidental and not causally

related. Based on these observations, the decreases in P450 and its related activities

were probably not a consequence of xanthine oxidase activity in rodents (Reiners et al..

1990; Cribb & Renton. 1993).

In a more recent in vivo study using pigs instead of rodents, multiple intravenous

(i.v.) doses of LPS (17 pg/kg) induced a mild acute-phase response in swine within 2 h

of the first injection (Monshouwer et al.. 1996a). Antipvrine plasma clearance was

decreased in the LPS treated pigs (control: 8.5±0.8; LPS: 2.2±0.7 ml/min/kg) and

decreased ethoxyresorufin O-deethylase. caffeine, 3-demethylase and testosterone 6P-

hydroxylase activities which corresponded to P450IA1. P450IA2 and P450IIIA

respectively, in rats. The P450IA and P450IIIA apoprotein concentrations were also

decreased. The results suggested that the effect on cytochrome P450 was relatively

non-selective in the pig and affects several P450 isozymes. High systemic

concentrations of TNF-a and IL-6 were associated with decreased antipvrine clearance

in LPS treated pigs. Microsomal 1-naphthol glucuronosyl transferase activity was not

altered in LPS treated pigs.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Endotoxin elicits a broad, non-specific cascade of events in vivo. a variety of

potent mediators and cytokines were secreted, primarily, by activated cytokines and

monocytes (Kielian & Blecha, 1995) The over-production of these effector molecules,

such as IL-1 and TNF-a. contributed to the pathophysiology of endotoxic shock.

Cellular recognition of LPS involved several different molecules, including a cluster of

differentiation antigen. CD 14. A thorough understanding of the interaction of LPS with

cells of the immune system is necessary before effective preventative or therapeutic

measures can be designed and tested to limit the host response to endotoxin. The CD 14

antigens function as a receptor for lipopolysaccharide binding protein complexes

(Kielian et al.. 1995). Lipopolysaccharide has a varying effects on CD14 expression in

vitro which required that the evaluation of CD14 expression in response to LPS with a

fully differentiated macrophage phenotype, the porcine alveolar macrophage. The

investigators found that LPS activation of porcine alveolar macrophages for 24 h

increased CD14-like receptor expression. The degree of CD14-like up-regulation was

related to LPS concentration. However, activation did not require the presence of serum

at concentrations of LPS.

Infection

Infection from viral, bacterial, or parasitic sources depressed drug metabolizing

activity of diseased animals (Myers & Kawalek. 1995). Pharmacokinetic changes were

often overlooked when drug toxicity or decreased efficacy occur during an infection,

especially in veterinary medicine. Influenza infections altered the metabolism of

theophylline and cytomegalovirus increased the toxicity of parathion. but not all viral

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. vaccines affected cytochrome P450 (Fisher et al.. 1981; Selgrade et al.. 1984). Both

heat-killed and formalin-fixed infection with Corynebacterium parvitm in rats and mice

decreased hepatic P450-mediated drug biotransformation (Farquar et al.. 1983: Soyka et

al., 1976). Malaria-infected rats have exhibited decreased ability to metabolize aniline,

ethylmorphine,/7-nitroanisole and hexobarbital (McCarthy et al.. 1970). Plasmodium

berghei were irradiated and injected into mice (Song et al.. 1995). There were

significant decreases in mouse hepatic cytochrome P450 and benzo(a)pyrene

hydroxylase activity, but no effect on N-demethylase activity. Antipvrine and

metronidazole were administered as a cocktail to young male Wistar rats to investigate

the effect of malaria infection due to the rodent parasite Plasmodium berghei and

Escherichia coli endotoxin-induced fever on the metabolism of the two compounds in

vivo (Kokwaro et al.. 1993). Malaria infection had no effect on clearance of antipyrine

or on formation clearance of any of its metabolites. However, the clearance of

metronidazole was reduced by approximately 20% compared with controls as a result of

decreased formation of hydroxymetronidazole. Fever decreased clearance of both

antipyrine and metronidazole by approximately 36% and 23%, respectively. These

results demonstrated that both malaria infection and fever influenced P450-dependent

drug metabolism and the effects seen were isozyme-selective Parasitic infection with

Fasciola hepatica or viral infection with influenza A and B, poliovirus, as

cytomegalovirus depressed mixed-function oxidase activity (Chang et al., 1978;

Descotes & Evreux. 1986; Descotes et al.. 1985; Kraemer & McClain. 1981; Kraemer

et al.. 1982; Maffei et al., 1981; Renton, 1981; Selgrade et al.. 1984). In mice infected

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. with Fasciola hepatica. there was a loss of respiratory control in isolated liver

mitochondria at 4 weeks post-infection (Somerville et al.. 1995). A similar time course

was demonstrated for a reduction in hepatic microsomal cytochrome P450 content. In

another parasitic study using Schistosoma mansoni. the intensity of infection determined

alterations in hepatic drug metabolism, as indicated by measuring zoxazolamine

paralysis times (Naik & Hasler. 1995).

TheA. pleuropneumoniae infection model was well characterized and has

become a standard respiratory disease model in pigs (Baarsch et al., 1995: Kamp & van

Leengoed, 1989). Hepatic microsomes. in vitro. from APP infected pigs were used to

determine enzyme activities for the model substrates: aniline-hydroxylation.

ethoxyresorufin O-deethylation. pentoxyresorufin O-depentylation and testosterone-

hydroxylation at the 20. 60. 1 la. 15a and 150 positions (Monshouwer et al., 1995b).

All oxidative enzyme activities were depressed by 33% twenty four hours after infection

and indicated that the depression was not selective. UDP-glucuronosyltransferase

activities were not affected. The dot blots showed a reduction in total mRNA in two

infected pigs, but this study was somewhat lacking in experimental design. The human

cytochrome P450IIIA4 cDNA probe was used to detect mRNA in a mixture of mRNA

on the dot blot. There was no control for the possibility that the IIIA4 cDNA probe

might cross-react with mRNA in other P450 subfamilies, resulting in a false positive.

Additionally, only two control and two treated pigs were used, which makes statistical

analysis difficult.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. In a subsequent experiment, the investigator attempted to verify in vitro findings

by performing an in vivo pharmacokinetic experiment with APP infected pigs using

model drugs (Monshouwer et al.. 1995a). The plasma clearance of antipyrine and

caffeine represented a decrease in oxidative drug metabolism and differed greatly from

the infected pigs compared to the controls (72% and 68%. respectively). The effect of

the infection on the glucuronidation of paracetamol was minimal in infected pigs (39%

depression). The clearance of indocyanine green and creatinine were unaffected which

indicates normal hepatic blood flow and renal excretion. Antipyrine. caffeine and

paracetamol were negligibly bound to plasma proteins so it was unlikely that the

decrease in clearance was from changes in protein binding. Cytochrome P450 enzymes

responsible for the reduction of oxidative metabolism were not pursued, but the results

suggested non-selective action in vivo. The possible mechanism for decreased

paracetamol disposition was not clear, because glucuronosyl activity was not suppressed

by the infection.

Actinobacillus Pleuropneumoniae

History

Actinobacillus pleuropneumoniae is the most common causative agent of swine

pleuropneumonia, a highly contagious and sometimes fatal, respiratory tract disease of

pigs known as porcine pleuropneumonia (Nicolet. 1994). The disease was first reported

in 1957 and was followed by worldwide reports of APP outbreaks in commercial swine

herds (Fedorka-Cray et al.. 1993a; Pattison et al., 1957). Morbidity approaches 100%

with 50% mortality, 55% increase in the feed conversion ratio and a 40% decrease in

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. average daily gain (Mackinnon. 1993). In controlled studies of swine infected with A.

pleuropneumoniae, 60% died 18 to 56 h after exposure (Kung et al., 1993a.b). Control

and prevention measures for porcine pleuropneumonia were often ineffective, because

of the complex nature of the disease (Fedorka-Cray et al.. 1993a: Maclnnes &

Rosendal. 1982; Sebunya & Saunders. 1983). Subsequently, pork producers have

suffered great economic loss.

Actinobacillus pleuropneumoniae is a facultative anaerobe that is non-spore

forming, encapsulated, nonmotile. Gram-negative pleomorphic coccobacillus (Kilian &

Biberstein. 1984). Biochemical characteristics of the organism include: urease positive:

able to ferment mannitol. xylose and ribose; with variable fermentation of lactose

(Kilian & Biberstein. 1984; Nicolet. 1994). A rough to smooth morphologic transition

may occur after passage in artificial medium. (Fedorka-Cray et al.. 1993a; Gunnarsson.

1979). Colonies exhibit P hemolysis on blood agar plates, but some isolates were non

hemolytic (Kamp & van Leengoed. 1989; Morrison et al., 1985; Sebunya & Saunders.

1983). Some field isolates have been identified with fimbria by negative staining

(Utrera& Pijoan. 1991).

Actinobacillus pleuropneumoniae was originally believed to be Haemophilus

parahaemolyticus. because of the requirement of nicotinamide adenine dinucleotide

(NAD) for growth (Biberstein et al., 1977; Kilian & Biberstein, 1984; Niven &

Levesque. 1988). In 1978. distinguishing biochemical and cultural characteristics were

identified between porcine and human strains and a new strain, Haemophilus

pleuropneumoniae, was named (Kilian et al., 1978). In 1983, DNA hybridization was

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. performed between Haemophilus pleuropneumoniae and Haemophilus influenza and the

percent binding indicated no measurable relationship (Pohl et al., 1983). The DNA

hybridization and phenotypic characteristics did indicate that Haemophilus

pleuropneumoniae was closely related to Actinobacillus lignieresi. The Haemophilus

pleuropneumoniae was transferred to the genus Actinobacillus with 2 biovars: a V

factor biovar 1 dependent on nicotinamide adenine dinucleotide and a V factor

independent biovar 2. The two strains were very similar except for the NAD

requirement and morbidity in biovar 2 induced pleuropneumonia was less than the

morbidity caused by strains of biovar 1 (Kilian et al.. 1978).

Epidemiology

Only pigs are known to be naturally susceptible to Actinobacillus

pleuropneumoniae (Fedorka-Cray et al.. 1993a). The organism has not been found in

rodents, birds, or humans and does not persist in the environment (Fedorka-Cray et al..

1993a; Nicolet. 1994). Serotypes 1. 5 and 7 are frequently found in the United States

and are common in the Midwest; serotypes 3. 4. 8 and 9 are infrequently found

(Fedorka-Cray et al.. 1993a; Sebunya & Saunders. 1983). Serotypes 1. 2. 3 5 and 7 are

common in Canada and serotypes 1, 2, 5. 7 and 9 are common in Europe (Rapp et al..

1985; Rosendal & Boyd, 1982; Sanford & Josephson. 1981; Schultz et al.. 1983). In a

recent study, bronchoalveolar lavage was performed in 128 pigs from five fattening

units showing acute pneumonia, subclinical pneumonia and chronic purulent pneumonia

(Tapner et al., 1996). Using cultural methods. APP could only be isolated from six

samples. In contrast, there were frequent positive reactions to APP antigens with

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. immunofluorescence microscopy. Thus, immunofluorescence was a better screening

method.

Occurrence of swine pleuropneumonia in previously non-infected herds can be

linked to introduction of asymptomatic carrier pigs (Greenway. 1981). Chronically

infected pigs that are asymptomatic may develop signs of the disease during transport

(Greenway. 1981). Actinobacillus pleuropneumoniae is spread by direct contact or by

aerosol transmission (Fedorka-Cray et al., 1993a). Nasal secretions can contain 109

colony forming units/milliter (CFU/ml) which quickly spreads the disease between pigs

(Rosendal & Mitchell. 1982). Stress factors of overcrowding, transportation and abrupt

temperature changes also increased the incidence of the disease (Sebunya et al., 1983b).

The increased susceptibility to APP can be predicted by the absence of toxin-

neutralizing antibodies to haemolysin and cytotoxin (Cruijsen et al.. 1995a.b). Autumn

through early spring is the period of greatest incidence of APP (Fedorka-Cray et al..

1993a.: Sebunya et al., 1983b).

Intense industrialization of pork production has been suggested as contributing

to the rapid rise of APP infected herds (Fedorka-Cray et al., 1993a). Pigs older than 12

weeks of age are the most vulnerable and immunologically naive herds are the most

severely affected (Sebunya et al., 1982.1983b: Shope. 1964). A recovered pig can

remain an asymptomatic carrier for months and provide low exposure of APP to the

herd. This allows the herd to establish a level of immunity that decreases the incidence

of new and acute infections. Chronically infected herds have some individuals that

remain seronegative. Seronegative sows did not pass on colostral immunity to suckling

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. piglets and the offspring will remain susceptible to infection. Piglets become vulnerable

when exposed to crowding and poor ventilation (Fedorka-Cray et al.. 1993a: Sebunya et

al.. 1982).

Signs, pathology and lesions

Actinobacillus pleuropneumoniae infection can cause death within 12 h after

exposure (Fedorka-Cray et al., 1993a). Clinical signs include: anorexia, fever, severe

respiratory distress, cyanosis, coughing and dyspnea. Discharge of blood tinged foam

from the nose and mouth is common in acutely infected pigs (Fedorka-Cray et al..

1993a). The severity of the disease is influenced by the virulence of the strain, size of

inoculum, protective immunity and stress (Greenway. 1981; Rosendal. 1985; Sebunya

et al.. 1983b; Shope. 1964; Jacobsen et al., 1996).

The disease can occur in the peracute. acute, subacute or chronic form (Fedorka-

Cray et al.. 1993a: Nicolet. 1994: Udeze et al., 1987). In the peracute form, death

occurs within 24 to 36 hours. Sudden death in piglets without previous clinical signs

has been reported (Fedorka-Cray et al.. 1993a; Sebunya et al.. 1983b; Shope, 1964).

The acute form may progress from severe clinical disease to death within a few days or

it may resolve into the chronic form depending on the severity of lung lesions and

response to antibiotic therapy (Fedorka-Cray et al., 1993a; Nicolet. 1994) In the early

stages of the disease, common microscopic changes included pulmonary necrosis,

hemorrhage, neutrophil infiltration, macrophage and platelet activation, vascular

thrombosis, widespread edema and fibrinous exudate (Bertram. 1985. 1986. 1988:

Liggett & Harrison. 1987). Following the acute response to APP infection, macrophage

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. infiltration, marked fibroplasia around areas of necrosis and fibrinous pieuritis were

characteristic in later stages of the disease (Hani et al.. 1973). Antibiotic therapy was

efficient only in the initial phase of the disease, when there was the best chance to

reduce mortality (Nicolet. 1994). Actinobacillus pleuropneumoniae was susceptible to

(3-lactams, mainly penicillin and cephalosporins: chloramphenicol: cotrimoxazole: and

tetracyclines, but resistance has developed to ampicillin. streptomycin, sulfonamides,

tetracyclines, chloramphenicol, tiamulin and a combination of lincomycin and

spectinomycin (Anderson & Williams, 1990; Gilbride & Rosendal. 1984; Hsu. 1990:

Vaillancourt et al.. 1988). Resistance was most frequently seen in serotypes 1.3 .5 and

7. Presently, the quinolone derivatives or the semisynthetic cephalosporin, ceftiofur

sodium, have been shown to be very effective after experimental challenge with APP

(Kobisch et al.. 1990; Stephano et al.. 1990).

Gross lesions common in acute APP infections consisted of serofibrinous

pieuritis and fibrinous and necrotizing hemorrhagic pneumonia (Sebunya et al.. 1983a).

Lesions were usually distributed bilaterally in the caudal lobes of the lungs (Sebunya et

al.. 1983a). In chronic infections, fibrinous pieuritis and necrotic foci that very in size

were evident. The necrotic foci, in recovered animals, serve as a nidus of infection for

months and infected animals were considered to be carriers (Fedorka-Cray et al..

1993a).

Sequential studies have been conducted to record histological lesions caused by

Actinobacillus pleuropneumoniae infection (Liggett & Harrison, 1987). Neutrophil

infiltration and fibrinous exudate are present in the early stages of the disease (Bertram,

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 1988). Bacteria occur as individual cells or groups throughout the exudate (Fedorka-

Cray et al., 1993a). Coagulative necrosis was evident 12 to 18 h after infection in areas

of concentrated suppuration (Liggett & Harrison. 1987). Macrophage infiltration and

areas of marked fibroplasia were evident around necrotic tissue as the disease

progressed (Bertram, 1988). Neutrophils occurring in various stages of degradation

suggested that vascular damage may be mediated by neutrophil degeneration and release

of lysomal contents (Liggett & Harrison. 1987). Sequestration of necrotic pulmonary

tissue and development of pleural adhesions were common sequellae to the acute

response (Bertram. 1988: Fedorka-Cray et al.. 1993a). Complete healing, locally

extensive areas of fibroplasia, or liquefactive necrosis may result after survival of acute

infections (Bertram. 1988).

Virulence factors

Capsule

Some of the 12 identified serotypes of APP cause more severe disease than

others (Brandreth & Smith. 1987: Fedorka-Cray et al., 1994: Jensen & Bertram. 1986:

Rosendal & Boyd. 1985). Inoculation with as few as 10,000 virulent bacteria can cause

death in 12 hours and 100 bacteria caused lung abscesses in swine (Perry et al., 1990).

Virulence factors on the cell surface or secreted by the bacteria were responsible for the

disease process.

The structure and chemical composition of the 12 APP serotypes have been

described (Fedorka-Cray et al., 1994). Capsules consist of polymers of two or three

sugars or amino acids, techoic acid polymers linked by phosphate diester bonds or

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. oligosaccharide polymers linked by phosphate bonds (Fedorka-Cray et al.. 1994). The

presence of phosphate or carboxylic acid creates a negative charge on the capsule and

was important for the evasion of APP cells from phagocytic cells. Serotype specificity

of APP was attributed to capsular antigens (Inzana. 1991; Inzana & Mathison. 1987).

The capsule protected the bacterium from host defenses, such as opsonization

and phagocytosis and complement mediated killing by classic and alternate pathways

(Inzana. 1991). However, not all encapsulated strains have been shown to be virulent

(Jacques et al.. 1988a: Jensen & Bertram. 1986: Rosendal & Boyd. 1985). Capsule

deficient mutants of APP serotypes 1 and 5 were less virulent in pigs than their

encapsulated parent strains (Inzana et al.. 1988: Rosendal & Maclnnes. 1990). Injection

of capsular material alone neither produced disease nor pulmonary lesions nor disease in

pigs (Fenwick et al.. 1986c). Purified capsule produced a poor antibody response, but

conjugation with protein improved immunogenicity and gave partial protection to

pleuropneumonia caused by APP (Inzana. 1990: Inzana et al.. 1988). Passive transfer of

non-specific capsular antiserum to swine provided a level of protection similar to

immunization with killed bacteria (Inzana et al.. 1988). Other virulence factors

accounted for the variation in virulence. The presence of lipopolysaccharides and

exotoxins were known to vary between A. pleuropneumoniae serotypes and strains

(Byrd & Kadis. 1989; Frey & Nicolet. 1990: Kamp & van Leengoed. 1989). Adhesins.

hemagglutinins, fimbriae and pili. played an unknown role in the pathogenesis of APP

(Fedorka-Cray et al.. 1994).

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Subsequently, to explore the attachment of APP to mucosal surfaces, an

investigator endobronchially inoculated seven pigs with APP serotype 1 and the pigs

developed subacute necrotizing pleuropneumonia (Narita et al., 1995). When the pigs

were treated with high doses of atropine (0.25 mg/kg) and xylacaine spray using a

bronchoscope, mucus secretion was suppressed along with ciliary activity. The animals

showed severe pleuropneumonia and two treated pigs died within 36 h after inoculating

320 CFU/2ml of APP serotype 1. The findings suggested that the attachment of APP to

the mucosal surfaces may be a critical in the pathogenesis of pneumonic lesions.

Lipopolysaccharide

Lipopolysaccharide is a macromolecule that is part of the outer membrane of

Gram-negative bacteria (Stanier. 1976). Lipopolvsaccharides are composed of a

complex lipid material, lipid A. which is attached to the core polysaccharide. The core

is comprised of sugars, including 2-keto-3-deoxyoctonate. attached to O-specific side

chains. The O-specific side chains protrude out from the cell surface of the bacterium

and endow strain specific antigenic properties on certain Gram-negative bacteria. O-

specific side chains have repeating units that convey a phenotypic characteristic of

smooth, side chains present, or rough, side chains absent. Each strain shares a common

core O antigen within each serotype. Serotype 5 can have both partially rough and

smooth strains, because the length of the side chain can vary (Altman et al.. 1990; Frey

& Nicolet. 1990). Serotype specific and cross reactive epitopes in the LPS have been

identified for APP (Fenwick & Osbum. 1986).

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Lipopolysaccharides have similar structures among the Gram-negative bacteria

and similar biological effects have been inferred (Fenwick. 1990). Lipopolysaccharide

induced the release of pro-inflammatory cytokine mediators. The mediators activate

various cells to include macrophages and neutrophils which played a primary role in

acute inflammation. Purified APP lipopolysaccharide induced non-necrotizing

pneumonia with neutrophil infiltration (Udeze et al.. 1987). The deleterious effect of

LPS was inflammation and not the associated necrotic lesion development (Idris et al..

1992; Udeze et al.. 1987). Necrotic lesion development seems to depend on the

presence of exotoxins that lyse neutrophils and release toxic substances that severely

damaged tissue.

Supporting evidence for the involvement of endotoxin with A.

pleuropneumoniae virulence was demonstrated in a study using pigs vaccinated with the

J5 bacterin from a mutant of Escherichia coli 0111: B4 (Udeze et al.. 1987). The

vaccinated pigs survived Actinobacillus pleuropneumoniae infection challenge, whereas

four of the five control non-vaccinated animals died following infection with APP.

Antibodies against lipid A and possibly to core antigens of LPS. may have a protective

effect against septicemia and death caused by pleuropneumonia infections (Udeze et al..

1987). J5 protection against septicemia and death in porcine pleuropneumonia was

indirect evidence that APP endotoxin contributed to pathogenicity. Lipopolysaccharide

isolated from A. pleuropneumoniae induced a pulmonary lesion and the severity of the

lesion may be a function of the composition of the LPS molecule. The heat stable

endotoxin and the heat labile extracellular hemolysins act in concert to produce lung

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. lesions. Endotoxin alone did not induce the full array of lung lesions observed in the

acute form of natural and experimental infections. Lipopolysaccharide and exotoxins

may act together as virulence factors in mediating the disease process (Fenwick &

Osbum. 1986; Fenwick et al., 1986a.b). Hemolysin and cytotoxin may also contribute

to the pathogenesis of lung lesions (Udeze et al., 1987).

Another virulence factor may be the affinity of APP endotoxin for porcine

respiratory tract secretions (Belanger et al., 1994). The affinity for a crude preparation

of porcine respiratory tract mucus of isolates of the Pasteurellaceae family

{Actinobacillus. Haemophilus and Pasteurella spp.) and of some unrelated Gram-

negative bacteria was examined. To identify the receptors recognized by the

lipopolysaccharidic adhesin of APP. affinity chromatography was used. Two

proteinaceous bands (10 and 11 kDA) were recovered. This suggested that two low-

molecular-mass proteins present in porcine respiratory tract secretions bind APP

endotoxin. In a latter study. LPS from all APP isolates tested, bound pig hemoglobin

and lipid A was involved in this binding (Belanger et al.. 1995). Some APP isolates

were able to use hemoglobin for growth. This indicated that binding of hemoglobin to

LPS might represent an important means by which APP acquires iron in vivo from

hemoglobin released from erythrocytes Iysed by the action of its hemolysins. Two

outer-membrane proteins were involved in the uptake of iron from transferrin by certain

Gram-negative bacteria, including Actinobacillus pleuropneumoniae. transferrin-

binding proteins 1 and 2 (Daban et al., 1996).

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Recently. 40 and 48 (kDa) outer membrane proteins have been identified from

Actinobacillus pleuropneumoniae. serotype 5 (Bunka et al. 1996; Cruz et al.. 1996).

Vaccine prepared from the outer membrane of serotype 5 provided protective immunity

in swine challenged with either serotype 5 or serotype 1. The outer membrane protein

was common to all 12 capsular serotypes of APP. but was not present in the outer

membranes of related species of Gram-negative swine pathogens. It was inconclusive

whether or not antibodies to the 48 kDa outer membrane antigen contribute to cross-

protective immunity to control porcine pleuropneumonia caused by APP. Live non

capsulated mutants of A. pleuropneumoniae may provide safe and cost-effective

protection against swine pleuropneumonia (Inzana et al. 1993). These observations

support the possibility that non-capsulated mutants of other encapsulated, toxin-

producing bacteria may also prove to be efficacious live-vaccine candidates.

Bacterins for Actinobacillus pleuropneumoniae have proven to be ineffective

(Fedorka-Cray et al., 1993b). Current bacterins may provide only partial protection in

swine following infection APP. Vaccination failure of whole cell bacterins has been

attributed to antigenic variation within a capsular serotype, due to antigenic variation

within LPS (Jolie et al., 1995). This lack of cross-protection, within a capsular subtypes

1A and IB. provides partial explanation for vaccination failures observed under field

conditions. The efficacy of a cell-free concentrate from mid-log phase growth cultures

of APP serotype 1 to four commercial bacterins were compared (Fedorka-Cray et al.

1993b). This cell-free preparation contained carbohydrate, endotoxin and protein and

had hemolytic and cytotoxic activity. Similar, but not identical, responses were

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. observed when antisera generated against the bacterins was used. This study indicated

that an acellular vaccine, containing multiple virulence factors, can provide complete

protection from mortality and significantly reduced morbidity to homologous challenge

(Fedorka-Cray et al., 1993b). Swine were protected by a subunit vaccine that did not

create unacceptable tissue reaction at the immunization site after challenge with APP.

serotype 1 (Walker et al.. 1995).

A study in mice used specific monoclonal antibodies (MoAbs) to Actinobacillus

pleuropneumoniae serotypes 1 and 2 which recognized serotype-specific antigens (Saze

et al.. 1994: Oishiet al., 1993). It was revealed that the two serotype-specific MoAbs

HI-18 and H22-7 recognized O-polysaccharides of the lipopolysaccharide from APP

serotypes 1 and 2. respectively. These results showed that LPS from APP bacterial cells

was one of the structural substances in the serotype-specific antigens and an important

component as one of the antigens protecting against the homologous serotype strain.

The combined vaccine of serotypes 1. 2 and 5 from APP was tested for its ability to

confer protection in pigs (Oishi et al., 1995). Clinical signs of pleuropneumonia were

not observed after intratracheal challenge with A. pleuropneumoniae.

Exotoxins

Bacterial exotoxins are soluble proteins secreted by live organisms (Rutter.

1988). Toxin release contributed to the disease process, especially for organisms

infecting mucosal surfaces (Shewin. 1988). The presence of exotoxins was

demonstrated by administering sterile culture supernatants of APP to pigs (Fedorka-

Cray et al.. 1994). Pulmonary lesions were similar to those found in natural infections.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Lung lesions were produced by all of the exotoxins, except one. PTX/ClvIII. which was

not essential for the pathogenicity of the organism (Fedorka-Cray et al., 1994). In an

attempt to gain insight into the events that take place during APP infection, the effects

of bacterial toxicity on porcine neutrophil functions and viability was evaluated (Udeze

et al.. 1992). Incubation of phagocytes with opsonized APP resulted in phagocylic

uptake of less than or equal to 4%. At the same bacterium-to-phagocyte ratio, levels of

lactate dehydrogenase activity of 74 and 81% were detected in the extracellular medium

after 1.5 and 3 h of incubation, respectively. Furthermore, the ingested bacteria were

not killed by the phagocytes. These effects were ascribed to hemolysin produced by the

bacteria, because the presence of hemolysin-neutralizing antibody prevented overt

cellular damage, significantly increased phagocytic uptake and resulted in an

approximately 10-fold decrease in the number of ingested bacteria. Cytolytic doses of

isolated hemolysin caused dose-related loss of cell viability, diminished bactericidal

activity of toxin-treated phagocytes for Escherichia coli and decreased the ability of the

phagocytes to undergo a respiratory burst upon stimulation with phorbol myristic

acetate. In contrast, sublytic doses of the hemolysin activated the phagocytes and

caused them to respond to phorbol myristic acetate with increased generation of

superoxide anion. Heated (100 degrees C. 5 min) hemolysin preparations did not

produce similar effects and it was concluded that the observed effects were not due to

contaminating endotoxin. The data indicated that APP hemolysin was a potent

antiphagocytic virulence factor by virtue of its leukocidal activity.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Two hemolytic and cytolytic toxins (Hly/Clyl and Hly/CIyll) were believed to

be important in disease progression and are related to the repeat toxin (RTX) family of

exotoxins of other Gram-negative microorganisms. These toxins include the leukotoxin

in Pasteurella haemolytica. the exotoxin in A. sais and the a hemolysin produced by

some strains of Escherichia coli (Devenish et al.. 1989: Lian et al., 1989). The derived

amino acid sequence of Hlyl showed 42% homology with the hemolysin of

Actinobacillus pleuropneumoniae serotype 5. 41% homology with the leukotoxin of

Pasteurella haemolytica and 56% homology with the Escherichia coli a-hemolysin

(Frey et ai. 1991). The 13 glycine-rich repeats and three hydrophobic areas of the Hlyl

sequence show more similarity to the E. coli a-hemolysin than to either the APP

serotype 5 hemolysin or the leukotoxin. while the last two were more similar to each

other. Two types of RTX hemolysins, therefore, seem to be present in APP. one (Hlvl)

resembling the a-hemolysin and a second more closely related to the leukotoxin. The

structural gene encoding Actinobacilluspleuropneumoniae-RTX-toxin I (ApxIA) was

cloned from serotype 10. as a major hemolytic and cytolytic toxin of the organism (Nagi

et al.. 1993a). The gene from serotype 10 was 98% identical to that from serotype 1 at

both the DNA and amino acid levels. Results of DNA sequencing showed serotypes 1.

9 and 11 to be the original form of the ApxIA gene and the allelic variant was found in

serotypes 5a. 5b and 10. The gene products, ApxIA proteins, have different secondary

structures at the carboxyl terminal region. Using the AppA gene encoding

Actinobacillus pleuropneumoniae-KYX-toxm II (ApxII) as a probe, it was demonstrated

by Southern hybridization that AppA-sequence was present in all 12 serotypes except

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 10 (Nagi et al., 1993b). After DNA sequencing, this region was determined to be part

of the HlylA gene that encoded a 105 kDa hemolysin (Apxl). The presence of the low

homology region was evident in serotype 10 compared to the AppA gene locus (HlylA)

found in serotypes 1, 5a. 5b. 9 and 11 which all had strong hemolytic activity with

sheep red blood cells and were highly virulent in mice. This suggested that Apxl may

be associated with the virulence phenotype of the organism. Another investigator

proposed a new and uniform designation for the App-RTX-toxins consisting of

hemolysins, cytolysins, pleurotoxins and their genes (Frey et al.. 1993). The

designation of Apxl. Actinobacillus pleuropneumoniae RTX-toxin I. was proposed for

serotypes 1. 5a. 5b. 9. 10 and 11, which was previously named hemolysin I (Hlyl) or

cytolysin I (Clyl). This protein was shown to be strongly hemolytic and cytotoxic

towards pig alveolar macrophages and neutrophils. The genes of the Apxl operon

would have the designations: ApxIC. ApxIA. ApxIB and ApxID for the activator, the

structural gene and the two secretion genes respectively. The designation of ApxII was

proposed for the RTX-toxin which was produced by all serotypes except 10 and was

previously named APP. HlvII. Clyll or Cyt. This protein is weakly hemolytic and

moderately cytotoxic. The genes of the ApxII operon would have the designations of

ApxIIC for the activator gene and ApxIIA for the structural toxin gene. In the ApxII

operon. no genes for secretion proteins have been found. Secretion of ApxII seemed to

occur via the products of the secretion genes ApxIB and ApxID of the Apxl operon.

The designation. ApxIII. was proposed for the non-hemolytic RTX-toxin of the

reference strains for serotypes 2. 3. 4. 6 and 8. which was previously named cytolysin

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Ill (Clylll). pleurotoxin (Ptx). or macrophage toxin. The previous studies and toxin

designations were corroborated by another study where structural analysis demonstrated

that intact Apxl operons, consisting of the four contiguous genes. ApxICABD, were

present in serotypes 1. 5. 9. 10 and 11 (Jansen et al., 1993a). However, Apxl operons

with a major deletion in the ApxICA genes were present in serotypes 2. 4, 6 . 7, 8 and

12; serotype 3 did not contain Apxl operon sequences. All Apxl operons were

transcriptionally active despite the partial deletion of the operon in some serotypes.

Next, the APP RTX-toxin III gene (ApxIII) was cloned (Jensen et al., 1993b). Two

genes. ApxIIIC and ApxIIIA. coded for proteins ApxIIIC and ApxIIIA. respectively,

which were 53 and 50% identical to the prototypic RTX proteins HlyC and HlyA of E.

coli. It was assumed that the ApxIIIA gene coded for the structural RTX-toxin and that

the ApxIIIC gene coded for its activator. In addition, it was found that ApxIII could be

secreted from E. coli by the heterologous RTX transporter proteins. HlyB and HlyD.

The deduced amino acid sequence of ApxIIIA was 50% identical to that of ApxIA and

41% identical to that of ApxIIA. Beside Apxl and ApxII. ApxIII was determined to be

the third RTX toxin produced by A. pleuropneumoniae.

The virulence of the RTX hemolysins (Apxl and ApxII) of the swine pathogen

Actinobacillus pleuropneumoniae was investigated using hemolysin-deficient mutants

(Tascon et al.. 1994). Two types of haemolysin mutants with single insertions of the

transposon were obtained from a serotype I strain producing both Apxl and ApxII. One

presented a complete loss of hemolytic activity because of the absence of Apxl and

ApxII production. The other displayed weaker hemolysis than the wild type and

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. produced only ApxII. In the non-hemolytic mutant, the transposon had inserted in

ApxIB. a gene involved in the exportation of Apxl and ApxII toxins. The weakly

hemolytic mutant resulted from the disruption of the structural gene for Apxl. Both

mutations in the Apxl operon were associated with a significant loss of virulence for

mice and pigs, demonstrating that hemolysins were involved in APP pathogenicity. The

non-hemolytic mutant was non-pathogenic and the weakly hemolytic mutant retained

some virulence for pigs, suggesting that both Apxl and ApxII were needed for full

virulence.

Actinobacillus pleuropneumoniae strains that secrete three different exotoxins

(Apxl. ApxII and ApxIII) have been implicated in the etiology of porcine

pleuropneumonia (Chang et al.. 1993). To understand the role of these toxins in the

pathogenesis of this disease, the hemolysin gene (ApxII )was cloned, which encodes a

110-kDa polypeptide with hemolytic and cytotoxic activity. To clone the third toxin

gene (ApxIII). a new genomic library using APP serotype 2 chromosomal DNA was

constructed. A series of five overlapping recombinant phage clones carrying the gene

(ApxIII) for this 120-kDa antigen were identified using a DNA probe containing

sequences from the Pasteurella haemolytica IktBD genes. Sequence analysis of a

region of the cloned DNA revealed four open reading frames encoding proteins with

predicted masses of 20.4, 112.5. 80.3 and 54.7 kDa. These genes, designated ApxIIC.

ApxIIIA. ApxIIIB and ApxIIID. respectively, were similar in sequence to the RTX

(repeat of toxin) toxin family. The toxin produced by the cloned gene kills BL-3 cells

and was not hemolytic in vitro. Mutants of Actinobacillus pleuropneumoniae serotype

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 1 that were devoid of RTX toxins did not activate or kill porcine neutrophils (Jansen et

ai, 1995; van de-Kerkhof et al... 1996). The Actinobacillus pleuropneumoniae RTX-

toxins Apxl. ApxII and ApxIII were important virulence factors of this swine pathogen

and emphasized the importance of Apxl and ApxII in pathogenesis. The Apx toxins

may be deleterious to defense cells of the host, enabling the bacterium to infect the host.

The hemolytic RTX toxins of 27 isolates of APP representing all serovars have

been isolated in Australia (Tarigan et ai, 1994.1996). The quantity of protein secreted

by these isolates was not significantly different between serovars. Hemolytic activity

was detected only in the unconcentrated supernatants from cultures of serovar 1 and 5

isolates. Hemolytic activity in supernatants of serovar 2. 3 and 7 isolates was detected

only after the supernatants were concentrated. The hemolytic activity of the culture

supernatant depended on the type of composition of media and adaptability of the

bacteria to in vitro culture. A low number of in vivo passage after several in vitro

passages cultures of APP characterized by waxy colonies, produced significantly

weaker hemolytic activity than initial APP.

Non-Swiss albino (CF1) male mice were used as a model to study the

pathological effects of specific toxic components of APP on the respiratory system

(Idris, et al., 1993). Mice were divided into five groups of ten each and were inoculated

by the intranasal route with whole-cell APP serotype 1. cell-free culture supernatant

fluid (CFCSF) containing hemolysin protein, purified lipopolysaccharide. heat-treated

CFCSF. or PBS solution. Pulmonary lesions were evaluated at 6 and 12 h after

inoculation. Mice inoculated with whole cells. CFCSF and LPS developed severe

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. purulent bronchiolitis and alveolitis. Focal pulmonary necrosis was also observed in

these three groups but was most consistent in the CFCSF-inoculated mice.

Ultrastructurally. lungs from mice inoculated with whole cells, LPS and CFCSF were

characterized by severe degenerative changes in type I and type II pneumocytes.

Alveolar spaces contained cellular debris and fibrin. Endothelial cells were swollen and

selected pulmonary capillaries were occluded with platelets and fibrin. Infiltrating

neutrophils were often swollen, vacuolated and degranulated. Although inoculation

with relatively large numbers of APP were required to kill mice, the mouse lung was

quite sensitive to the toxic components produced by these bacteria. The elaboration of

these two toxic components by APP may be responsible for the characteristic

pulmonary inflammatory and necrotic lesions observed in infected swine.

Fluoroquinolones

Biotransformation

The predominant site of fluoroquinolone biotransformation is the piperazine ring

(Figures 1.1 and 1.2). (Neu. 1989. 1992). The oxoquinolones represent the major

metabolites of ciprofloxacin, enoxacin and norfloxacin (Lode et al.. 1990). It has been

postulated that fluoroquinolones producing 4-oxo metabolites were stereochemically

similar to theophylline or caffeine and inhibit methylxanthine metabolism (Robson,

1992: Sakar et ai. 1990; Staib et ai, 1989; Fuhr et al.. 1992,1993). The potential for

interaction with theophylline was not identical for all gyrase inhibitors (Sorgel &

Kinzig, 1993). Enoxacin is the strongest inhibitor of theophylline and caffeine

metabolism, followed by tosufloxacin. ciprofloxacin and pefloxacin. Fleroxacin.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. ofloxacin, rufloxacin and sparfloxacin have none or negligible effects. A likely

mechanism for this interaction is the inhibition of subsets of the cytochrome P450

enzyme. Piperazine ring-cleaved compounds and naphthyridine nuclei were shown to

be most active inhibitors of cytochrome P450IA2 (Figure 1.1). The effect of various

substituents was also tested, leading to an equation that predicted inhibition of 3-

caffeine 3-demethylation. Piperazine ring-cleaved compounds were more inhibitory

than parent compounds and a nitrogen at position 4 or 7, respectively, reduced or

increased inhibitory activity. Data on interactions of rifampin and cyclosporine with

gyrase inhibitors were conflicting. Rifampin increased the clearance of fleroxacin but

did not change the elimination half-life significantly. Although norfloxacin may

interfere with metabolism of the S enantiomer of warfarin, fleroxacin did not affect

pharmacokinetic parameters of either the R or S enantiomer or the anticoagulant

response.

Ciprofloxacin is the main metabolite of enrofloxacin resulting from ethyl

dealkylation at the para nitrogen on the piperazinyl ring (Figure 1.2) (Outman &

Nightingale. 1989). Desethylene-ciprofloxacin (Ml) resulted from dealkylation of the

ethyl group from the piperazinyl ring. Sulfo-ciprofloxacin (M2) was produced when the

para nitrogen on the piperazinyl ring was sulfated. Oxo-ciprofloxacin (M3) occurred

with the oxidation of the ortho position in relation to the para nitrogen on the 3' carbon

of the piperazinyl ring. Formyl-ciprofloxacin (M4) was formed by an aldehyde group

on the para nitrogen of the piperazinyl ring.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. OH Quinolone Structure Piperazinyl Ring

Fluoroquinolones Basic Structure

Naphthyridine Pyridopyrimidine

OH OH

HN HN CH •C Moiety Enoxacin Pipemidic Acid

O O O O

OH OH

HN CH}

Lomefloxacin Enrofloxacin Figure 1.1. The basic structure of the quinolones relates to the inhibition of methylxanthine metabolism. The N-C=N-C-N-C moiety is found in pipemidic acid and enoxacin. but not lomefloxacin or enrofloxacin.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. o o

O O

CH3 OH Enrofloxacin N N HN

Ciprofloxacin

F OH O O

N OH OH o=s N 'N

Sulfo-ciprofloxacin (M2)

O O Formyi-ciprofloxacin (M4)

'N HN O O O S' OH Oxo-ciprofloxacin(M3) NH

NH2

Desethylene-ciprofloxacin(Ml)

Figure 1.2. The fluoroquinolone, enrofloxacin, and some of its metabolites.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Flavin-containing monooxvgenase

Usually, flavin-containing monooxygenases catalyze the formation of N-oxides

from tertiary aliphatic amines like the 4-methvl-1-piperazinyl gyrase inhibitors (Sorgel.

1989). Like the cytochrome P450's. flavin-containing monooxygenases are oxidative

enzymes involved in Phase I biotransformations and can be referred to as mixed-

fiinction amine oxidases (Sipes & Gandolfi. 1991). The FAD-flavoprotein is present in

the endoplasmic reticulum and is capable of oxidizing nucleophilic nitrogen using

NADPH and oxygen. Flavin-containing monoxvgenase competes with the cytochrome

P450 system in the oxidation of amines, where FMO activity is high in humans and

pigs, but low in rats. Tertiary amines are converted to amine oxides, secondary amines

are converted to hydroxyl amines and nitrones and primary amines are converted to

hydroxy lam ines and oximes. The catalytic mechanism for flavin-containing

monoxvgenase is known in some detail for porcine liver (Poulsen & Ziegler. 1979:

Beaty & Ballou. 1980. 1981 a.b). The flavin-containing monooxygenases are not under

the same regulatory control as cytochrome P450's (Sipes & Gandolfi. 1991). While

P450's can be induced by phenobarbital and 3-methylcholanthrene. FMO was

repressed. Steroid sex hormones seem to regulate FMO enzymes with testosterone

decreasing and progesterone increasing the cellular concentrations. Unlike P450's.

xenobiotic substrates were not oxygenated for the reduction of flavin by NADPH or for

the reaction of dioxygen with the reduced flavin (Poulsen & Ziegler. 1979: Beaty &

Ballou. 1980. 1981 a.b). The key to FMO catalytic activity was the 4a-

hydroperoxyflavin intermediate and was formed by the reaction of oxygen with

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. reduced flavin. The 4a-hydroperoxvflavin intermediate was stable, which was a feature

unique to this type of monoxvgenase. The hydroperoxyflavin was also the oxygenating

intermediate and oxygen was transferred from the hydroperoxyflavin to the substrate.

The oxygenation reaction was identical in a model mechanism with similar products to

the oxidation of nucleophiles by synthetic protein-free N-5-alkyI. 4a-hydroperoxyflavin

(Ziegler. 1988). There was no evidence for intermediates during oxygen transfer in

either the porcine model or enzymatic reactions. This suggested that activation of

substrate was not required and explains the nature of products formed and for the broad

range of xenobiotics oxygenated by flavo-protein monooxygenases. Therefore, only

xenobiotics with electron-rich centers can serve as substrates. However, the substrate

must gain access to the enzyme-bound hydroperoxyflavin. Hydroperoxyflavin was

common to all forms of the enzyme, so it was evident that steric differences in access

may be responsible for differences in substrate specificity.

Substrate specificity of the enzyme-bound oxygenating intermediate should be

limited to organic nitrogen compounds with functional groups that are readily oxidized

by organic peroxides (Ziegler. 1988). Hydroperoxyflavin was not a strong oxidant for

the attack of nitrogen atoms in amides, carbamides. imines. nitrones. oximes.

isocynates, nitriles. or heterocyclic aromatic amines (Poulsen & Ziegler. 1979; Beaty &

Ballou. 1980. 1981a,b). Amines, hydroxylamines. hydrazines and sulfur functional

groups were more susceptible to oxidation and xenobiotics containing these groups

were suitable substrates. Amine functional groups are quite common in alkaloids and

synthetic drugs which makes the number of substrates in this category very large. Most

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. all lipophillic acyclic (nortryptyline. promazine) or cyclic (perazine, nicotine) secondary

and tertiary amines, respectively, tested were substrates. Flavin-containing

monoxvgenase had a loose specificity for amines and precise fit into the catalytic cavity

was not essential for biotransformation of substrates. Subsequently, the enzyme

catalyzes both acyclic and cyclic amines with equal efficiency.

The piperazinyl ring on enrofloxacin has two tertiary nitrogens (Figure 1.2).

The most likely position for N-oxidation and dealkylation by FMO would be the

nitrogen at the para position, possibly because there would be less steric hindrance. The

metabolism of enrofloxacin by FMO is supported by work with tertiary amines related

to brompheniramine, an antihistamine (Cashman et al.. 1993). The metabolism of

racemic, (D)- and (L)-brompheniramine was studied in microsomes and with highly

purified FMO from porcine liver. The main metabolite was the metabolite containing

aliphatic nitrogen N-oxide. but some N-demethylation was observed. Furthermore,

brompheniramine was extensively N-oxygenated by the highly purified FMO from hog

liver. N-oxygenation of bromapheniramine was enantioselective in both microsomes

and highly purified FMO. The Km for N-oxygenation of (D)-brompheniramine was

markedly lower than the Km for (L)-brompheniramine. (D)- and (L)-zimeldine were

less conformationally flexible model compounds of brompheniramine and both

compounds were found to be steroselectively N-oxygenated by the highly purified FMO

from hog liver. The results suggested that (D)-brompheniramine and (L)-zimeldine

have the aliphatic tertiary amine nitrogen atom and aromatic ring center at a defined

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. distance and geometry and were more efficiently N-oxygenated than their respective

isomers.

Antimicrobial mechanism of action

The primary target of quinolone activity in prokaryotic cells was DNA gyrase

(Vance-Bryan et al., 1990). This enzyme, a type II topoisomerases. was required for

DNA replication, transcription of certain operons. repair and recombination. In vitro.

the quinolones inhibit gyrase mediated DNA supercoiling and promote double stranded

DNA breakage at specific sites. New details of the molecular interactions of quinolones

with their target DNA gyrase and DNA have come from the nucleotide sequences of the

gyrA genes from resistant mutants of Escherichia coli and wild-type strains of other

bacteria and studies of gyrase A tryptic fragments (Hooper & Wolfson 1991). The

importance of an amino-terminal domain in quinolone action was supported by

observations of alterations in DNA super-twisting that were associated with altered

quinolone susceptibility, possibly by indirect effects on DNA gyrase expression.

Specific binding of relevant concentrations of norfloxacin to a complex of DNA gyrase

and DNA in the presence of ATP. the cooperativity of DNA binding and the crystalline

structure of nalidixic acid led to a model in which quinolones bind cooperatively to a

pocket of single-strand DNA created by DNA gyrase. Quinolones vary in their relative

activity against DNA gyrase and its eukaryotic homolog. topoisomerase II. The action

of the fluoroquinolones on the eukaryotic enzyme was associated with genotoxicity.

Inhibition of bacterial DNA synthesis by quinolones may correlate with minimum

inhibitory concentrations (MICs) in some species, but comparisons of drug

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. accumulation and inhibition of DNA synthesis in permeabilized cells among species

have been difficult to interpret. In addition to interaction with DNA gyrase. the specific

factors necessary for bacterial killing by quinolones include oxygen and new protein

synthesis.

Pharmacokinetic characteristics

The fluoroquinolones are characterized by a large volume of distribution, which

is reflected by good tissue penetration and long biological half-life in dogs, pigs and

humans (Greene & Budberg. 1993; Kung et al.. 1993a.b; Lode et al.. 1990; Vancutsem

et al.. 1990). The gastrointestinal absorption in mammals is rapid and substantial; the

duration of action is long and the excretion mainly through the kidney (Vancutsem et

al.. 1990). Their adverse effects were not severe when compared to the beneficial

features fluoroquinolones exhibit. The targets for adverse effects are: central nervous

system, urinary tract. DNA (genotoxicity). skin (phototoxicity), reproductive, digestive

tract and the juvenile cartilage (Takayama et al.. 1995; Vancutsem et al.. 1990). Neither

blood concentration nor pH affected low serum protein binding (Greene & Budberg.

1993; Kung et al.. 1993a.b; Lode et al.. 1990). Low serum protein binding promoted

rapid elimination by renal and extrarenal mechanisms with high total and renal

clearances. There was transluminal enteric excretion as well as nephron tubular

secretion (Wolfson & Hooper. 1991: Vance-Bryan et al.. 1990). The transluminal

elimination of ciprofloxacin implied that the drug was eliminated across the intestinal

wall without significant biliary elimination (<1%) (Bergan. 1989). In fact, transluminal

elimination compensates for loss of renal elimination. All fluoroquinolones, except

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. pefloxacin and enrofloxacin. undergo limited metabolism (Wolfson & Hooper. 1991:

Vance-Bryan et al. 1990). Water solubility promotes excellent bioavailability and

rapid distribution. In humans, pharmacokinetic characteristics include a large volume

of distribution for fluoroquinolones (1.74 to 5.0 L/kg), a two compartment model after

oral dosing and a three compartment model after i.v. dosing (Lode et al. 1990). The

two peripheral compartments consist of one with a rapidly equilibrating high clearance

rate and the other slowly equilibrating with a slow clearance rate that prolongs the half-

life (Lode et al. 1990).

The large volume of distribution is not counterbalanced by rapid elimination of

quinolones for most species. In pigs, the half-life is 6 to 8 h for the elimination of

ciprofloxacin, which is much slower than other reported half lives (Kung et al.

1993a.b). Ciprofloxacin may have potential clinical use in veterinary medicine, because

of its breadth and intensity of activity against Gram-negative pathogens and no cross

resistance with P-Iactams or aminoglycosides occurs (Nouws et al. 1988). Pigs were

administered an i.v. injection of 3.06 mg/kg of ciprofloxacin. The volume of

distribution, elimination half-life and total clearance were: 3.83 L/kg. 2.57 h and 17.3

ml/min/kg respectively. Twenty four hours after i.v. dosing, the urinary drug

concentration in pigs was 8.9 pg/ml and plasma protein binding for ciprofloxacin in

pigs was 23.6 %. The renal clearance of ciprofloxacin was 10 times higher than the

creatinine clearance and indicated a predominately tubular secretion pattern of the drug.

Urinary recovery for pigs following i.v. administration was 47.9%. No glucuronide

conjugates of ciprofloxacin or its metabolites (M, or M2) were detected in the plasma or

100

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. urine of the pigs (Nouws et al., 1988). The low protein binding of ciprofloxacin was

attributed to interference of fatty acids in the drug binding by albumin or fewer binding

sites on albumin and a i-acid glycoprotein (Belpaire. 1986).

The relationship of the physicochemical properties of gyrase inhibitors to their

hepatic and renal elimination pathways was analyzed (Sorgel & Kinzig, 1993). Luminal

fluid concentrations of gyrase inhibitors were affected by an active process and

inhibited by agents, such as probenecid, that inhibit tubular secretion of anions (Bergan.

1989; Sorgel & Kinzig. 1993). Probenecid may inhibit base transport in the proximal

tubule and inhibit quinolone transport as well. Available evidence suggested that all

gyrase inhibitors were secreted as anions by the proximal tubules. Cimetidine. which is

cationic at physiologic pH. inhibited base transport in the proximal tubule and inhibit

base transport of gyrase inhibitors. Reabsorption also affected tubular concentrations.

The N4'-methylated derivatives were the most lipophilic and addition or removal of the

methyl group can. but did not always, affect reabsorption. The data indicated that all

gyrase inhibitors undergo tubular secretion as either acids or bases and that some also

were significantly reabsorbed. Hepatic handling and resultant excretion of metabolites

were also influenced by the presence or absence of N4'-methylation. A step in the

hepatic handling of N4'-methylated gyrase inhibitors that leads to N4'-oxidation has not

yet been found in rufloxacin.

Enrofloxacin in veterinary medicine

In the USA. approved use of enrofloxacin is limited to dogs; approval for use in

food-animals is currently being sought for several fluoroquinolones (Greene &

101

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Budberg, 1993: Vancutsem et al., 1990). even though extra-label use of other approved

fluoroquinolones has been banned by the U.S. Food and Drug Administration. The

activities of danofloxacin. a novel fluoroquinolone and two other antimicrobials were

determined in vitro against field isolates of seven Mycoplasma species of veterinary

importance isolated from cattle, swine and poultry in five European countries (Cooper

et al., 1993; Giles et al., 1991; McGuirk et ai. 1992). It was concluded that

danofloxacin is highly active in vitro for the antibiotic control of all of the Mycoplasma

species tested and thus showed great potential for the treatment of respiratory and other

infections caused by Mycoplasma species in cattle, pigs and poultry. Marbofloxacin is a

new fluoroquinolone that was developed exclusively for veterinary use (Spreng et ai.

1995) Marbofloxaicn showed a broad spectrum of activity for the treatment of Gram-

negative and Gram-positive bacterial diseases in dogs and cats. The post-antibiotic

effect durations were found to be almost equal to the durations of enrofloxacin and

ciprofloxacin. Marbofloxacin has been recently approved in 1997 in the United

Kingdom for salmonellosis and colibacillosis in cattle (Dr. Michael Myers, personal

communication).

Enrofloxacin is available in some countries as a injectable for parenteral use and

as a food additive (Greene & Budberg. 1993). Enrofloxacin is the first antibacterial of

this family to be available to veterinary medicine in the United States (Vancutsem et al..

1990). In swine, quinolones are effective for treating difficult respiratory diseases:

mycoplasmal pneumonia and arthritis; enteric infections such as diarrhea of suckling,

weaning or fattening pigs (coli diarrhea); septicemia caused by pathogenic Escherichia

102

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. coli (edema disease) or Salmonella spp.; mastitis-metritis-agaiactia and urinary tract

infections (Vancutsem et al., 1990; Wendt. & Sobestiansky 1995). Enrofloxacin is

effective for the control of diseases caused by Escherichia coli and Haemophilus.

Mycoplasma, Pasteurella, Eubacterium suis, Actinobacillus suis, Actinomyces

pyogenes, Corynebacterium pseudotuberculosis. Erysipelothrix rhusiopathiae.

Haemophilus pctrasuis, Haemophilus somnus, Pasteurella haemolytica, Pasteurella

multocida, Rhodococcus equi. Streptococcus equi. Streptococcus suis. Streptococcus

zooepidemicus and Salmonella spp in the United States (Hariharan et al., 1995; Prescott

& Yielding. 1990; Vancutsem et al., 1990; Wendt. & Sobestiansky 1995).

Streptococcus suis types 1 and 2 were most commonly isolated via direct swabbing of

palatine tonsils of healthy nursery pigs on 35 farms throughout the United States (Dee et

al.. 1993). All isolates of Streptococcus suis were susceptible to enrofloxacin. 97% of

the isolates were susceptible to ceftiofur and 94% were susceptible to ampicillin.

However, only 80% of the isolates were susceptible to penicillin and only 18% were

susceptible to tetracycline.

While vaccine protection has not been rewarding for the control of porcine

pleuropneumonia caused by APP. antimicrobial therapy has proven to be effective .

Treatment for infection usually lasts 8 to 10 days in swine using quinolones. In

controlled studies of swine infected with APP, 60% of the pigs died 18 to 56 h after

experimental inoculation (Glawischnig et al., 1989; Schroder. 1989). Enrofloxacin was

administered intramuscularly for 3 days at 2.5 to 5 mg/kg beginning 8 h post-infection

or 5 mg/kg starting 12 h post-infection. Clinical improvement began 12 h after therapy

103

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. was initiated and recovery was facilitated in all treated animals (Bauditz. 1987;

Stephano et al.. 1987).

In a recent study, the pharmacokinetic parameters of enrofloxacin in clinically

normal dogs and mice and drug pharmacodynamics in neutropenic mice with

Escherichia coli or Staphylococcal spp. infections were investigated (Meinen et al..

1995; Walker et al.. 1992). Enrofloxacin dosages ranged from 0.78 to 50 mg/kg of

body weight in dogs and 6.25 to 200 mg/kg in mice. It was determined that

enrofloxacin total dose rather than the dose frequency was significant in determining

drug efficacy. Pharmacokinetic parameters were analyzed by multivariate stepwise

linear regression analysis. The calculations revealed that the area under the

concentration-time curve was more important than the time above minimum inhibitory

concentration. So. enrofloxacin killing of Escherichia coli and Staphylococcus was

plasma concentration-dependent and not time-dependent.

104

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. CHAPTER II

INFECTION MODEL

Introduction

Previous experiments by other investigators were designed to demonstrate

efficacy and pharmacokinetic changes using non-infected animals. However, animals

may have altered drug disposition and clearance when infected with pathogenic

organisms. The infectious disease may alter the drug pharmacokinetic changes and

efficacy studies often do not examine drug pharmacokinetic parameters. Therefore,

evidence of a potential problem will not be detected. Few studies have explored the

possibility of drastically increased toxicity or reduced efficacy in domestic animals to

the same extent as rodent and human studies (Monshouwer. 1996: Monshouwer et al..

1995a.b. 1996a.b: Myers & Kawalek. 1995: Pijperset al.. 1990.1991: Short. 1986:

Short et al.. 1986). Endotoxin has been shown to reduce the elimination rate of

antipyrine and trimethoprim in swine and decreased the total clearance of tiliazad

mesylate in beef calves (Ladefoged, 1979: Rose et al.. 1993).

Target animal safety problems could arise in the drug development and approval

process whereby therapeutic doses are actually toxic in the infected animal (Ladefoged.

1979: Rose et al.. 1993). Conversely, increased rate of clearance could indicate a need

for more frequent dosing. Doses that were previously efficacious may not be sufficient

to treat an infected animal. Human food safety is a concern. However, increased drug

clearance and decreased half-life in food animals would result in decreased residue

concentrations that are well below tolerance concentrations and withdrawal times.

105

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. The goal of the infection study was 1) to characterize pharmacokinetic changes

associated with APP infection in swine after receiving enrofloxacin and 2) to evaluate

the pulmonary infection model compared to the endotoxin model and 3) to characterize

the systemic level of cytokines that may be associated with pharmacokinetic changes.

Plasma concentrations of IL-6 were determined from 0 to 72 h after infection. Plasma

concentrations of IL-ip were determined for 0. 2, 6 and 12 h after infection. Plasma

concentrations of enrofloxacin and ciprofloxacin were determined for pharmacokinetic

analysis from 24 to 72 h after infection. Urine enrofloxacin and ciprofloxacin were also

measured and normalized with urine creatinine concentrations for samples collected 48

and 72 h after infection. Gross necropsies were performed on all pigs which included

lung weights and tissues collection for histopathology.

Materials and Methods

Animal management

The experiment consisted of 24 crossbred. Landrace x Yorkshire, castrated pigs

that were procured from a local vendor. All of the pigs were weighed prior to

intravenous catheterization and were within a 25 to 35 kg weight range. Housing

consisted of a heated building with a concrete floor and all animals were fed 3 kg/day

from a single batch of a commercial corn-based ration without antibiotics. The pigs had

been fed a commercial ration that was medicated with tylosin before procurement. All

pigs were examined upon arrival and were clinically healthy at the start of the study.

All procedures were approved by the Food and Drug Administration, Center for

106

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Veterinary Medicine, Office of Research. Institutional Animal Care and Use

Committee.

Experimental design

The design consisted of a 2 x 2 factorial of treatments groups (Table 2.1).

Actinobacillus pleuropneumoniae (gift from Dr. Michael P. Murtaugh. University of

Minnesota. St. Paul. Minnesota) was one treatment. The second treatment was

dexamethasone (0.5 mg/kg, Phoenix Pharmaceuticals. St. Joseph. Missouri). The

bacteria were administered bv endotrachael infusion at zero hour into two of the four

groups as a comparison to endotoxin that was administered by i.v. bolus in the next

experiment. Intranasal inoculation in previous studies had resulted in a low infection

rate while endotrachael inoculation produced a 100% infection rate. Four groups of six

pigs were intravenously catheterized and placed in stainless steel metabolism cages over

a one month period. Two groups were procured at a time and one group was placed in

metabolism cages for four days followed by the second group. This process was

repeated with another two groups of pigs. The Actinobacillus pleuropneumoniae was

administered as a single endotrachael infusion to two groups while the other two groups

received a sham saline infusion (Table 2.1). One infected group and one sham group

received i.v. dexamethasone at -18 h . 0 h and 12 h . A bolus dose of 5 mg/kg

enrofloxacin (Baytril . Bayer, Shawnee Mission. Kansas) was administered

intravenously 24 h after saline or bacterial infusion to all four groups of pigs.

107

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table 2.1. Experimental design for the Actinobacillus pleuropneumoniae infection study.

Treatment Sham Infection Infection

Sham Infection Infection No Dexamethasone

No Dexamethasone No Dexamethasone

Sham Infection Infection Dexamethasone

Dexamethasone Dexamethasone

108

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Bacteria

Animals were endobronchially inoculated with Actinobacillus

pleuropneumoniae serotype 1. strain L91-2. a field strain isolated from an outbreak in

Iowa(Baarsch et. a i. 1995: van Leengoed et. al.. 1987. 1989. 1990). The virulence of

strain L91-2 was reduced by serial in vitro passage on solid growth media. Single

colonies were passaged four times or until atypical morphology was evident. Colonies

were creamier in appearance and two to three times larger and had a more rapid growth

rate compared to non-passed microorganisms.

Logarithmically growing bacteria were employed to prepare inocula for the pigs.

Cultures of APP bacteria were grown on agar plates from frozen stock (-70°C) that had

been preserved in broth culture and 80% glycerol. The agar media consisted of 43

grams of Casman's agar (Difco Laboratories, Detroit. Michigan) dissolved in 1000 ml

of water and autoclaved for 20 minutes. The media was allowed to cool to 50°C and 50

ml of heat inactivated horse serum was added. After the addition of 2 ml (5 g/L) of

sterile NAD (Sigma. St. Louis. Missouri) the agar was poured into sterile plates. After

incubating at 37°C for 12 hours, a single colony wras picked from the Casman's plate

and inoculated into 3% tryptic soy broth (Difco Laboratories. Detroit. Michigan)

containing 1% NZ amine (Sigma, St. Louis. Missouri), 0.1% yeast extract and 0.001%

NAD and incubated at 37°C for 4-6 h while shaking at 215 rpm. The NAD was added

to the broth after autoclaving and just before inoculation of the bacteria.

The cells were washed with 5% bovine serum albumin (BSA) in phosphate

buffered saline (PBS. pH 7.4) after centrifugation at 1500g for 10 minutes. The washed

109

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. cells were diluted to 0.4 optical density (550 run) which corresponded to approximately

2.0 x 10 CFU/ml. Viable cell-counts before inoculation were confirmed by plating on

Casman's agar and reading the plate counts on the day following endobronchial

inoculation. The cells were further diluted in PBS-BSA to about 2 x 104 CFU/ml. Six

milliliters of the 2 x 104 CFU/ml bacterial inoculum was delivered through a rigid

polypropylene tubing (1 m x 1 mm I.D.) into the bronchial lumen using a 12 ml syringe.

Air was flushed into the polypropylene tubing after the inoculation of the bacteria.

Non-infected animals received 6 ml of PBS-BSA.

Blood collection

A cranial vena cava catheter was implanted in each animal 24 h prior to bacterial

inoculation or saline inoculation. Plastic tubing (0.6 mm I.D. x 1 m) was inserted

through a 14 gauge (14 cm in length) stainless steel cannula (Brocht et al.. 1989; van

Leengoed et al.. 1987). The animals were restrained without anesthesia while inserting

the cannula 2.5 cm lateral to the point of the sternum at a 45° angle. The catheter was

secured at the point of skin penetration with surgical silk. The end of the catheter was

extended to the back of the neck and protected with 15 cm of Elastikon2' (Johnson &

S) Johnson. Raritan. New' Jersey) and two rolls of Vetrap (3M Animal Care. St. Paul.

Minnesota). A 19 gauge tubing adapter (Becton/Dickson. Sandy. Utah) was inserted

into the end of the catheter and fitted with an injection cap (Becton/Dickson, Sandy.

Utah).

Blood samples were collected through a saline-heparin lock (10 units/ml. Sigma.

St. Louis, Missouri) in order to measure plasma concentrations of enrofloxacin.

110

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. ciprofloxacin (Sigma, St. Louis. Missouri) IL-6 and TNF-a. Cytokine blood samples

were collected in heparin tubes (Monovette. Sarstedt, Germany) at 0. 1. 2. 3,4, 6. 8, 10.

12.24, 36. 48. 60 and 72 h after sham infection or infection. High-performance liquid

chromatography (HPLC) samples were also drawn in heparin tubes at 0. 0.25. 0.5. 1. 2.

3, 4. 6, 8, 12. 24, 30 and 48 h after i.v. administration of enrofloxacin at 24 h post

infection or sham infection. The samples were placed on ice before transporting to the

laboratory and centrifuged (Jouan CR 412. Winchester. Virginia) at 1500g for 10

minutes. The centrifuged plasma was harvested and aliquoted for storage at -70°C.

Pharmacokinetic analysis

Plasma sample preparation

Plasma samples were analyzed for enrofloxacin and ciprofloxacin by mixing 100

pi of sample with 300 pi of a 2: 1 mixture of 0.05 N sodium hydroxide (Sigma. St.

Louis. Missouri) and acetonitrile (HPLC grade. Burdick & Jackson. Muskegon.

Michigan) containing 333 pg/L of pipemidic acid (Tyczkowska et al.. 1994). The final

internal standard concentration of the fortified samples was 250 ng/ml pipemidic acid.

After vortexing for 10-15 seconds, a 200 pi aliquot was placed in a 30.000 molecular

weight cut-off microfilter (Microcon 30®. Millipore. Burlington. Massachusetts) and

centrifuged (Eppendorf 5402. Brinkman Instruments, Westbury, New York) at 14.000g

in a 45° fixed-angle rotar for 1 h . Approximately 150 pi was recovered and placed in

tapered glass sample vials (Sulpelco. Bellefonte. Pennsylvania). Ten microliters of the

clear ultrafiltrate was injected into the liquid chromatograph.

I ll

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Urine sample preparation

Urine samples were collected 24 h after infection or sham infection and at 0. 24

and 48 hours after enrofloxacin administration. The urine samples were aliquoted into

cryovials and stored at -70°C. After thawing at room temperature, sample dilution was

accomplished by adding 100 pi of the sample to 400 pi of diluent (2: 1 mixture of 0.05

N. sodium hydroxide and acetonitrile) in siliconized microcentrifuge tubes (Costar,

Cambridge, Massachusetts) and mixed by vortexing. A 50 pi aliquot was taken from

the 1:4 urine dilution and added to a sample vial containing 950 pi of the following

mixture: 750 pi of 0.05 N sodium hydroxide and acetonitrile (2: 1): 200 pi of distilled

water with 263 pg/L of pipemidic acid. The pipemidic acid had been added from a 5

mg/L stock solution that was diluted with the 2: 1 mixture of 0.05 N sodium hydroxide

and acetonitrile. The pipemidic acid, water and sodium hydroxide/acetonitrile were

mixed in a single batch prior to addition to the sample vial. The final urine dilution in

the sample vial was 1: 99 after mixing the 50 pi of the 1:4 diluted urine to the 950 pi of

internal standard, water and 2: 1 mixture of 0.5 N sodium hydroxide/acetonitrile. The 2

ml glass sample vials (Sulpelco. Bellefonte, Pennsylvania) were placed in the HPLC for

10 pi injections.

Urine creatinine

Urine creatinine was used to normalize the urine concentrations of enrofloxacin

and ciprofloxacin (Palmisano et. al.. 1995). This approach avoided the need to collect

and measure the volume of 24 hour urine samples, because of the occasional

contamination with drinking water or accidental loss of a portion of the 24 hour urine

112

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. sample. Urine creatinine was assayed using a Urine Creatinine Kit (Sigma, St. Louis,

Missouri). The urine samples were first diluted ten to thirty times with water. Working

standard dilutions were prepared by diluting 150 mg/L stock standard with water

resulting in a standard curve consisting of 0. 25. 50, 75. 100 and 150 mg/L. Sample and

standards were prepared in triplicate by adding 30 pi to 300 pi of alkaline picrate

solution into each well of a microplate including the standard curve wells on each plate.

The plates were mixed for 30 seconds and let stand at room temperature for 10 minutes.

The plates were read at 500 nm using a microplate spectrophotometer (Spectra Max

250. Molecular Devices. Sunnyvale. California)

The standard curve on each of the four test plates was linear, "y" (absorbance)

versus “c" (concentration. mg/L): y=0.007 c + 0.221. R=0.9995; y=0.007 c + 0.189,

R=0.9995; y=0.007 c + 0.191. R=1.0: and y=0.007 c + 0.185. R=0.9995. The

coefficient of variation was less than 7% and 10% for the standar ds and samples,

respectively.

Recovery of the internal and external standards

Pooled pig plasma from an unrelated study was used to explore recovery of

ciprofloxacin and enrofloxacin. Ten serial dilutions (1: 1) of ciprofloxacin and

enrofloxacin were prepared in the range of 313 pg/L to 80 mg/L using water. The final

concentrations of the 10 standards ranged from 15.6 pg/L to 4 mg/L after adding 50 pi

of each stock standard to 950 pi of pooled pig plasma. The fortified plasma samples

were analyzed for enrofloxacin and ciprofloxacin by mixing 100 pi of the fortified

plasma with 300 pi of a diluent consisting of: a 2: 1 mixture of 0.05 N sodium

113

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. hydroxide and acetonitrile with 333 pg/L pipemidic acid as the internal standard

(Tyczkowska et al.. 1994). The final internal standard concentration of the fortified

samples was 250 pg/L pipemidic acid. After vortexing for 10-15 seconds, a 250 pi

aliquot was placed in a 30.000 molecular weight cut-off microfilter and centrifuged at

14.000g in a 45° fixed-angle rotary for lh. Approximately, 200 pi of the ultrafiltrate

was recovered and placed in tapered glass sample vials. Ten microliters of the clear

ultrafiltrate was injected into the liquid chromatograph Average analyte recovery from

five replicates at each of the 10 concentrations ranged from 91% to 104% for pipemidic

acid. 94% to 107% for ciprofloxacin and 94% to 106% for enrofloxacin. The plasma

peak height counts were compared to the average of five replicates of aqueous internal

standard and external standards that were prepared in water without microfiltration. The

coefficients of variation for each of the 10 concentration recoveries ranged from 1.9% to

11% for the percent of recovery of pipemidic acid, ciprofloxacin and enrofloxacin.

Aqueous standard curves for plasma

Aqueous standard curves for plasma were constructed by first preparing

enrofloxacin and ciprofloxacin working standards consisting of eleven dilutions: 0.

0.156. 0.313. 0.625. 1.25. 2.5. 5.0. 10.0. 20. 40 and 80 mg/L. Each working standard

was diluted 20 times to final concentrations ofO, 0.0078. 0.0156. 0.0313. 0.0625. 0.125,

0.25, 0.5. 1. 2 and 4 mg/L. for enrofloxacin and ciprofloxacin. The final dilution for

HPLC analysis was prepared by adding 50 pi of working standard to 950 pi of the

following mixture: 750 pi of 0.05 N sodium hydroxide and acetonitrile (2: 1); 200 pi of

distilled water with 263 pg/L of pipemidic acid. The pipemidic acid had been added

114

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. from a 5 mg/L stock solution that was diluted with the 2: 1 mixture of 0.05 N sodium

hydroxide and acetonitrile. The pipemidic acid, water and sodium

hydroxide/acetonitrile were mixed in a single batch prior to addition to the sample vial.

The final internal standard concentration of the standards and fortified samples was 250

ng/ml of pipemidic acid. The standards were not filtered through the 30.000 molecular

weight cut-off filters.

The linearity of the response, "y" (ratio of peak height counts) versus ”c '

(concentration. mg/L) were tested over eight standard solutions. The typical response

standard curves for plasma samples were linear as represented by the equations: y=12.8

c + 0.135. R=0.9995 for enrofloxacin; and y=10.2 c - 0.323. R=0.9984 for

ciprofloxacin. Five replicates of each of the 10 standard concentrations (7.8 pg/L to 4

mg/L) for ciprofloxacin and enrofloxacin yielded coefficients of variation ranging from

2% to 6% for ciprofloxacin and 0.8% to 13% for enrofloxacin.

Aqueous standard curves for urine

Aqueous standard curves for urine were constructed by first preparing

desethylene-ciprofloxacin. enrofloxacin and ciprofloxacin stock standards.

Desethylene-ciprofloxacin stock standard (6.25 mg/L) was diluted (1:1) into the

enrofloxacin and ciprofloxacin stock standard (20 mg/L). Working standards were

prepared by simultaneous 1:1 serial dilutions of the three standards in the stock solution.

The working standards concentrations were: 0. 0.0244. 0.0488. 0.0975. 0.195. 0.39.

0.78. 1.56 and 3.13 mg/L for desethylene-ciprofloxacin and corresponding dilutions: 0.

115

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 0.078. 0.156. 0.313. 0.625, 1.25. 2.5, 5.0 and 10.0 mg/L of enrofloxacin and

ciprofloxacin.

Each working standard was diluted 20 times with 0.05 N sodium

hydroxide/acetonitrile (2:1) to final concentrations of 0. 3.9. 7.8. 15.6. 31.3. 62.5. 125.

250 and 500 pg/L, for enrofloxacin and ciprofloxacin and 0. 1.22. 2.44. 4.88. 9.75. 19.5.

39. 78 and 156 pg/L. for desethylene-ciprofloxacin. The final solution for HPLC

analysis was prepared by adding 50 pi of working standard to 950 pi of the following

mixture: 750 ul of 0.05 N sodium hydroxide and acetonitrile (2: 1): 200 pi of distilled

water with 263 pg/L of pipemidic acid. The pipemidic acid had been added from a 5

mg/L stock solution that was diluted with the 2: 1 mixture of 0.05 N sodium hydroxide

and acetonitrile. The pipemidic acid, water and sodium hydroxide/acetonitrile were

mixed in a single batch prior to addition to the sample vial. The final internal standard

concentration of the standards and fortified samples was 250 ng/ml of pipemidic acid.

The internal standard, water and sodium hydroxide/acetonitrile were mixed in a single

batch prior to addition to the sample vial. The standards were not filtered through the

30.000 molecular weight cut-off filters.

The linearity of the response, "v” (ratio of peak height counts using the internal

standard, pipemidic acid) versus concentration, "c” (mgT. for desethylene-

ciprofloxacin, ciprofloxacin and enrofloxacin) were tested over eight standard dilutions.

The typical response standard curves for urine samples were linear as represented by the

equations: y=16.0 c - 0.0379. R=0.9997 for enrofloxacin; y=l 1.8 c + 0.00253.

116

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. R=0.9997 for ciprofloxacin; and y=123 c + 0.0434, R=0.9997 for desethylene-

ciprofloxacin.

Five replicates of each of the 8 concentrations (3.9 - 500 pg/L for ciprofloxacin

and enrofloxacin and 1.22 - 156 pg/L for desethylene-ciprofloxacin) of standard yielded

coefficients of variation ranging from 0.7% to 2.9% for desethylene-ciprofloxacin, 2%

to 11 % for ciprofloxacin and 1 % to 9% for enrofloxacin. respectively. The minimum

detection concentrations for pipemidic acid, ciprofloxacin, enrofloxacin. and

desethylene-ciprofloxacin were 33. 10. 12 and 1 ng/ml, respectively. The actual

minimum detection level was 40, 49 and 4 ng/ml for ciprofloxacin, enrofloxacin and

desethylene-ciprofloxacin. respectively because of a four-fold plasma dilution. The

actual minimum detection limit for urine was 200. 240 and 20 ng/ml for ciprofloxacin,

enrofloxacin and desethylene-ciprofloxacin. respectively because of a 20-fold urine

dilution. The minimum detection limit was based on a signal-to-noise ratio of 3.

High-performance liquid chromatography

Ten microliters injections of samples and standards were loaded into a

Rheodvne injector valve using a an autosampler that was incorporated into the liquid

chromatograph (Perkin Elmer Integral 4000. Perkin Elmer. Norwalk, Connecticut). The

column was a reverse phase C-18 column with dimensions of 250 mm c 3.2 mm and a 5

pM particle size protected by a guard column (Primasphere^. Phenomenex, Torrance.

California). The mobile phase for the plasma samples consisted of 19% acetonitrile and

81% 0.15 M citric acid (Sigma. St. Louis. Missouri) with a flow rate o f 0.5 ml/min with

a 10 minute run time for plasma. The mobile phase for the urine samples consisted of

117

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 18% acetonitrile and 82% 0.15 M citric acid with a flow rate of 0.5 ml/min with a 14

minute run time. The UV detector was set at 280 nm and the fluorescence detector (LS-

4, Perkin Elmer. Norwalk, Connecticut) settings were 276 nm and 450 nm for excitation

and emission, respectively. The peak height data was integrated by Turbochrom9

(Version 4.2. Perkin Elmer-Nelson, San Jose. California). The resulting plasma

concentrations were then subjected to nonlinear regression analysis using a

noncompartmental model (WinNonlin Version 1.1. Apex, North Carolina).

Interleukin-6 bioassay

Interleukin-6 assay was performed as previously described using the murine IL-

6 dependent 7TD1 cell line (ATCC No. CRL 1851. Rockville. Maryland) to assay the

swine plasma (Myers et al.. 1992). The cells were grown in Ultraculture media (500

ml. Bio-Whittaker. Walkersville, Maryland) with supplements of HEPES (5 ml. 1 M.

Sigma. St. Louis. Missouri), gentamicin (1 ml. 50 mg/ml. Bio-Whittaker. Walkersville,

Maryland), sodium bicarbonate (5 ml. 7.5%. Sigma. St. Louis. Missouri), fungizone (2

ml. 250 mg/ml, GIBCO, Grand Island, New York) and glutamine (10.3 ml. 200 mM.

Sigma. St. Louis. Missouri). The media was further supplemented with 10 units of IL-

6/mI and 50 pM of 2-mercaptoethanol to maintain the cells.

The cells were washed three times in assay media that did not contain IL-6. A

standard curve was prepared for each 96-well microplate (Costar. Cambridge.

Massachusetts). A standard curve (33.3, 25, 10. 5. 3.33. 2.5. 1 and 0 units per/ml) was

prepared for each microplate by dilutions. Specific activity of the human IL-6 standard

o was 1.375 x 10 units/mg of protein. The samples were diluted 1: 39 with assay media

118

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. and 100 pi of the diluted sample or standard was added, in duplicate, to the appropriate

wells. The 7TD1 cells were diluted with assay media to a concentration of 2-10 cells x

104 cells/ml and 100 pi of cells was added to each well of the microplate. The plates

were incubated for 68 hour at 37°C in 5% C 02. Four hours before termination. 20 pi of

Alamar blue dye (Alamar Biosciences, Sacramento, California) was added to each well

and the plate was incubated at 37°C for 4 hours. The activity of the IL-6 was assessed

by use of a fluorometer (Cytofluor 2350. Millipore. New Bedford. Massachusetts) with

settings of 530 nm excitation and 590 nm emission.

The standard curve was plotted as a log-logit transformation using Cytocalc

(Millipore. New Bedford. Massachusetts). The log-logit transformations gave 12 linear

response curves. ”y" (absorbance) versus "c" (concentration, pg/ml). for each plate:

y = l.57 c - 5.34. R=0.95; v=l.49 c - 5.12. R=0.94; y=1.41 c - 4.82. R=0.91; y = l.50 c -

4.95. R=0.92; y=1.38 c - 4.70. R=0.91: y=1.40 c - 4.76. R=0.90; v=1.27 c -4.45.

R=0.98; y=l .44 c - 4.98. R=0.99; y=l .38 c -4.72. R=0.99; v=1.45 c -5.05. R=0.98:

y=l .33 c - 4.63. R=0.98 and y=1.38 c - 4.81. R=0.99. The sample values were

interpolated from the standard curve on each of the microplates and the data was plotted

using SigmaPlot (Version 3.0. Jandel Scientific. San Rafael. California).

Interleukin-ip ELISA assay

Microplates that were coated with mouse monoclonal antibody to IL-1 p

(Interleukin-ip ELISA Test K.it. Genzyme Diagnostics. Cambridge, Massachusetts)

were used to measure IL-1P in pig plasma at 0, 2. 6 and 12 hours. A 100 pi aliquot of

standard or sample was added to each well in duplicate. A standard curve was prepared

119

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. to include 0. 4. 64. 256, 512 and 1024 pg/ml of IL-1 p using polypropylene culture

tubes. The serial dilutions of the standards were accomplished with sample diluent that

was supplied in the kit and were mixed by vortexing. The undiluted plasma was added

directly to the test wells.

The test wells were covered with plate sealer and incubated for 30 minutes at

37°C to allow IL-1P to bind with the monoclonal antibody. The liquid was aspirated

from the test wells after incubation. The wells were washed five times with wash

reagent followed by aspiration after each wash. After the last wash, the plates were

blotted with paper towels to remove excess liquid. Reconstituted IL-1P rabbit

polyclonal antibody (100 pi) was added to each well. The plates were covered with

plate sealer and incubated for 30 minutes at 37°C. The test wells were washed and

aspirated five times and 100 pi of IL-1 (3 conjugate (peroxidase anti-rabbit IgG reagent)

was added to each well. The plates were incubated for 15 minutes at 37°C and washed

five times.

Finally. 100 pi of peroxidase substrate and chromagen (tetramethylbenzidine)

reagent was added to each well and the plates were incubated for 30 minutes at room

temperature. A blue color was produced in the presence of the peroxide enzyme. The

color reaction was stopped by the addition of acid (50 pi) which changes the blue color

to yellow. The intensity of the yellow color was directly proportional to the amount of

IL-ip present in the sample or standard. The plates were read at 450 nm absorbance on

a microplate spectrophotometer (Spectra Max 250. Molecular Devices. Sunnyvale.

California) within 30 minutes after stopping the reaction. A standard curve was plotted

120

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. using Softmax Pro (Version 1.2, Molecular Devices. Sunnyvale. California) and the

plasma sample concentrations of IL-ip were interpolated from the standard curve on

each plate. The response curves of the standard dilutions were nonlinear, "y”

(absorbance) versus "c” (concentration, pg/ml): y=0.12 + 0.006 c - (2.5 x 10-6)c\

R=0.9995 and y=0.175 + 0.009 c - (5 x 10-6)c2. R=0.9995.

Health observations, necropsies and histopathology

Health observations were made during the entire study period of 4 days. Gross

necropsies were performed on all animals in the infection study. The pigs were

euthanized with Euthasol® which contained sodium pentobarbital (Delmarva

Laboratories. Inc.. Bristol. Tennessee). Brain and spinal cord were not examined. Fresh

liver (100 to 200 grams) was collected and frozen at -80°C if required to perform

Northern or Western blots. Lung weights and gross necropsy observations were

recorded on a necropsy form. The color, texture, location and size of gross lesions were

also noted on the necropsy form. After trimming the trachea from the lung at the level

of the cranial bronchi the lung was weighed before sampling for histopathology.

The following tissues (1-2 mm thick up to 4 x 4 cm) were collected for

histopathology: right bronchial lymph node, middle mediastinal lymph node, mesenteric

lymph node, lung, two sections of heart from the left ventricle and septum, three

sections of liver from different lobes, one section of the middle region of the spleen and

one section of each kidney. The lungs were covered with a paper towel during fixation

in the specimen jar. If gross abnormalities were observed in other organs or tissues, a

sample was collected for histopathology. The tissues were placed in a 4 L specimen jar

121

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. containing 10% buffered formalin. pH 7.4. (Sigma. St. Louis. Missouri) in a 1: 20 ratio

of tissues to formalin. The tissues were allowed to fix in the formalin for 24 hours and

the spent formalin was decanted and replaced with fresh formalin. The tissues were

allowed to completely fix for at least one week before transporting to the Department of

Veterinary Pathology. College of Veterinary Medicine. Louisiana State University,

Baton Rouge. Louisiana for processing and microscopic examination. The tissues were

sealed and shipped to Louisiana State University in whirl pack plastic bags with a

minimum of formalin. Selected tissues were processed by routine methods for light

microscopy.

Statistics

Pharmacokinetic parameters, urine enrofloxacin and ciprofloxacin

concentrations. IL-1 and IL-6 plasma concentrations and lung weights were analyzed by

Two-Way ANOVA. General Linear Model, using SigmaStat. Version 2.0 and

SigmaPlot. Version 3.0 (Jandel Scientific. San Rafael. California). Pharmacokinetic

parameters that were statistically analyzed were: : Area Under Curve. 0 h to last

sample. (AUC q.* pg h /L); Area Under Curve. 0 h to infinity. (AUC,pg h /L);

Mean Residence Time, (MRT. h ); Terminal half-life. (t1/2 h ); Total Body Clearance

(Cltot. L/hr/kg); Volume of Distribution at Steady State (Vss. L/kg) and Volume of

Distribution of the elimination phase (Vp, L/kg)) and Elimination Rate Constant (p.

1/h). Bacterial infection and dexamethasone treatments were the main effects with

infection by dexamethasone as the interaction. Two-way ANOVA was performed after

ranking the raw data, P<0.01. except for the comparison of total urine creatinine.

122

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. ciprofloxacin and enrofloxacin. Urine for the control and dexamethasone groups was

collected and the volume measured, but urine volumes for the infected and infected-

dexamethasone groups were not measured. AH pairwise multiple comparisons were

made using the Student-Newman-Keuls test with P<0.01. except for total urine

creatinine, ciprofloxacin and enrofloxacin which was done using the t-Test. P<0.01.

after ranking the raw data.

Results

Animal observations and pathology

The infected pigs became lethargic and dyspnic within 2 hours after inoculation

withActinobacillus pleuropneumoniae. Three infected pigs vomited and all infected

pigs were anorectic. After 24 hours, the infected pigs were still depressed but ate some

of their ration. Breathing was labored in all of the infected pigs until the time of

necropsy. In contrast, all of the non-infected pigs were eating and drinking and did not

exhibit labored breathing after sham inoculation.

Five of the non-infected pigs showed focal to extensive areas of hemorrhage and

the lesions were raised above the surface of the lung. The remaining 7 non-infected

pigs had normal lungs (Figure 2.1). Gross pathology observations in all of the infected

pigs included fibrinous pleuropneumonia with necrosis and hemorrhage in a focal to

multifocal pattern (Figure 2.2). The pulmonary lesions were raised above the surface

and observed predominately on the right lung in the middle and caudal lobes. The rank

means of the lung/body weight (g/kg) ratio of the infected pigs was increased and

significantly different (PO.Ol) from the non-infected pigs (Table 2.2; Figure 2.3).

123

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Histopathology of the non-infected pigs revealed slight to moderate, chronic

interstitial pneumonia that was not indicative of Actinobacillus pleuropneumoniae

infection (Figure 2.4). The infected pigs had necrotizing extensive, severe,

fibrinopurulent pneumonia with edema and fibrinopurulent pleuritis (Figures 2.5 and

2.6). One of the infected pigs died within 24 hours after inoculation. The cytokine

results were analyzed for this pig. but not the pharmacokinetic changes because plasma

samples were not collected. The gross and microscopic lesions in the infected pigs were

compatible with Actinobacillus pleuropneumoniae infection. Lung cultures of the

infected and non-infected pigs on agar plates provided only isolates that were

contaminants instead of the characteristic Actinobacillus pleuropneumoniae colonies.

Enrofloxacin administration at 24 h after infection may have prevented the recovery of

viable bacteria from the lung..

Plasma interleukin-6

Plasma interleukin-6 concentrations were elevated and significantly different in

the non-infected pigs versus the infected pigs based on area under the curve form 0 to

72 h (PO.Ol) (Table 2.3; Figures 2.7 and 2.8). Comparison of the infected group

versus infected-dexamethasone group was not significantly different (PO .O l) (Figure

2.8). Control group and dexamethasone group interleukin-6 concentrations remained at

baseline over the 72 h period (Figure 2.7).

Plasma interleukin-1

Plasma interleukin-1 concentrations were not statistically different among the four

treatment groups in the endotoxin study. The IL-1 plasma concentrations of

124

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. STUDY 318.14 PIG 27 12/7/95 GROUP I NON-INFECTED NO DEXAMETHASONE

Figure 2.1. Dorsal view of the trachea, heart and lungs of a control pig (non-infected. no-dexamethasone).

125

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. STUDY 318.14 PIG 1 11/8/95 GROUP III INFECTED NO DEXAMETHASONE

Figure 2.2. Dorsal view of the trachea, heart and lung in an infected pig (no- dexamethasone). The gross lesion (5 x 6 cm) on the right caudal lung lobe (arrow) described as necrotizing fibrinopurulent pneumonia that is consistent with Actinobacillus pleuropneumoniae infection.

126

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table 2.2. Lung/body weight (g/kg) ratios in control, dexamethasone. infected and infected-dexamethasone treatment groups. Control Dexamethasone Infected Infected- Dexamethasone LW/BW (g/kg) 8.49 8.82 12.3 12.7 (min-max) (7.41-9.44) (7.58-20.3) (10.3-14.7) (9.5-16.7) The lungs weights were normalized with body weights. LW/BW; the ratio of the lung weight/total body weight. Values are raw data medians and (min-max) represents the range of raw data within a group.

127

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Figure 2.3. Rank means±SD of iung/body weight ratios in control, infected, dexamethasone and infected-dexamethasone treatment groups. Values with identical superscripts are not significantly different. P< 0.01. abUsed to compare non-infected pigs with infected pigs, (n= 12) (Two-way analysis of variance performed after ranking the raw data. PO.Ol).

128

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. without prohibited reproduction Further owner. copyright the of permission with Reproduced

Rank Means of Lung Weight/Body Weight Ratios 20 24 2 i 22 12 16 10 18 14 4 0 6 si ] ]

\ 1 | j i i Control Dexamethasone Infected Infected Dexamethasone Control Treatm ent Groups ent Treatm 129 Inf-Dex b Figure 2.4. Photomicrograph of a normal section of lung from a control pig with slight, chronic interstitial pneumonia.

130

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Figure 2.5. Photomicrograph of a section of lung from an infected pig with necrotizing, fibrinopurulent pneumonia.

131

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Figure 2.6. Photomicrograph of a section of lung from an infected pig with necrotizing, fibrinopurulent pneumonia with alveolar edema, bronchitis and bronchopneumonia.

132

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. control, dexamethasone, endotoxin and endotoxin-dexamethasone groups remained at

baseline levels over the 12 h period after endotoxin administration.

Plasma enrofloxacin pharmacokinetic changes

Area under the curve

The rank means of no-dexamethasone pigs versus dexamethasone treated pigs

were significantly different for both area under the curve for zero to infinity AUC(inf)

and area under the curve from zero to the last measured time point AUC(last) (Table

2.4: Figure 2.9). The no-dexamethasone pigs rank means were greater (PO.Ol) than the

dexamethasone treated pigs rank means (Figure 2.10). The ratio of AUC(last) to

AUC(inf) was lower for the control group compared to the dexamethasone. infected and

infected-dexamethasone groups, 0.76, 0.82. 0.86 and 0.91 respectively (Figure 2.11).

This is expected, because the control group has a longer half-life than the other three

treatment groups which increases the percentage of the AUC from the last measured

time point to infinity within the area of AUC(inf) (Table 2.4: Figures 2.10 and 2.11).

The larger area under the curve from the last measured time point to infinity in the non-

infected and infected pigs should not bias pharmacokinetic calculations because the last

measured time point at 36 and 48 h are above the 0.05 mg/L limit of detection for

plasma enrofloxacin and the elimination phase was determined over four time points

from a beginning time point of 3 to 12 h to an ending time point of 36 to 48 h.

1 JJ

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Clearance, volume of distribution, mean residence time, half-life and elimination rate constant

Clearance was significantly greater (PO .O l) for the dexamethasone treated pigs

compared to the no-dexamethasone pigs (Table 2.5: Figure 2.12). Volume of

distribution based on the elimination phase (Vp) and volume of distribution at steady

state (Vss) were both significantly less (PO .O l) for the infected pigs compared to the

non-infected pigs (Table 2.5: Figures 2.13 and 2.14). Mean residence time and half-life

were significantly less (PO .01) for the infected pigs compared to non-infected pigs

(Table 2.5: Figure 2.15). The elimination rate constant (P) was significantly greater

(PO .O l) for the dexamethasone treated pigs compared to the no-dexamethasone pigs

(Table 2.5). The elimination rate constant (P) was also significantly greater (PO .01)

for the infected pigs compared to the non-infected pigs (Table 2.5).

Urine enrofloxacin/creatinine and ciprofloxacin/creatinine ratios

Ciprofloxacin and enrofloxacin were both detected in the 24 and 48 h urine samples

(Tables 2.6 and 2.7). The ciprofloxacin and enrofloxacin peaks did not appear in the 0 h

blank sample (Figure 2.16). Ciprofloxacin was the only metabolite that was measured

in the urine of the infection study pigs (Figure 2.16). The four other possible lesser

metabolites were oxo-ciprofloxacin. desethylene-ciprofloxacin. oxo-enrofloxacin and

desethylene-enrofloxacin (Figure 2.17). Oxo-enrofloxacin is a suspect peak found in

the urine of a control pig between the pipemidic acid and ciprofloxacin at about 7

minutes retention time in the infection study (Figures 2.16 and 2.17). The oxo-

enrofloxacin suspect peak corresponded to a peak detected in a separate analysis using a

134

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 120 H —•— Control —^— Dexamethasone —▼— Infected —•"— Infected-Dexamethasone g 100 r vi r

80 f-

S 0 60 r u vo1 s

JU 40 r £■« 4> L

c ssVi 20 r

°t

0 20 40 60 80 Time (h)

Figure 2.7. Mean plasma concentration of interleukin-6 collected at 0, 1. 2. 3. 4. 6. 8, 10. 12. 24. 36. 48, 60. and 72 hours after infection or sham infection.

135

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table 2.3. Area under the curv e for plasma interleukin-6 in pigs. Control Dexamethasone Infected Infected- Dexamethasone

AUC0_>72 hr 32.3 38.6 467 1158 (units/ml x h) (0-340.1) (0-335.6) (2.11-946.7) (39.27-6014.9) (min-max) A U C o ^ hr: area under the curve from 0 to 72 h. Values are raw data medians and (min-max) represents the range of raw data within a group.

136

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Figure 2.8. Rank means of area under the curve for plasma interleukin-6. Values with identical superscripts are not significantly different. P< 0.01. *\B w Used to compare non-infected pigs with infected pigs. (n=12) (Two-way analysis of variance performed after ranking the raw data. P<0.01).

137

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. without prohibited reproduction Further owner. copyright the of permission with Reproduced

Rank Means of Area Under Girvc for Plasma Interleukin-6 20 25 0 i 30 10 15 0 5 i i i 1 i ! : i : ------oto Dxmtaoe netd Inf-Dex Infected Dexamethasone Control Treatment GroupsTreatment 138 1 ; -yy-

■\ T ; \ Control Dexamethasone Infected -J Infected-Dexamethasone " m

su w © U u cs X s© : L.o s V s

I \ / \ 0.1 h \ \ /

\ y

-y y - o 10 20 30 40 70 Time (h)

Figure 2.9. Semilogrithmic plot of mean plasma concentrations of enrofloxacin measured at 0. 0.25. 0.5. 1. 2, 3, 4, 6, 8. 12. 24. 36. and 48 h after i.v. enrofloxacin (5 mg/kg BW) administration and 24 h after infection or sham infection.

139

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table 2.4. Area under the curve (mg/L x h) for plasma enrofloxacin in control, dexamethasone. infected and infected-dexamethasone treatment groups. Area Under the Control Dexamethasone Infected Infected- Curve Dexamethasone (mg/L x h) AUC(inf) 31.1 19.5 31.1 14.2 (min-max) (21.3-32.2) (12.2-27.1) (23.1-45.0) (11.3-32.4) AUC(last) 22.8 14.8 28.3 13.0 (min-max) (17.1-25.3) (10.4-22.8) (19.9-34.5) (10.4-26.7) AUC(inf) area under the curve from zero to infinity AUC(last). area under the curve from zero to the last measured hour. Values are raw data medians and (min-max) represents the range of raw data within a group.

140

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Figure 2.10. Rank means±SD of area under the curve for plasma enrofloxacin in control, infected, dexamethasone and infected-dexamethasone treatment groups. AUC(inf) area under the curve from zero to infinity. AUC(last). area under the curve from zero to the last measured hour. Values with identical superscripts are not significantly different. P< 0.01. abUsed to compare no-dexamethasone pigs with dexamethasone treated pigs for AUC(inf) (n—12) (Two-way analysis of variance performed after ranking the raw data. PO.Ol). ^Used to compare no-dexamethasone pigs with dexamethasone treated pigs for AUC(last) (n=12) (Two-way analysis of variance performed after ranking the raw data. P<0.01).

141

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. without prohibited reproduction Further owner. copyright the of permission with Reproduced

Rank Means of Area Under Curve for Plasma Enrofloxacin 20 22 24 28 26 30 io i io 12 j 14 J J - 0 2 j 4 6] 8 - ] i J I 1 i ! l I i I ! Uif AUQlast) AUQinf) Control Infected Dexamethasone Infected-Dexamethasone Treatment Groups Treatment 142 Figure 2.11. Means of area under the curve ratios for plasma enrofloxacin in control, infected, dexamethasone and infected-dexamethasone treatment groups. AUC(inf). area under the curve from zero to infinity. AUC(Iast), area under the curve from zero to the last measured hour.

143

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. without prohibited reproduction Further owner. copyright the of permission with Reproduced

Means of AUC(last)/AUC(int) Ratios for Enrofloxacin 0.0 0.4 0.6 0.2 0.8 Control Dexamethasone Infected Inf-Dex Inf-Dex Infected Dexamethasone Control Treatment Groups Treatment 144 5.13 0.359 (8.25-19.1) (11.3-27.3) (3.33-5.97) (0.154-0.443) (0.0369-0.0840) 3.04 4.85 21.2 13.6 3.20 0.161 Infected lnfected-Dexamethasone 0.0560aU 0.0679bH (12.8-19.2) (18.5-30.1) (2.59-4.71) (3.50-6.42) (2.60-4.84)) (0.111-0.216) 24.2 7.75 0.259 0.0516bA (15.9-25.8) (20.5-36.9) (0.185-0.409) a 31.3 5.52 7.61 5.47 21.4 17.2 14.6 10.2 0.161 Control Dexamethasone 0.0360“ (17.7-29.9) (4.33-6.89) (4.40-8.60) (0.0261-0.0414) (0.0318-0.0744) (0.0472-0.0706) li/2 (h) li/2 P P ( 1/h) V«(L/kg) V ptfA g) (min-max) MRTinr(h) (min-max) (min-max) (min-max) (26,1-44.2) (min-max) (4.25-6.73) (4.32-9.52) (min-max) (0.155-0.234) Parameters C\„ (L/h/kg) Pharmacokinetic Table 2.5. Table Plasma pharmacokinetic parameters ofenrofloxacin in control, infected, dexamethasone and infected-dexamethasone variance performed after ranking the raw data, to to comparecompareP<0.01). thethe AHUsed AHUsed non-inlectednon-inlected pigs with the infected pigs after ranking the afterdata (n=12)ranking ('Two-way analysisthe raw data,of variance P<0.01). performed Values are raw data medians and (min-max) represents the range Valuesof raw withdata withinidentical a group. superscripts are not significantly different, P< 0.01. ,lbUsed to compare ,lbUsed the no-dexamethasone pigs with the dexamethasone treated pigs after ranking the dataTwo-way ( analysis 12) (n= of mean residence time from zero half-lifeto infinity;of the terminalt1/2, phase and |), elimination rate constant. Cll01, total body clearance;volume Cll01, ofdistributionV() based on the terminal volumephase; ofVss, distribution at steady state; MR r il)t, treatment groups. -t- C/|

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Figure 2.12. Rank means±SD of clearance for plasma enrofloxacin in control, infected, dexamethasone and infected-dexamethasone treatment groups. Values with identical superscripts are not significantly different. P< 0.01. abUsed to compare no-dexamethasone pigs with dexamethasone treated pigs (n=12) (Two-way analysis of variance performed after ranking the raw data. PO.Ol).

146

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. without prohibited reproduction Further owner. copyright the of permission with Reproduced

Rank Means of Enrofloxacin Oearance 20 24] 10 12 16 14 18 4 0 ? 6 8 oto Dxmtaoe netd Inf-Dex Infected Dexamethasone Control Treatment Groups Treatment 147 b Figure 2.13. Rank means±SD for volume of distribution of the elimination phase for plasma enrofloxacin in control, infected, dexamethasone and infected-dexamethasone treatment groups. Values with identical superscripts are not significantly different. P< 0.01. Used to compare non-infected pigs with infected pigs. (n=12) (Two-way analysis of variance performed after ranking the raw data, P<0.01).

148

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. without prohibited reproduction Further owner. copyright the of permission with Reproduced

Rank Means of Volume of Distribution for the Enrofloxacin Elimination Phase 20 22 2 ; 12 0 i 10 1 14 ^ 18 6 ^ 16 0 i 4 6 ; 8

^ i ] I I I

Control Dexamethasone Infected Infected Dexamethasone Control Treatment Groups Treatment 149 B Inf-Dex B Figure 2.14. Rank means±SD of volume of distribution at steady state for plasma enrofloxacin in control, infected, dexamethasone and infected-dexamethasone treatment groups. Values with identical superscripts are not significantly different, P< 0.01. Used to compare non-infected pigs with infected pigs (n=l2) (Two-way analysis of variance performed after ranking the raw data. P<0.01).

150

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. without prohibited reproduction Further owner. copyright the of permission with Reproduced

Rank Means of Volume of Distribution at Steady State for Enrofloxacin 20 22 24 12 i 14 18 10 16 2 4 6 j- 0 8

\ ] i i l | i i i

Control Dexamethasone Infected Infected Dexamethasone Control Tream ent Groups ent Tream 151 B ______Inf-Dex B I ______I Figure 2.15. Rank means±SD of mean residence time (MRT) and half-life (tt/2) for plasma enrofloxacin in control, infected, dexamethasone and infected-dexamethasone treatment groups. Values with identical superscripts are not significantly different. P< 0.01. ABUsed to compare non-infected pigs with infected pigs for mean residence time (h) (n=12) (Two-way analysis of variance performed after ranking the raw data, PO.Ol). CD Used to compare non-infected " pigs with infected pigs for half-life w (h) (n=12) (Two- way analysis of variance performed after ranking the raw data. PO.Ol).

152

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. without prohibited reproduction Further owner. copyright the of permission with Reproduced

Rank Means of Mean Residence Time and Half-Life for Enrofloxacin 0 i 20 22 1 4 2 26 28 30 10 10 2 i 12 1 4 1 16 18 - 0 4 I 2 ' 6 | J 8 i i ^ J i

\ 7 i i i

i ! I

Pharmacokinetic Parameter Pharmacokinetic R Half-Life MRT B B 153 Control Infection- Dexamethasone Infection- Infected Dexamethasone D D Standard Oh 48 h Ciprofloxacin 8e+5 - Enrofloxacin

s 3 6e+5 L Suspect Oxo-enrofloxacin r O U \ 11 Pipemidic Acid ^ 1 JS

a. 4e+5 r

2e+5 r

-» 0 1 ? j 4 5 6 7 8 9 10 11 12 Time (min) Figure 2.16. Chromatographs of standards (ciprofloxacin and enrofloxacin) and the internal standard (pipemidic acid); 0 h urine sample; and 48 h urine samples in a control pig.

154

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Enrofloxacin

O O o o

Oxo-enrofloxacin Ciprofloxacin

Desethylene-enrofloxacin Oxo-ciprofloxacin

Desethyiene-ciprofloxacin Figure 2.17. Enrofloxacin and the major metabolite, ciprofloxacin, with four minor metabolites.

155

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table 2.6. Total urine creatinine, enrofloxacin and ciprofloxacin in control and dexamethasone treatment groups for the infection study. Control Dexamethasone

24 h Creat (mg) 510a OO o 00 (min-max) (139-962) (336-1037) 48 h Creat (mg) 1204a 1002a (min-max) (798-1417) (459-1044) 24 h Enro (mg) 10.7° 13.5° (min-max) (6.64-14.3) (8.28-17.7) 48 h Enro (mg) 8.46c 4.45d (min-max) (5.08-19.0) (2.91-6.12) 24 h Cipro (mg) 11.0e 9.92° (min-max) (4.24-12.9) (7.50-23.6) 48 h Cipro (mg) 8.45c 3.45f (min-max) (4.74-11.1) (3.04-6.53) Creat (mg), total urine creatinine: Enro (mg), total urine enrofloxacin: and Cipro (mg), total urine ciprofloxacin. Values are raw data medians and (min-max) represents the range of raw data within a group. Values with identical superscripts are not significantly different. P< 0.01. “Used to compare the control group with the dexamethasone group for total urine creatinine at 24 and 48 h (n=6) (t Test after ranking the raw' data. PO.Ol). cdUsed to compare the control group with the dexamethasone group for total urine enrofloxacin at 24 and 48 h (n=6) (t Test after ranking the raw data. PO.Ol). ctUsed to compare the control group with the dexamethasone group for total urine ciprofloxacin at 24 and 48 h (n=6) (t Test after ranking the raw data, PO.Ol).

156

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table 2.7. Urine enrofloxacin/creatinine and ciprofloxacin/creatinine ratios in control, infected, dexamethasone and infected-dexamethasone treatment groups. Control Dexamethasone Infected Infected- Dexamethasone 24 h Enro/Creat (pg/mg) 22.5 16.5 18.2 14.7 (min-max) (14.1-47.8) (14.5-28.4) (11.5-25.8) (7.28-32.0) 48 h Enro/Creat 8.58 9.17 (pg/mg) 4.75 3.81 (min-max) (5.71-27.9) (2.80-6.33) (4.89-15.1) (0.828-5.05) 24 h Cipro/Creat 18.5 19.3 20.4 13.1 (pg/mg) (min-max) (10.8-47.5) (7.24-29.5) (7.54-24.7) (9.17-16.9) 48 h Cipro/Creat 7.52 4.46 10.8 6.41 (pg/mg) (min-max) (5.32-47.6) (0.575-6.69) (4.27-14.2) (1.11-9.69) Enro/Creat (pg/mg). ratio of urine ciprofloxacin to urine creatinine. Cipro/Creat (pg/mg), ratio of urine enrofloxacin to urine creatinine. Values are raw data medians and (min-max) represents the range of raw data within a group.

157

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Figure 2.18. Rank means±SD of urine enrofloxacin/creatinine ratios in control, infected, dexamethasone and infected-dexamethasone treatment groups at 24. and 48 h after enrofloxacin administration. Values with identical superscripts are not significantly different. P< 0.01. aUsed to compare no-dexamethasone pigs with dexamethasone treated pigs and non- infected pigs with infected pigs at 24 h (n= 12) (Two-way analysis of variance performed after ranking the raw data. P<0.01). cdUsed to compare no-dexamethasone pigs with dexamethasone treated pigs at 48 h (n=12) (Two-way analysis of variance performed after ranking the raw data. PO.Ol)

158

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Control 28 Dexamethasone Infected 26 - Infected-Dexamethasone

24 i J 22 J £ ; "8 20 j 1 is i u S 16 -I I ! 1 1 4 j cl ! I 12 i u 3 ’ o 10 f vi S 8 J

J 6

0 24 h 48 h Hour

159

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Figure 2.19. Rank means±SD of urine ciprofloxacin/creatinine (pg/mg) ratios in control, infected, dexamethasone and infected-dexamethasone treatment groups at 24 and 48 h after enrofloxacin administration. Values with identical superscripts are not significantly different. P< 0.01. aUsed to compare no-dexamethasone pigs with dexamethasone treated pigs and non- infected with infected pigs at 24 h (n=12) (Two-way analysis of variance performed after ranking the raw data. P<0.01). '’Used to compare no-dexamethasone pigs with dexamethasone treated pigs and non- infected pigs with infected pigs at 48 h (n=12) (Two-way analysis of variance performed after ranking the raw data. PO.Ol).

160

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 28 i Control Dexamethasone »! Infected I Infected-Dexamethasone 24 \

.1 20 s •c 1 18 U ‘3 16 § i ? 14 1

f , : , •C 2 o 10 QA £ 8

s 6

0 24 h 48 h Hour

161

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. different column and mobile phase, but using a control urine sample from this study

(Dr. Joseph Kawalek. unpublished data). Desethylene-ciprofloxacin was used to make a

previous standard curve, but was not detected in the urine samples. A peak in the 24

and 48 h urine samples did co-elute at the same time (6.3 min) as desethylene-

ciprofloxacin and was between the unknown peak (7 minutes retention time) and

pipemidic acid. This peak was an endogenous product, because it appeared in the zero

hour urine samples and had an absorption maxima at 250 nm. Flouroquionolones have

a characteristic absorption maxima at about 280 nm. but not at 250 nm. In vitro studies

with liver microsomes have supported this observation where oxo-ciprofloxacin.

desethylene-ciprofloxacin and desethylene-enrofloxacin were not detected (Dr. Joseph

Kawalek. unpublished. FDA/CVM final report 275.16). Ciprofloxacin was detected in

urine after a short incubation and oxo-enrofloxacin was detected after a prolonged

incubation of porcine hepatic microsomes from pigs unrelated to this study (Dr. Joseph

Kawalek. unpublished. FDA/C VM final report 275.16).

Total urine creatinine is not significantly different for the control group

compared to the dexamethasone group (Table 2.6). Total urine enrofloxacin is not

significantly different at 24 h. but the total urine enrofloxacin was significantly less for

the dexamethasone group compared to the control group at 48 h (Table 2.6). Total urine

ciprofloxacin is not significantly different at 24 h. but the total urine ciprofloxacin was

significantly less for the dexamethasone group compared to the control group at 48 h

(Table 2.6).

162

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. The urine enrofloxacin/creatinine ratios are not significantly different in the 24 h

urine samples (Table 2.7; Figure 2.18). The urine enrofloxacin/creatinine ratios were

significantly less in the dexamethasone treated pigs compared to the no-dexamethasone

pigs at 48 h (Table 2.7; Figure 2.18). The urine ciprofloxacin/creatinine ratios are not

significantly different in the 24 and 48 h urine samples (Table 2.7; Figure 2.19)

Discussion

The infected animals developed signs of APP infection within 2 h which was

verified by gross pathology and histopatholgy (Figures 2.1-2.6). The establishment of

APP infection was further supported by increased lung weights and increased plasma

interleukin-6 in the infected pigs (Table 2.2; Figure 2.3). There was only a slight but

statistically different elevation of plasma IL-6 in infected pigs. This proved that IL-6

was released systemically. but returned to baseline by 48 (Table 2.3; Figures 2.7 and

2.8). The intensity and duration of IL-6 release was much less than expected and it is

unlikely that cytochrome P450's w'ere suppressed. Nonetheless, the signs, pathology

and systemic IL-6 release all demonstrated that the APP disease model was standardized

and reproducible in this study.

Statistical analysis was a challenge because many pharmacokinetic parameters,

such as volume of distribution, half-life, mean residence time and clearance, do not have

a normal distribution which prevents parametric testing (Dyke & Sams. 1994).

Parametric tests are based on larger sample sizes of 30 or more observations (Dyke &

Sams. 1994). The infection study data had 6 animals per group or 11 to 12 animals. If

there was no interaction, the two infected groups, and two dexamethasone groups could

163

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. be pooled which allowed two-way analysis of variance. The ranking of raw data was

necessary, because the two by two factorial designs could not be applied to the non-

parametric Kruskal-Wallis test to detect differences among three or more groups. A

distribution free test was not available that would allow analysis of a two by two

factorial by non-parametric methods. Therefore, a compromise approach was used to

rank the data and perform two-way analysis of variance on the ranked data with a

P<0.01 to guard against Type II error or false positives.

Pharmacokinetic parameters were calculated on the assumption that the

extrapolated area under the curve from the last measured time point to infinity did not

bias the calculation of clearance, volume of distribution, mean residence time and half-

life. When the AUC(inf) was compared to AUC(last). the outcome of the statistical

analysis was the same (Table 2.3; Figure 2.10). The ratios of AUC(last)/AUC(inf)

among the control, dexamethasone. endotoxin and endotoxin-dexamethasone treatment

groups were acceptable, because the area under the curve from the last measured time

points to infinity was calculated based on values above the limit of detection for

enrofloxacin (Figure 2.11). A long half-life may be associated with a large extrapolated

area under the curve which is not important provided that the error of the last measured

time point is minimal. Error around the last measured time point may be of little

consequence because an extrapolated area under the curve could contribute 10% of the

total area under the curve, but only contribute 5% error with a bias of 50% (Purves.

1992). This means that the subsequent calculations of pharmacokinetic parameters were

164

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. not strongly biased due to error associated with enrofloxacin plasma concentrations

around the limit of detection.

Pharmacokinetic parameters were calculated from the dose. AUC(inf).

AUCM(inf) and P (elimination constant) for clearance. Cl=dose/AUC; volume of

distribution of the elimination phase; Vp=dose/(P x AUC); volume of distribution at

steady state. Vss=(dose/AUC) x (AUMC/AUC)=C1 x MRT; mean residence time.

MRT=AUMC/AUC: and terminal half-life, t1/r>=0.693/p=(0.693 x Vp)/Cl. Clearance is

a measure of efficiency of removal of a chemical from an organ or the body. The

volume of distribution during the terminal phase. Vp. is the proportionality constant

between the amount of drug in the body post distribution and the plasma concentration

during the elimination phase. Volume of distribution based on the terminal phase (Vp)

depends on the rate of elimination from the central compartment (Ritschel & Denson.

1991). Volume of distribution at steady state. Vss. is the proportionality constant

between the amount of drug in the body and the plasma concentration at steady state for

intravenous bolus or constant-rate infusion administration of a drug. Mean residence

time is the average amount o f time that all molecules spend in the body or the mean

transit time. Plasma half-life is the amount of time for a 50% reduction in the

concentration of a xenobiotic in the plasma which is a constant if elimination is

independent from the dose.

Volume of distribution based on the elimination phase will be influenced in

disease-altered elimination, whereas distribution changes independent of elimination are

reflected by Vss (Ritschel & Denson, 1991). With decreasing Vp, the concentration in

165

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. plasma increases (Ritschel & Denson. 1991). The volume of distribution at steady state

is affected by both plasma and tissue binding, decreasing as the free fraction in plasma

and tissues increases and the bound fraction decreases. The volume of distribution for

both the elimination phase and steady state were decreased for the infected animals but

not for dexamethasone treated animals (Table 2.5; Figures 2.13 and 2.14). Volume of

distribution of the elimination phase. Vp decreases as plasma concentration increases

(Ritschel & Denson. 1991). Decreased Vss would indicate decreased plasma and tissue

protein binding and increased plasma and tissue free fraction (Ritschel & Denson.

1991). As the free fraction in plasma increases, plasma clearance should increase

(Ritschel & Denson. 1991). At steady state, distribution is in equilibrium with

elimination and the amount of drug bound to plasma and tissue protein is at a

maximum. At ail other times, the amount of drug bound to tissue protein depends upon

the time course of the drug during distribution or elimination phases.

The infected pigs had a significantly larger elimination rate constant compared

to the non-infected pigs (Table 2.5). The elimination rate constant increased while the

AUC remained the same, and the volume of distribution of the elimination phase

decreased for the infected pigs (Figures 2.10 and 2.13). This explanation is further

supported by the decreased half-life of the infected pigs versus the non-infected pigs

where the elimination rate constant is related to half-life by the equation: t|/2=0.693/p

(Figure 2.15). As the elimination rate constant increases, the half-life decreases. Mean

residence time would be expected to parallel half-life where the MRT of the infected

pigs was less than the non-infected pigs. The literature value for ciprofloxacin half-life

166

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. was 6 to 8 h in pigs , which is much less than the enrofloxacin median half-life (21.4 h)

or MRT (31.3 h) of the control group (Table 2.5; Figure 2.15) (Rung et ai. 1993a.b).

Clearance in the infected pigs was not altered as expected if there was an

increase in the free fraction of plasma enrofloxacin with a decreased volume of

distribution. The free fraction of enrofloxacin may have increased without an increase

in clearance because a decrease in volume of distribution could have offset an increase

in the elimination rate constant, p. where clearance is mathematically related to volume

of distribution and elimination rate constant by the equation: Cl=Vyp (Table 2.5). The

elimination rate constant, p. was significantly greater for the infected pigs compared to

the non-infected pigs (Table 2.5). Thus, volume of distribution of the elimination phase

in the infected pigs was affected by an increase in the elimination rate constant

(Vp=dose/(P x AUC). However, volume of distribution at steady state was also

significantly decreased for the infected pigs, but is independent of the elimination rate

constant. This suggests that some other factor, such as decreased protein binding in

tissues and increased protein binding in plasma, is also a component of the decreased

volume of distribution for the infected pigs. The loss of plasma proteins to the alveolar

space may also account for the decreased volume of distribution as observed in the

photomicrograph of a section of lung from an infected pig (Figure 2.6).

In addition to protein binding, several disease conditions can affect the

elimination rate constant and Vp such as alterations in blood flow and pH changes for

healthy and diseased animals (Ritschel & Denson, 1991). The onset and duration of

action of a drug is dependent on the rate or extent of distribution and a change in the

167

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. apparent volume of distribution may change the drug concentration in plasma, tissues

and at the receptor site. The volume of distribution may be reduced by reduced blood

supply to peripheral tissues in shock and the concentration at the site of action may be

diminished. The infected pigs may have had poor pulmonary perfusion and respiratory

acidosis due to labored breathing caused by pulmonary infection (Figures 2.5 and 2.6).

Enrofloxacin is amphoteric with acid and base properties. A small change in acid-base

balance may have an amplified effect on the degree of ionization of weak organic acids

and bases. Increased ionization could reduce the tissue penetration by affecting the

solubility of enrofloxacin and result in decreased volume of distribution. Depending on

pH. enrofloxacin could bind to albumin as an acid or to a,-glycoprotein as a base and be

displaced in plasma or tissues. However, neither the blood concentration, nor pH

affected the low serum protein binding of other fluoroquinolones (Greene & Budberg.

1993). Low serum protein binding would promote rapid elimination by renal and

extrarenal mechanisms. In pigs, low plasma protein binding (23.6%) of ciprofloxacin

was previously attributed to interference by fatty acids with albumin or more binding

sites on plasma proteins such as albumin and a |-acid glycoprotein (Nouws et al.. 1988:

Belpaire. 1986). In humans. AGP plasma concentration can increase as much as two

fold during the acute-phase respose (Kremer et. al., 1988). Although AGP has an

affinity for basic drugs, it can bind acidic drugs (Kremer et. a*.. 1988). The acidic drugs

which exhibit a high or intermedicate affinity to AGP do not contain a carboxyl moiety

and share a common binding site on human serum albumin, called site I or the warfarin

site. In comparison, all drugs having a low affinity or no affinity to AGP exhibit

168

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. carboxyl groups and bind specifically to another human serum albumin site, called site

II or the diazepam site (Kremer et. al.. 1988). Enrofloxacin is an amphoteric compound

with a basic nitrogen in the piperazinyl ring and an acidic carboxyl group on the other

end of the molecule (Figure 1.2). The decreased volumes of distribution in the infected

pigs may occur subsequent to the release of AGP. The basic nitrogens on the

piperazinyl ring may preferentially bind to AGP molecules because it takes longer for

albumin to decrease in the acute-phase response while AGP is increasing.

While enrofloxacin volumes of distribution are not altered in pigs receiving

dexamethasone. clearance for the dexamethasone treated pigs compared to the no-

dexamethasone pigs was increased (Table 2.5; Figures 2.13 and 2.14). The reason for

the increase in clearance may be related to increased glomerular filtration rate. A five

day regimen of adrenocorticotropin (ACTH) and glucocorticoids (GC) raised blood

pressure, caused marked antinatriuresis. expansion of extracellular fluid and plasma

volume, and altered renal function in humans (Connell, et al.. 1987). Both treatments

raised glomerular filtration rate (GFR). as demonstrated by inulin clearance. Creatinine

clearance was not changed and it proved to be an unreliable index of GFR during steroid

administration. Filtration fraction also increased during both ACTH and GC treatment,

and renal blood flow fell only during cortisol treatment. Effective renal plasma flow,

measured by para-aminohippurate clearance, remained unchanged. The mechanism for

changes in GFR may be a reduction in the availability of angiotensin II at glomerular

receptor sites, because dexamethasone increases the activity of aminopeptidase as

shown in human glomerluar epithelial cells (Stefanovic et al.. 1991). Aminopeptidase

169

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. splits off the N-terminal Asp from angiotensin II and inactivates angiotensin II.

Therefore, the increased clearance of enrofloxacin in the dexamethasone treated pigs

could be due to increased GFR and filtration fraction.

The major metabolite, ciprofloxacin and a suspect peak, oxo-enrofloxacin. were

found in the urine (Figures 2.16 and 2.17). The peak corresponding to oxo-

enrofloxacin. a minor metabolite, is intriguing because it contradicts the absence of

metabolites in another investigation where ciprofloxacin was administered to pigs

(Nouws et. al., 1988) (Figures 2.16 and 2.17). There was no statistical difference

among the treatment groups at 24 h for the total urine enrofloxacin and ciprofloxacin;

and urine enrofloxacin/creatinine and ciprofloxacin/creatinine ratios (Tables 2.6 and 2.7

Figures 2.18 and 2.19). However, the 48 h sample had a reduced

enrofloxacin/creatinine ratio for the dexamethasone treated pigs compared to the no-

dexamethasone pigs, but the 48 h ciprofloxacin/creatinine ratio was unchanged. The

reduced enrofloxacin/creatinine ratio could be due to the depletion of enrofloxacin in

the first 24 h of urine collection, because the clearance of enrofloxacin is increased in

dexamethasone treated pigs. Creatinine clearance would be expected to increase with

increased GFR after dexamethasone treatment, but total urine creatinines were not

significantly different (Table 2.6). However, it has already been stated that creatinine

clearance is an unreliable index during steroid treatment (Connell et al.. 1987). It is

curious that a concomitant decrease in the ciprofloxacin/creatinine ratio was not seen in

the dexamethasone treated pigs, where a lack of statistical significance could be due to

small sample size or lack of biological significance.

170

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Apparently, there was no effect of APP infection on either the

enrofloxacin/creatinine ratio or the ciprofloxacin/creatinine ratio in infected pigs (Tables

2.6 and 2.7; Figures 2.18 and 2.19). The hypothesis in this study included the alteration

of metabolism in conjunction with other pharmacokinetic changes that have already-

been described. There was a small systemic elevation of interleukin-6, but interleukin-1

was not systemically elevated. Therefore, it is probable that metabolism of enrofloxacin

to ciprofloxacin was not affected by infection, because there was no increase in the

enrofloxacin/creatinine ratio and a decrease in the ciprofloxacin/creatinine ratio in

infected pigs. Any changes in metabolism of enrofloxacin to ciprofloxacin would have

been easily observed because of the large amount of ciprofloxacin excreted in the urine

of control animals. Whatever the metabolic pathway, cytochrome P450 or flavin-

containing monooxygenase, is responsible for the dealkylation of enrofloxacin to

ciprofloxacin, it is still intact in the APP infected and dexamethasone treated pigs.

In contrast to the absence of metabolic changes in this study, a previous

investigation reported the suppression of oxidative hepatic biotransformation in pigs

infected with APP (Monshouwer et al., 1995a). The hepatic clearance of antipyrine and

caffeine were decreased by 72 and 68%, respectively. Renal function and hepatic blood

flow were not affected. The APP infected pigs in the Netherlands did not exhibit a

change in microsomal uridine diphosphate glucuronyl transferase activities

(Monshouwer et al.. 1995b). No glucuronide conjugates of ciprofloxacin or its

metabolites were detected in the urine of calves and piglets (Nouws et. al.. 1988).

Additionally, the urine samples were subjected to a mixture of 0.05 N sodium hydroxide

171

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. and acetonitrile in a two to one ratio, respectively. This may have been sufficient to

hydrolyze any enrofloxacin and ciprofloxacin urine conjugates as reported in a study of

fenoprofen and oxprenolol in horses (Delbeke & Debackere. 1994). Hydrolysis of

fenoprofen conjugates was accomplished by treating urine samples with a final sodium

hydroxide concentration of 0.025 N. The final concentration of sodium hydroxide that

was used to prepare urine samples for HPLC analysis in this study was 0.032 N.

Therefore, glucuronated enrofloxacin and ciprofloxacin metabolites were not measured

in this study.

The different results found in the present study compared to the Netherlands

study are not necessarily contradictory. The previous study was done at a different

location using a different genetic base of pigs that were infected with a different

serotype of APP that may have differed in virulence. No change in clearance and

metabolism was found in this study compared to decreased oxidative metabolism of

antipyrine and caffeine in the Netherlands study. The antipyrine and caffeine were used

as model drugs to test oxidative biotransformation in the infected pigs. Instead of

model drugs, a therapeutic drug, enrofloxacin. was tested in this study. Enrofloxacin

clearance was not altered by APP infection. Enrofloxacin can be biotransformed to

oxygenated metabolites but this is not an important pathway in pigs (Dr. Joseph

Kawalek. unpublished; Nouws et al.. 1988). Dealkylation results in the major

metabolite, ciprofloxacin. Oxidation of enrofloxacin to the suspect minor metabolite,

oxo-enrofloxacin. requires confirmation by mass-spectroscopy.

172

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. CHAPTER III

ENDOTOXIN MODEL

Introduction

Endotoxin derived form Eschericia coli has been used as a model for septic

shock in swine (Myers et. al. 1997; Monshouwer et al., 1996a). The administration of

intravenous endotoxin in this study and any associated pharmacokinetic changes was

intended to provide a comparison to Actinobacillus pleuropneumoniae infection and the

subsequent plueropneumonia. Drug disposition and metabolism can both be affected by

infection and endotoxin challenge. Other toxins besides endotoxins are certainly

present in porcine pleuropneumonia and may contribute to pathological changes.

However, endotoxin has been used extensively as a model of septic shock and the acute-

phase response. Endotoxin is not directly cytotoxic to tissue, but it does induce release

of TNF-a and IL-6 (Edwards et al.. 1994). A CD14 leukocyte receptor appears to

initiate TNF-a release after binding endotoxin to LPS binding protein.

Viral, bacterial or parasitic infections and inflammatory stimuli from endotoxin

or turpentine induce physiological, hematological and metabolic changes that

characterize the acute-phase response (Ballmeret al.. 1991; van Miert. 1990). The

acute-phase response may be triggered by cytokines released from macrophages-

monocytes. Interleukins la and ip. TNFs a and P and interferons a and P are

cytokines that inhibit oxidative and conjugative drug metabolism in laboratory animals

(Craig et al.. 1989b; Ghezzi et al., 1986a.b; Morgan & Norman. 1990; Renton &

Knickle. 1990). Furthermore, active infection with bacterial proliferation is not

173

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. necessary for changes to occur in drug biotransformation. Endotoxins from Gram-

negative and positive bacterial cell walls are potent inhibitors of hepatic mixed-function

oxidase (Azri & Renton. 1987: Gorodischer et al.. 1976; Lavicky et al.. 1988: Sasaki et

al.. 1984: Sonawane et al.. 1982; Stanley et al.. 1988; Williams & Szentivanyi. 1983:

Yoshida et al., 1982). Escherichia coli. Bordatellapertussis. diphtheria toxoid and

tetanus toxoid all contain cell wall endotoxins that alter P450 activity (Ashner et al..

1992: Fantuzzi et al.. 1994a.b).

A previous study compared an LPS model to an Actinobacillus

pleuropneumoniae model and demonstrated a distinct similarity with respect to effects

on hepatic drug metabolism (Monshouwer et. al.. 1996a). The experimentally induced

APP infection in pigs was reported to induce a mild acute-phase response and inhibition

of P450. The plasma clearance of caffeine and antipyrine were shown to be decreased

by 70% at 24 h after inoculation (Monshouwer et al.. 1995a.b).

The goal of the endotoxin study was 1) to characterize pharmacokinetic changes

associated with endotoxin administration in swine after receiving enrofloxacin and 2) to

evaluate the endotoxin model as a substitute for the APP infection model or other Gram-

negative-pathogens. Plasma concentrations of IL-6 were determined from 0 to 9 h after

endotoxin administration. Aspartate aminotransferase plasma concentrations were

determined for 0. 24 and 96 h after endotoxin infusion. Plasma concentrations of

enrofloxacin and ciprofloxacin were determined for pharmacokinetic analysis from 24

to 96 h after endotoxin infusion. Urine enrofloxacin and ciprofloxacin were also

174

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. measured and normalized with urine creatinine concentrations. Gross necropsies were

performed and included lung weights.

Materials and Methods

Animal management

The experiment utilized 24 crossbred. Hampshire x Yorkshire, castrated pigs

that were procured from a local vendor. All of the pigs were weighed prior to

intravenous catheterization and were within a 25 to 35 kg weight range. Housing

consisted of a heated building with a concrete floor and all animals were fed 3 kg/day

from a single batch of a commercial com-based ration without antibiotics. The pigs had

been fed a commercial ration that was medicated with tylosin before procurement. All

pigs were examined upon arrival and were clinically healthy at the start of the study.

Experimental design

The design consisted of a 2 x 2 factorial of treatment groups (Table 3.1).

Eschericici coli 055: B5 endotoxin (2 pg/kg. Sigma. St. Louis. Missouri) was dissolved

in about 5 ml of phosphate buffered saline (0.0067 M of phosphate. 0.15 M sodium

chloride. pH 7.4; Sigma. St. Louis. Missouri) as the first treatment. This endotoxin dose

(2 pg/kg) was previously determined by previous studies at the Center for Veterinary

Medicine. The second treatment was dexamethasone (0.5 mg/kg. Phoenix

Pharmaceuticals. St. Joseph Missouri). Endotoxin was infused by i.v. bolus at zero hour

into two of the four groups as a comparison to APP infection. Two groups (12 pigs)

were procured at a time and one group was placed in stainless steel metabolism cages

175

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table 3.1. Experimental design for the endotoxin study.

Treatment Sham Infusion Endotoxin

Sham Infusion Endotoxin No Dexamethasone

No Dexamethasone No Dexamethasone

Sham Infusion Endotoxin Dexamethasone

Dexamethasone Dexamethasone

176

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. for four days followed by the second group. This process was repeated with another

two groups of pigs over a one month period. The endotoxin treatment was administered

as single i.v. injection to two groups while the other two groups received a sham saline

infusion (Table 3.1). One endotoxin group and one sham group received i.v.

dexamethasone at -18 h . 0 h and 12 h. A bolus dose of 5 mg/kg enrofloxacin (Baytril .

Bayer. Shawnee Mission. Kansas) was administered intravenously 24 h after saline or

endotoxin infusion to all four groups of pigs.

Blood collection

Intravenous cannulation and blood collection was identical to the infection

study. Cytokine blood samples were collected in heparin tubes (Monovette. Sarstedt.

Germany) at 0. I. 3. 6 and 9 h after endotoxin and sham infusion. Plasma samples for

HPLC analysis were collected in EDTA tubes (Monovette. Sarstedt. Germany) at 0.

0.25. 0.5. 1. 2. 3. 4. 6. 8. 12, 24. 36. 48. 60 and 72 h after enrofloxacin infusion.

Pharmacokinetic analysis

Urine and plasma sample preparation

Plasma sample preparation was identical to the infection study. Urine samples

were collected at 24 hour intervals starting 24 h after endotoxin infusion or saline at 0.

24. 48 and 96 hours.

Urine creatinine

Urine creatinine preparation was identical to the infection study. A plot of the

standard curves for creatinine gave a linear response, “y” (absorbance) versus "c”

(concentration. mg/L): y=0.007 c + 0.197. R= 1.0. y=0.008 c + 0.219. R=0.9995.

177

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. y=0.007 c + 0.203. R=1.0. y=0.007 c + 0.21. R= 1.0 and y=0.008 c + 0.207. R=0.9995

where the coefficient of variation was less than 5% and 10% for the standards and

samples, respectively.

Aqueous standard curves for urine and plasma

Aqueous standard curves for plasma and urine were constructed by first

preparing desethylene-ciprofloxacin, enrofloxacin and ciprofloxacin stock standards

which was similar to the infection study.

The linearity of the response, "y“ (ratio of peak height counts using the internal

standard, pipemidic acid) versus "c" (concentration. mg/L) were tested over eight

standard solutions of desethylene-ciprofloxacin, ciprofloxacin and enrofloxacin. The

typical response standard curves for plasma samples were represented by the linear

equations: y=17.1 c - 0.00478. R=0.9997 for enrofloxacin; y=12.3 c - 0.0216. R=0.9997

for ciprofloxacin: and y=127 c + 0.0366, R=0.9997 for desethylene-ciprofloxacin. The

typical response standard curves for urine samples were linear and represented by the

same equations that were presented in the infection study: y=16.0 c - 0.00379.

R=0.9996 for enrofloxacin: y= 11.8c- 0.00253, R=0.9997 for ciprofloxacin and y=124

c + 0.0434. R=0.9997 for desethylene-ciprofloxacin.

Five replicates of each of the 8 concentrations (3.9 - 500 pg/L for ciprofloxacin

and enrofloxacin and 1.22 - 156 pg/L for desethylene-ciprofloxacin) of standard

resulted in a coefficient of variation ranging from 0.7% to 9% for the plasma standard

curves and 0.7% to 11% for the urine standards curves to include enrofloxacin.

ciprofloxacin and desethylene-ciprofloxacin.

178

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. High-performance liquid chromatography

High-performance liquid chromatography analysis was identical to the infection

study for urine and plasma.

Interleukin-6 bioassay

Interleukin-6 assay was performed as previously described in the infection

study. The standard curve was plotted as a log-logit transformation using Cytocalc

(Millipore. New Bedford. Massachusetts). The log-logit transformations gave linear

response, "y" (absorbance) versus “c" (concentration, pg/ml). curves for each of the five

microplates: y=2.74 c - 9.48. R=0.9180; y —1.52 c - 1.01. R=0.986; y=2.76 c - 9.85.

R=0.917; y=2.51 c - 8.85. R=0.903 and y=2.42 c - 8.67. R=0.903.

Plasma aspartate aminotransferase assay

The plasma aspartate aminotransferase (AST) assay was performed using an

AST/GOT Transaminase Diagnostic Test Kit to measure plasma AST at 0. 24 and 48 h

after endotoxin (Sigma. St. Louis. Missouri). The AST assay was undertaken as an

indicator of liver damage after endotoxin administration. All four groups were tested by

adding 50 pi of plasma or standard to 250 pi of substrate reagent. The mixture was

vortexed and placed in a 37°C water bath for 1 hour. A color reagent (250 pi) was

added after incubation. The mixture was vortexed and incubated at room temperature

for twenty minutes. Next, a 2.5 ml aliquot of 0.4 N sodium hydroxide was added and

the test tube was mixed by inversion. After waiting 5 minutes, a 200 pi aliquot sample

mixture was pipetted, in triplicate, into a 96 well microplate. The results were read at

179

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 503 nm absorbance using a microplate spectrophotometer (Spectra Max 250. Molecular

Devices. Sunnyvale. California).

A standard curve was prepared for each microplate by diluting standards with

water and substrate to the following concentrations: 0. 9.6. 26.4. 45.6. 69.6 and 103.7

lU/ml where 0.48 international unit (IU) was equivalent to one Sigmund-Frankel unit.

The standards were prepared for reading on the microplate spectrophotometer by adding

50 pi of standard to 250 pi of substrate reagent. The mixture was vortexed and

incubated at room temperature for 20 minutes. A 2.5 ml aliquot of 0.4 N sodium

hydroxide was added and the test tube was mixed by inversion. After a 5 minute wait,

triplicate 300 pi aliquots of each standard were transferred into the microplates. The

resulting standard curves were nonlinear, "y” (absorbance) versus "c" (concentration.

lU/ml): y={-0.603/[l + (c/255)0827]} + 0.801. R=0.999: y={-0.607)/[l + (c/273)0 723]} +

0.801. R=0.999; y={-0.573)/[l + (c /227)0856]} + 0.741. R=0.9995 and y={-0.641/[l +

(c /283)0 8-9]} + 0.831. R=0.999. The coefficient of variation for the standards and

samples was less than 3% and 11%, respectively. The sample values were interpolated

from the standard curves using Softmax Pro (Version 1.2. Molecular Devices.

Sunnyvale. California).

Health observations and necropsies

Health observations were made during the entire study period of 4 days. Gross

necropsies were performed on all animals in the infection study. The pigs were

euthanized with Euthasol® which contained sodium pentobarbital (Delmarva

Laboratories, Inc.. Bristol. Tennessee). Brain and spinal cord were not examined. Fresh

180

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. liver (100 to 200 grams) was collected and frozen at -80°C for Northern or Western

blots, if required. Lung weights and gross necropsy observations were recorded on a

necropsy form. The color, texture. location and size of gross lesions were also noted on

the necropsy form. After trimming the trachea from the lung at the level of the cranial

bronchi, the lung was weighed.

Statistics

Statistical analyses of pharmacokinetic parameters, urine creatinine,

enrofloxacin and ciprofloxacin concentrations. IL-6 plasma concentrations, plasma

aspartate transaminase and lung weights were similar to the infection study. One-way

analysis of variance was performed where the predominant statistical difference was due

t the endotoxin group or an endotoxin and dexamethasone interaction.

Results

Animal observations and pathology

The endotoxin treated pigs became depressed and dyspnic within 30 to 60

minutes after i.v. injection. One endotoxin treated pig vomited. Signs of labored

breathing and depression were reduced 24 h after endotoxin administration, but all pigs

were anorectic. At 48 h. all pigs were more active and consumed almost all of their

ration. At 72 h, all endotoxin treated pigs appeared normal and had finished their ration.

There were no gross lesions present in the lungs at necropsy. No significant

differences (PO.Ol) in the lung/body weight ratio (g/kg) were found among control,

dexamethasone, endotoxin and endotoxin-dexamethasone treatment groups (Table 3.2;

181

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Figure 3.1). However, there was a significant interaction (P<0.01) between

dexamethasone and endotoxin. Histopathology was not done in the endotoxin study.

Plasma aspartate aminotransferase was elevated in endotoxin treated pigs at 24 h

and returned to baseline levels at 96 h after endotoxin administration (Table 3.3). The

rank means at 0 and 96 h for plasma aminotransferase were not significantly different

for no-dexamethasone pigs versus dexamethasone treated pigs, and no-endotoxin pigs

versus endotoxin treated pigs (Figure 3.2). The rank means for plasma aminotransferase

at 24 h were significantly greater (PO.Ol) for the no-dexamethasone pigs versus

dexamethasone treated pigs and the endotoxin treated pigs versus no-endotoxin pigs.

Plasma interleukin-6

The mean plasma interleukin-6 concentration was markedly elevated for the

endotoxin treatment group compared to the control, dexamethasone and endotoxin-

dexamethasone treatment groups (Figure 3.3). Rank means o f area under the curve

from zero to 9 h (A U C o^) were significantly greater (P<0.01) for the endotoxin versus

the no-endotoxin pigs (Table 3.4; Figure 3.4).

Interleukin-6 synthesis is increased by tumor necrosis factor-a (Edwards et al.,

1994). Usually, tumor necrosis factor is released from macrophages and monocytes as a

proximal-mediator before the release IL-1 and IL-6 in septic shock. Endotoxin

increases serum TNF-a concentrations by enhancing TNF-a gene transcription

approximately 10-fold, cellular TNF-a mRNA 100-fold and a 10,000-fold increase in

the quantity of protein secreted from activated monocytes. Dexamethasone and other

glucocorticoids inhibit TNF-a mRNA accumulation (Beutler et al., 1986; Han et al.,

182

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 1990b; Luedke et al.. 1990). Glucocorticoids act both at the transcriptional and post-

transcriptional level to block TNF-a synthesis (Han et al.. 1990a; Trinchieri. 1991).

This mechanism of TNF-a release that is stimulated by endotoxin and inhibited by

glucocorticoids can explain why the IL-6 plasma concentration was decreased in the

endotoxin-dexamethasone group compared to the endotoxin group (Figures 3.3 and

3.4).

Plasma enrofloxacin pharmacokinetic changes

Area under the curve

The area under the curve for the endotoxin treatment groups was significantly

greater (P<0.01) compared to control, dexamethasone and endotoxin-dexamethasone

treatment groups for both area under the curve from zero to infinity, AUC(inf), and area

under the curve from zero to the last measured time point AUC(last) (Table 3.5: Figures

3.5 and 3.6). There was a significant interaction (PO.Ol) between endotoxin and

dexamethasone for AUC(last) values. The AUC(last)/AUC(inf) ratios were 0.86. 0.92.

0.89, 0.93 and 0.86 for control, dexamethasone. endotoxin and endotoxin-

dexamethasone groups, respectively (Figure 3.7). Therefore, the area of the

extrapolated curve from the last measured time point to infinity was less than 14%.

which should not bias pharmacokinetic calculations. Furthermore, the one-way analysis

of variance of AUC(last) and AUC(inf) produced identical results where the endotoxin

group was significantly different from the control, dexamethasone and endotoxin-

dexamethasone groups.

183

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Clearance, volume of distribution, mean residence time, half-life and elimination rate constant

Clearance was significantly greater (PO.Ol) for the dexamethasone treated pigs

compared to the no-dexamethasone pigs by two-way analysis of variance (Table 3.6:

Figure 3.8). Clearance was significantly greater (PO.Ol) for the endotoxin group

compared to the control, dexamethasone and endotoxin-dexamethasone groups by one

way analysis of variance (Figure 3.8). Volume of distribution based on the elimination

phase (Vp) and volume of distribution at steady state (Vss) were both significantly

greater (PO.Ol) for dexamethasone treated pigs compared to the no-dexamethasone

pigs (Table 3.6; Figures 3.9 and 3.10). Mean residence time was significantly greater

(PO.Ol) for the endotoxin group compared to control, dexamethasone and endotoxin-

dexamethasone groups and there was a significant interaction (PO.Ol) between

endotoxin and dexamethasone (Table 3.6: Figure 3.11). There was no significant

difference (PO.Ol) in half-life among the control, dexamethasone. endotoxin and

endotoxin-dexamethasone treatment groups (Table 3.6: Figure 3.11).

Urine enrofloxacin/creatinine and ciprofloxacin/creatinine ratios

Ciprofloxacin was the major enrofloxacin metabolite that was detected in the

urine of the endotoxin study pigs as observed in the infection study (Figures 2.17 and

3.12). The minor metabolite, oxo-enrofloxacin. was a suspect peak in the urine but oxo-

enrofloxacin ratios were not calculated (Figure 2.17 and 3.12). The suspect oxo-

enrofloxacin peak had a similar retention time (7 min) as the suspect oxo-enrofloxacin

peak in the infection study (Figures 2.17 and 3.12). Ciprofloxacin and enrofloxacin

184

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. were both detected in the 24. 48 and 72 h urine samples (Tables 3.7 and 3.8). The

ciprofloxacin, oxo-enrofloxacin and enrofloxacin peaks did not appear in the 0 h blank

samples (Figure 3.12). Desethylene-ciprofloxacin was not detected in the urine samples

even though an unknown endogenous product co-eluted at the same time (6.3 min) as

desethylene-ciprofloxacin (Figure 3.12). As in the infection study, this unknown peak

was determined not to be desethylene-ciprofloxacin because it co-eluted with an

endogenous peak in the 0 h urine sample and had an ultraviolet absorption maximum at

250 nm. The characteristic fluoroquinolone absorption maxima occurs around 280 nm.

Total urine creatinine was not significantly different among the control,

dexamethasone. endotoxin and endotoxin-dexamethasone groups at 24, 48 and 72 h

(Table 3.7). Total urine enrofloxacin and ciprofloxacin were not significantly different

at 24 and 48 h (Table 3.7). Total urine enrofloxacin and ciprofloxacin were

significantly greater (P<0.01) for the endotoxin group compared to the control,

dexamethasone and endotoxin-dexamethasone groups at 72 h by one-way analysis of

variance (Table 3.7).

The urine enrofloxacin/creatinine ratios were not significantly different (PO.Ol)

for the 24 h urine samples (Table 3.7; Figure 3.13). The enrofloxacin/creatinine ratio of

the 48 h urine samples for the endotoxin group was significantly greater (PO .O l) than

the control, dexamethasone. and endotoxin-dexamethasone treatment groups. There

was a significant interaction (PO .O l) between endotoxin and dexamethasone (Table

3.7; Figure 3.13). The 48 h urine samples also had a significantly lower (PO.Ol)

185

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table 3.2. Lung/body weight (g/kg) ratios in control, dexamethasone. endotoxin and endotoxin-dexamethasone treatment groups. The lung weights were normalized with body weights. Control Dexamethasone Endotoxin Endotoxin- Dexamethasone

LW/BW (g/kg) 10.8 9.36 10.1 10.5 (min-max) ______(8.84-11.9) (8.38-10.5) (8.21-10.5) (9.68-12.2) LW/BW; the ratio of the lung weight to total body weight. Values are raw data medians and (min-max) represents the range of raw data within a treatment group.

186

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Figure 3.1. Rank means±SD of lung/body weight ratios in control, endotoxin, dexamethasone and endotoxin-dexamethasone treatment groups. Values with identical superscripts are not significantly different. P< 0.01 *. indicates a significant interaction between endotoxin and dexamethasone. aUsed to compare control, dexamethasone, endotoxin and endotoxin-dexamethasone treatment groups (n=6) (One-way analysis of variance performed after ranking the raw data. P<0.01).

187

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. without prohibited reproduction Further owner. copyright the of permission with Reproduced

Rank Means of Lung Weight/Body Weight Ratios 20 j 22 : 24 -I26 -I28 0 i 10 T 30 8 J 18 i 4 - 6 \ 2 J o ' i ' 8

\ i : I .': I ; | i t i

j I I

- , oto DxmtaoeEdtxn End-Dex Dexamethasone Endotoxin Control J ------...... : — TreatmentGroups - - 188 - - - J — — V. ,. /V'.'

- - - Table 3.3. Plasma aspartate aminotransferase in control, endotoxin, dexamethasone and endotoxin-dexamethasone treatment groups AST Control Dexamethasone Endotoxin Endotoxin- Dexamethasone (IU/ml) Oh 13.9 10.2 17.1 13.4 (min-max) (9.74-25.4) (3.72-12.7) (12.3-20.1) (9.27-23.3) 24 h 18.9 11.4 41.2 16.2 (min-max) (13.5-21.7) (6.80-13.9) (17.7-708) (11.4-33.9) 96 h 16.7 13.3 18.1 15.9 (min-max) (8.76-21.9) (6.64-16.8) (13.5-29.6) (10.9-18.6) AST (IU/ml). plasma aspartate aminotransferase Values are means and (min-max) represents the range of raw data within a group.

189

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Figure 3.2. Rank means±SD of plasma aspartate aminotransferase in control, endotoxin, dexamethasone and endotoxin-dexamethasone treatment groups at 0. 24 and 72 h after endotoxin administration. Values with identical superscripts are not significantly different. P< 0.01 aUsed to compare no-dexamethasone pigs with dexamethasone treated pigs and no endotoxin pigs with endotoxin treated pigs at 0 h (n=12) (Two-way analysis of variance performed after ranking the raw data. P.0.01). tdUsed to compare no-dexamethasone pigs with dexamethasone treated pigs at 24 h (n=12) (Two-way analysis of variance performed after ranking the raw data. P<0.01). CDUsed to compare no-endotoxin pigs with endotoxin treated pigs at 24 h (n=12) (Two- way analysis of variance performed after ranking the raw data. P<0.01). eUsed to compare no-dexamethasone pigs with dexamethasone treated pigs and no endotoxin pigs with endotoxin treated pigs at 96 h (n=l2) (Two-way analysis of variance performed after ranking the raw data. PO.Ol).

190

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. without prohibited reproduction Further owner. copyright the of permission with Reproduced

Rank Means of Plasma Aspartate Aminotransferase 5 1 25 30 15 10 h24 h Oh cC 191 dC Hour cD Endotoxin-Dexamethasone Dexamethasone Control Endotoxin dD 96h Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. without prohibited reproduction Further owner. copyright the of permission with Reproduced hours after endotoxin (2 pg/kg BW) administration at 0 h. 0 at administration BW) pg/kg (2 endotoxin after hours Figure 3.3. Mean plasma concentration of interleukin-6 measured at 0. at measured interleukin-6 of concentration plasma Mean 3.3. Figure Mean Plasma Interleukin-6 Concentration (units/nil) 2000 2500 3000 50 r 3500 1000 1500 500 0 2 —~— Dexamethasone • Control—•— 192 Endotoxin-Dexamethasone Endotoxin 4 Time (h) Time 6 8 1.3.6 and 9 and 10 Table 3.4. Area under the curve for plasma interleukin-6 in pigs in control, dexamethasone. endotoxin and endotoxin-dexamethasone treatment groups. Area Under the Control Dexamethasone Endotoxin Endotoxin- Curve Dexamethasone (units/ml x h) AUCo_9h 387 203 17243 1100 (min-max) (44.4-489) (131-440) (1447-23579) (483-16748) AUC q^ q h; area under the curve from 0 to 9 h. Values are raw data medians and (min-max) represents the range of raw data within a group.

193

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Figure 3.4. Rank means±SD of area under the curve for plasma interleukin-6 in control, endotoxin, dexamethasone and endotoxin-dexamethasone treatment groups. Values with identical superscripts are not significantly different. P< 0.01). Used to compare no-endotoxin pigs with endotoxin treated pigs (n=12) (Two-way analysis of variance performed after ranking the raw data. P<0.01).

194

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. without prohibited reproduction Further owner. copyright the of permission with Reproduced

Rank Means of Area Under Curve for Plasma lnterleukin-6 20 25 0 3 10 15 0 5 oto DxmtaoeEdtxn End-Dex DexamethasoneEndotoxin Control Treatment Groups Treatment 195 Control Dexamethasone -J Endotoxin

'Bfmid S, Endotoxin-Dexamethasone s

■hm -w2 s u y s o U #c "3 «s X £ ”0im S U a: cn a:

ca u 0.1

0 10 20 30 40 70 Time (h)

Figure 3.5. Semilogrithmic plot of mean plasma concentrations of enrofloxacin measured at 0. 0.25. 0.5. 1. 2, 3, 4, 6, 8. 12. 24. 36. 48. 60. and 72 h after i.v. enrofloxacin (5 mg/kg BW) administration and 24 h after endotoxin or sham endotoxin

196

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table 3.5. Area under the curve (mg/L x h) for plasma enrofloxacin in control, dexamethasone. endotoxin and endotoxin-dexamethasone treatment groups. Area Under the Control Dexamethasone Endotoxin Endotoxin- Curve (mg/L x h) Dexamethasone

AUC(in0 11.2 9.43 22.4 9.22 (min-max) (9.13-14.2) (8.41-13.7) (18.7-36.6) (6.20-13.2) A U C d^ 10.4 8.47 21.2 7.71 (min-max) (8.02-13.2) (7.05-12.0) (16.0-34.6) (5.65-11.7) AUC,inr). area under the curve from zero to infinity AUC(Iast). area under the curve from zero to the last measured hour. Values are raw data medians and (min-max) represents the range of raw data within a group.

197

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Figure 3.6. Rank means±SD of area under the curve for plasma enrofloxacin in control, endotoxin, dexamethasone and endotoxin-dexamethasone treatment groups, comparing AUC(last) with AUC(inf). AUC(inf). area under the curve from zero to infinity AUC(last). area under the curve from zero to the last measured hour. Values with identical superscripts are not significantly different. P<0.01. * indicates an interaction between endotoxin and dexamethasone. abUsed to compare the endotoxin group with the control, dexamethasone and endotoxin- dexamethasone group for AUC(inf) (n=6) (One-way analysis of variance performed after ranking the raw data. P<0.01). cd • Used to compare the endotoxin group with the control, dexamethasone and endotoxin- dexamethasone group for AUC(last) (n=6) (One-way analysis of variance performed after ranking the raw data, P<0.01). CD Used to compare w endotoxin-dexamethasone group with control and dexamethasone groups for AUC(last) (n=6).(One-way analysis of variance performed after ranking the raw data. P<0.01).

198

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. without prohibited reproduction Further owner. copyright the of permission with Reproduced

Rank Means of Area Under Curve for Plasma Enrofloxacin 20 22 26 24 28 30 14 10 2 1 12 6 i16 18 4 0 2 6 8

- -

U(n) AUC(last) AUC(inf) Control Endotoxin-Dexamethasone Endotoxin Dexamethasone Treatm ent Groups ent Treatm b 199 cC D* cD Figure 3.7. Means of area under the curve ratios for plasma enrofloxacin in control, endotoxin, dexamethasone and endotoxin-dexamethasone treatment groups. AUC(inf) area under the curve from zero to infinity AUC(last), area under the curve from zero to the last measured hour.

200

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. without prohibited reproduction Further owner. copyright the of permission with Reproduced

Means of AUC(last)/AUC(inf) Ratios for Enrofloxacin 0.0 0.4 0.2 0.6 0.8 Control Dexamethasone Endotoxin Dexamethasone Endotoxin Control Treatment Groups Treatment 201 End-Dex 13.3 11.8 9.21 7.07 (8.46-14.7) (5.69-9.55) (9.07-17.4) (7.24-11.6) (0.379-0.806) Dexamethasone 16.2 21.6 4.62 4.37 0.223 0.542 Endotoxin Endotoxin- (9.70-20.4) (2.59-6.25) (14.1-28.0) (2.52-7.13) (0.137-0.267) 12.7 8.64 (6.03-10.5) (0.366-0.594) (0.0491-0.0844) (0.0340-0.0714) (0.0473-0.0819) 10.5 12.7 5.12 7.16 0.447 0.531 0.0658“ 0.0624“ 0.0432“ 0.0589“ Control Dexamethasone (4.01-6.14) (5.52-7.42) (8.75-13.6) (10.8-16.9) (4.60-8.56) (0.062-0.100) (h) 1/2 1 P(1/h) MRT (h) 12.0 V„(L/kg) 6.11 Vss(L/kg) (min-max) (6.96-11.3) (8.22-14.1) (min-max) (min-max) (min-max) (min-max) (min-max) (0.353-0.548) Parameters Cll0, (L/li/kg) Cll0, Pharmacokinetic (n=6) (One-way analysis ofvariance performed after ranking the raw data, P<0.01). mean residence time;half-life t!/2, and elimination(i, rate constant. Values are raw data medians and (min-max) represents the range Valuesof rawwith data identicalwithin a superscriptsgroup. are not significantly different, to “Used compare P< 0.01. the endotoxin group with the control, dexamethasone and endotoxin-dexamethasone groups after ranking the data CIU)1, total body clearance; volumeV,, ofdistribution CIU)1, based on the terminal phase;volume ofVss, distribution at steady state; MRT, treatment groups. Table 3.6. Plasma pharmacokinetic parameters ofenrofloxacin in control, endotoxin, dexamethasone and endotoxin-dexamethasone t o o t o

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Figure 3.8. Rank means±SD of clearance for plasma enrofloxacin in control, endotoxin, dexamethasone and endotoxin-dexamethasone treatment groups. Values with identical superscripts are not significantly different. P< 0.01. abUsed to compare no-dexamethasone pigs with dexamethasone treated pigs (n=12) (Two-way analysis of variance performed after ranking the raw data. P<0.01). ■\B * w Used to compare the endotoxin group with the control, dexamethasone and endotoxin-dexamethasone groups (n=6) (One-way analysis of variance performed after ranking the raw data. PO .O l).

203

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. without prohibited reproduction Further owner. copyright the of permission with Reproduced

Rank Means of Enrofloxacin O 0 i 20 22 4 - 24 2 1 14 - 18 16] ,oi 0 4j 6 ] 2 8 i ] ] ] j t ! i i I i ! i i oto Dexamethasone Endotoxin Control aA Treatment Groups bA 204 aB -T- End-Dex ; .y • ...y y y '-r.-.y; 7. . : i '.V •' ,;; C - r •*- <■ /•. bA ■K \: - V >‘..w ■ j - \ : ! *. ... . r: I Figure 3.9. Rank means±SD of volume of distribution of the elimination phase for plasma enrofloxacin in control, endotoxin, dexamethasone and endotoxin- dexamethasone treatment groups. Values with identical superscripts are not significantly different. P< 0.01. abUsed to compare no-dexamethasone pigs with dexamethasone treated pigs (n=l2) (Two-way analysis of variance performed after ranking the raw data. PO .O l).

205

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. without prohibited reproduction Further owner. copyright the of permission with Reproduced

Rank Means ofVolniiii of Distribution for the Enrofloxacin Elimination Phase 20 2 J 22 0 1 2 i 12 14^ 1 16 18 - 4] 0 2 ] 6 1 8

- - 1 I I i i i i ! I i I

oto CfexarnsthasmeControl Endctoxin Treatment Groups Treatment 206 End-Ctex b Figure 3.10. Rank means±SD of volume of distribution at steady state for plasma enrofloxacin in control, endotoxin, dexamethasone and endotoxin-dexamethasone treatment groups. Values with identical superscripts are not significantly different. P< 0.01. abUsed to compare no-dexamethasone pigs with dexamethasone treated pigs (n=12) (Two-way analysis of variance performed after ranking the raw data. P.O.01).

207

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. without prohibited reproduction Further owner. copyright the of permission with Reproduced

Rank Means of Volume of Distribution at Steady State for Enrofloxacin 20 I 22 4 i 24 10 12 16 14 1 18 | 4 0 6 8

- \ t | j i i i I l

oto DxmtaoeEdtxn End-Dex Dexamethasone Control Endotoxin Treament Groups Treament 208 Figure 3.11. Rank means±SD of mean residence time (MRT) and half-life (tl/2) for plasma enrofloxacin in control, endotoxin, dexamethasone and endotoxin- dexamethasone treatment groups. Values with identical superscripts are not significantly different. P< 0.01. *. indicates a significant interaction between endotoxin and dexamethasone. abUsed to compare the endotoxin group with control, dexamethasone and endotoxin- dexamethasone groups for mean residence time (n=6) (One-way analysis of variance performed after ranking the raw data, PO.Ol). cUsed to compare no-dexamethasone pigs with dexamethasone treated pigs and no endotoxin pigs with endotoxin treated pigs for half-life (n=12) (Two-way analysis of variance performed after ranking the raw data. P<0.01).

209

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. without prohibited reproduction Further owner. copyright the of permission with Reproduced

Rank Means of Mean Residence Time and Half-Life for Enrofloxacin 0 j 20 25 30 15 0 a R Half-Life Parameter Pharmacokinetic MRT 210 Control Dexamethasone Endotoxin-Dexamethasone Endotoxin l.2e+6

Standard Oh Ciprofloxacin 72 h l.0e+6 !■

Suspect Oxo-enrofloxacin Enrofloxacin Pipemidic Acid

8.0e+5 r

s \ ' J i i 3 11 I'i i 5 11' ' ■ ! i i1 i 'i ■ i 1 l * 11 •3 6.0e+5 j - i ! M Vs I I ■ M i \ -a; 3 v ' v \ r\ 1 1 i i Su ■ '" V [i rv—

4.0e+5 r

2.0e+5

0 1 i 4 5 6 7 8 9 10 11 12 Tim e (m in)

Figure 3.12. Chromatographs of standards (ciprofloxacin and enrofloxacin) and the internal standard (pipemidic acid); 0 h urine sample: and 72 h urine samples in an endotoxin treated pig.

211

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table 3.7. Total urine creatinine, enrofloxacin and ciprofloxacin in control, endotoxin, dexamethasone and endotoxin-dexamethasone treatment groups. Creat (mg), total urine creatinine; Enro (mg), total urine enrofloxacin; and Cipro (mg), total urine ciprofloxacin. Values are raw data medians and (min-max) represents the range of raw data within a group. Values with identical superscripts are not significantly different. P< 0.01. aUsed to compare control, dexamethasone. endotoxin and endotoxin-dexamethasone treatment groups for total urine creatinine at 24. 48 and 72 h (n=6) (One-way analysis of variance performed after ranking the raw data. P<0.01). ^ Used to compare the endotoxin group with the control, dexamethasone and endotoxin- dexamethasone groups for total urine enrofloxacin at 24. 48 and 72 h (n=6) (One-way analysis of variance performed after ranking the raw data. P<0.01). uUsed to compare the endotoxin group with the control, dexamethasone and endotoxin- dexamethasone groups for total urine ciprofloxacin at 24. 48 and 72 h (n=6) (One-way analysis of variance performed after ranking the raw data. P<0.01).

212

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Control Dexamethasone Endotoxin Endotoxin- Dexamethasone

24 h Creat (mg) I223a 832a 751a 1042a (min-max) (60.8-1539) (354-1292) (6.94-1161) (289-1409) 48 h Creat (mg) 987a 1128a 856a 1022a (min-max) (723-1488) (854-1697) (218-1148) 879-1373) 72 h Creat (mg) 922a 73 2a 1209a III 4a (min-max) (353-1414) (445-1241) (488-1790) (580-1843) 24 h Enro (mg) 10.4° 8.14° 8.86° 7.60° (min-max) (8.82-13.0) (5.59-15.6) (0.003-16.3) (4.09-8.87) 48 h Enro (mg) 4.15c 4.03c 5.39° 2.04c (min-max) (2.31-4.98) (1.74-9.06) (0.951-11.3) (1.13-3.31) 72 h Enro (mg) 1.19C 1.04c 2.38d 0.746° (min-max) (0.468-1.49) (0.715-1.92) (1.60-4.57) (0.589-0.957) 24 h Cipro (mg) 11.3° 12.5° 6.37c 15.4° (min-max) (8.36-14.0) (7.90-18.6) (0.027-18.2) (8.75-17.2) 48 h Cipro (mg) 4.54e 5.07e 8.60° 4.32e (min-max) (3.39-5.52) (3.40-9.86) (1.91-11.0) (2.49-6.25) 72 h Cipro (mg) 1.23e 1.15C 4.311' 1.88° (min-max) (0.468-2.27) (0.201-2.11) (3.38-5.93) (0.743-4.72)

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table 3.8. Urine ciprofloxacin/creatinine and enrofloxacin/creatinine ratios in control. endotoxin, dexamethasone and endotoxin-dexamethasone treatment groups.

Control Dexamethasone Endotoxin Endotoxin- Dexamethasone

24 h Enro/Creat (pg/mg) 9.84 14.4 10.7 6.77 (min-max) (5.73-11.4) (5.15-19.5) (0.383-14.1) (2.91-30.7) 48 h Enro/Creat (pg/mg) 3.78 3.53 6.45 2.06 (min-max) (2.89-5.38) (2.03-5.87) (4.36-9.85) (0.826-3.26) 72 h Enro/Creat (pg/mg) 1.23 1.07 2.19 0.586 (min-max) (0.923-3.03) (1.00-1.71) (1.50-4.99) (0.378-1.40) 24 h Cipro/Creat (pg/mg) 9.22 20.3 8.31 15.7 (min-max) (6.91-12.1) (7.81-22.3) (3.85-15.7) (6.21-50.3) 48 h Cipro/Creat (pg/mg) 4.01 4.07 9.16 4.17 (min-max) (3.01-7.02) (3.83-6.40) (7.01-11.2) (2.07-7.11) 72 h Cipro/Creat (pg/mg) 1.99 1.07 4.34 2.31 (min-max) (0-2.70) (0.451-1.70) (2.40-6.92) (0.705-2.90) Enro/Creat (pg/mg). ratio of urine enrofloxacin/urine creatinine. Cipro/Creat (pg/mg). ratio of urine enrofloxacin to urine creatinine. Values are raw data medians and (min-max) represents the range of raw data within a group.

214

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Figure 3.13. Rank means±SD of urine enrofloxacin/creatinine ratios in control, endotoxin, dexamethasone and endotoxin-dexamethasone treatment groups at 24.48 and 72 h after enrofloxacin administration. Values with identical superscripts are not significantly different. P< 0.01 *. indicates a significant interaction between endotoxin and dexamethasone. aUsed to compare no-dexamethasone pigs with dexamethasone treated pigs and no endotoxin pigs with endotoxin treated pigs at 24 h (n=12) (Two-way analysis of variance performed after ranking the raw data. P<0.01). udUsed to compare the endotoxin group with control, dexamethasone and endotoxin- dexamethasone groups at 48 h (n=6) (One-way analysis of variance performed after ranking the raw data. P<0.01). CD Used w to compare the endotoxin-dexamethasone group with the control group at 48 h (n=6) (One-way analysis of variance performed after ranking the raw data. PO.Ol). l'tUsed to compare the endotoxin-dexamethasone group with the control, dexamethasone and endotoxin groups at 72 h (n=6) (One-way analysis of variance performed after ranking the raw data. P<0.01).

215

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. without prohibited reproduction Further owner. copyright the of permission with Reproduced

Rank Means of Urine Enrofloxacin/Crea ' 8 ' e Ratios 25 5 3 10 15 0 24 h 48 h 48 24h Control Dexamethasone Endotoxin-Dexamethasone Endotoxin cC 216 Hour D* cD e e 72h f* Figure 3.14. Rank means±SD of urine ciprofloxacin/creatinine ratios in control, endotoxin, dexamethasone and endotoxin-dexamethasone treatment groups at 24. 48 and 72 h after enrofloxacin administration. Values with identical superscripts are not significantly different. P< 0.01. abUsed to compare no-dexamethasone pigs with dexamethasone treated pigs at 24 h (n=12) (Two-way analysis of variance performed after ranking the raw data. P<0.01). LliUsed to compare the endotoxin group with the control, dexamethasone and endotoxin- dexamethasone groups at 48 h (n=6) (One-way analysis of variance performed after ranking the raw data. P<0.01). dUsed to compare endotoxin group with control, dexamethasone and endotoxin- dexamethasone groups at 72 h (n=6) (One-way analysis of variance performed after ranking the raw data. P<0.01).

217

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. without prohibited reproduction Further owner. copyright the of permission with Reproduced

Rank Means of Ciprofloxacin/Creatinine Ratios 20 25 0 ] 30 35 10 15 0

1 \ i 24 h 24 li a as®!®*® Endotoxm-Dexamethasone Control Endotoxin Dexamethasone 218 f r u [fo 48 h h 48 72h enrofloxacin/creatinine ratio for the endotoxin-dexamethasone group compared to the

control group (Table 3.7; Figure 3.13). The 72 h enrofloxacin/creatinine ratios for the

endotoxin-dexamethasone group were significantly less (P<0.01) for the endotoxin-

dexamethasone group compared to the control, dexamethasone and endotoxin groups

(Table 3.7; Figure 3.13). There was a significant interaction (P<0.01) between

endotoxin and dexamethasone (Table 3.7; Figure 3.13).

The ciprofloxacin/creatinine ratios were significantly greater (P<0.01) for the

dexamethasone treated pigs compared to no-dexamethasone pigs in the 24 h urine

samples (Table 3.8; Figure 3.14). The ciprofloxacin/creatinine ratio was significantly

greater (PO .O l) for the endotoxin group compared to the control, dexamethasone and

endotoxin-dexamethasone groups in the 48 and 72 h urine samples (Table 3.8; Figure

3.14).

Discussion

All of the endotoxin treated pigs exhibited signs of labored breathing and

lethargy within two hours which confirmed that the pigs received enough endotoxin to

induce systemic illness. The plasma AST concentrations were elevated at 24 hours and

returned to baseline by 96 h after endotoxin administration. This meant that the liver

injury present at 24 h was repaired by 96 h. Transient liver injury is logical because the

plasma interleukin-6 concentrations were significantly elevated in the endotoxin treated

pigs at 3 h but had decreased to near baseline by 9 h (Table 4.3; Figures 3.3 and 3.4).

Therefore, the stimulus for liver injury and possible suppression of cytochrome P450's

was absent after 9 h.

219

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Pharmacokinetic parameters were calculated on the assumption, similar to the

infection study, that the extrapolated area under the curve from the last measured time

point to infinity did not bias the calculation of clearance, volume of distribution, mean

residence time and half-life. When the AUC(inf) was compared to AUC(last). the

outcome of the statistical analysis was the same, based on one-way analysis of variance

(Table 3.5: Figures 3.5 and 3.6. Unlike the infection study for AUC(last) and AUC(inf).

the endotoxin group was significantly greater than the control, dexamethasone and

endotoxin-dexamethasone groups (Figure 3.6). The biological significance of the

endotoxin and dexamethasone interaction for AUC(last) can not be readily explained

based on AUC data (Figure 3.6). The dexamethasone treatment seems to counteract the

endotoxin response. The ratios of AUC(last)/AUC(inf) among the control,

dexamethasone. endotoxin and endotoxin-dexamethasone treatment groups were

acceptable because less than 10% of the total area was extrapolated (Figure 3.7). The

calculation of pharmacokinetic parameters based on AUC(inf) is still valid .

The volume of distribution of enrofloxacin for both the elimination phase and

steady state was increased for the dexamethasone treated animals but not the endotoxin

treated animals compared to the no dexamethasone and no-endotoxin pigs (Table 3.5:

Figures 3.9 and 3.10). Increased Vp may indicate decreased plasma protein binding and

increased free fraction while the increased Vss could indicate increased plasma and

tissue protein binding and decreased free fraction for enrofloxacin (Ritschel & Denson.

1991). Thus, the free fraction in the plasma and tissues should decrease and the

clearance of enrofloxacin in the endotoxin group should increase for the dexamethasone

220

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. treated pigs (Figures 3.8 - 3.10). The increased clearance in the dexamethasone treated

pigs is more likely due to increased GFR and filtration fraction as a glucocorticoid

effect on the kidney (Connell et al.. 1987: Stefanovic et al.. 1991). The rate of

elimination from the central compartment and the AUC could both contribute to a

change in Vp in the dexamethasone treated pigs, because the elimination rate constant

and AUC are both inversely related to the volume of distribution of the elimination

phase by the equation: Vp=dose/([3 x AUC). A decrease in one or both parameters could

increase Vp. The AUC did decrease for the dexamethasone treated pigs but the

elimination rate constant did not change, which could account for the increase in Vp

(Table 3.6: Figures 3.9 and 3.10).

The half-life for the endotoxin group was not significantly different from the

control, dexamethasone and endotoxin-dexamethasone groups (Figure 3.11). Mean

residence time would be expected to parallel half-life., but MRT of the endotoxin group

is greater than the control, dexamethasone and endotoxin-dexamethasone groups

(Figure 3.11). The graphical profiles are similar even though the statistical outcomes

are different (Figure 3.11).

The most likely explanation for increased protein binding in tissues leading to

increased volumes of distribution in dexamethasone treated pigs would be lack of

interference by fatty acids with albumin and more binding sites on albumin or a,-acid

glycoprotein (Greene & Budberg, 1993). This assumes that other factors, such as

enrofloxacin blood concentration or pH. did not affect the serum protein binding of

enrofloxacin. While volumes of distribution were altered for the dexamethasone treated

221

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. pigs, the volumes of distribution are not altered for the endotoxin treated pigs compared

to the no-endotoxin pigs (Table 3.5; Figures 3.9 and 3.10). In the infection study, the

volumes of distribution were decreased in the infected pigs and unchanged for the

dexamethasone treated pigs (Figures 2.13 and 2.14). In comparison to the infection

study, the failure of endotoxin to change the volume of distribution in the endotoxin

treated pigs can not be readily explained. There are many more toxins associated with

APP infection than would be associated with endotoxin administration, and the

endotoxin was administered as a single i.v. dose.

Ciprofloxacin and suspect oxo-enrofloxacin were found in the urine(Figures

2.17 and 3.12). This is consistent with the infection study where the suspect lesser

metabolite, oxo-enrofloxacin. was also detected in the urine after i.v. administration of

enrofloxacin. There was no significant difference among the treatment groups at 24 h

for the urine enrofloxacin/creatinine ratio, but the urine ciprofloxacin/creatinine ratio

was significantly greater for the dexamethasone treated pigs compared to the no-

dexamethasone pigs at 24 h (Table 3.7 and 3.8; Figures 3.13 and 3.14). The effect of

dexamethasone on GFR and clearance was not evident at 24 h for the urine

enrofloxacin/creatinine ratio, but the increased urine ciprofloxacin/creatinine ratio in the

dexamethasone treated pigs at 24 h might be explained by increased clearance related to

augmented GFR (Figures 3.13 and 3.14).

At 48 h. the urine enrofloxacin/creatinine ratio was greatest for the endotoxin

group compared to control, dexamethasone and endotoxin-dexamethasone groups; and

the endotoxin-dexamethasone was less than the control and dexamethasone groups.

222

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. (Table 3.7; Figure 3.13). The interaction between endotoxin and dexamethasone in the

48 h urine enrofloxacin/creatinine ratios was probably due to GFR “washout” in the

dexamethasone group and peripheral vasodilatation followed by renal vasoconstriction

in the endotoxin group (Figure 3.13). In other words, the endotoxin group may have

developed hypotension associated with endotoxic shock, with a subsequent renal

vasoconstriction to maintain homeostasis. In fact, endotoxin treated pigs had a flushed,

red skin until they were necropsied. This supports a physiological effect of endotoxin

on enrofloxacin clearance rather than a metabolic effect. The urine

ciprofloxacin/creatinine ratio in the endotoxin group should have decreased relative to

the control group, if metabolism of enrofloxacin had been decreased (Tables 3.7 and

3.8; Figure 3.14). The ciprofloxacin/creatinine ratio for the endotoxin group was

actually increased at 48 and 72 h which means that there was no liver injury present to

cause a decrease in total cytochrome P450 protein (Figure 3.14). Liver damage for the

endotoxin treated pigs was transient as indicated by plasma AST (Figure 3.2). At 24 h

after endotoxin administration, the dexamethasone treated pigs had decreased plasma

AST concentrations compared to the no-dexamethasone pigs while the endotoxin

treated pigs had increased plasma AST concentrations compared to no-endotoxin pigs.

This implies a sparing effect of dexamethasone on liver damage.

Two or more AST isozymes are located in the cytosol and the mitochondria and

catalyze the conversion o f aspartate and alpha-ketoglutarate to glutamate and

oxaloacetate (Duncan & Prasse. 1986). The enzyme occurs in almost all cells. It is

considered to be a diagnostic enzyme for liver and muscle disease, because of high

223

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. activity in these tissues. The plasma AST half-life for swine is about 18 hours. Plasma

AST activity increases with changes in heptocellular permeability due to sublethal

injury and necrosis. The magnitude of the increase approximates the number of cells

affected in acute disease. In acute hepatic sublethal injury or necrosis and high serum

AST. hepatic insufficiency may be minimal. Consequently, the 24 h

enrofloxacin/creatinine and ciprofloxacin/creatinine ratios were elevated for the

endotoxin group 48 h after endotoxin administration which is 24 h after AST elevation.

By 48 h. liver damage (plasma AST concentration) was on the decline which would

suggest that any depression of total cytochrome P450 protein content was increasing.

This seems reasonable, because total cytochrome P450 content and enzyme activities

were decreased 24 h after five doses of endotoxin (17 pg/kg) in the Netherlands study

(Monshouwer et. al.. 1996a) compared to a single i.v. dose (2 pg/kg) used in this study.

Resolving liver damage and elevated urine ciprofloxacin/creatinine ratio point to a

physiological mechanism for decreased enrofloxacin clearance in the endotoxin group.

A decrease in enrofloxacin conversion to ciprofloxacin is not likely in the endotoxin

treated pigs.

Dexamethasone depressed IL-6 concentrations after endotoxin administration

(Table 3.4; Figures 3.3 and 3.4). Then, the effect of endotoxin could have depended

upon concurrent dexamethasone concentration. Therefore, the endotoxin treated pigs

showed no changes in clearance or volume of distribution of enrofloxacin. The changes

in urine enrofloxacin/creatinine and ciprofloxacin/creatinine ratios at 48 h may be the

224

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. result of enrofloxacin and ciprofloxacin "washout7' due to increased GFR (Figures 3.13

and 3.14).

At 72 h, the urine enrofloxacin/creatinine and ciprofloxacin/creatinine ratios

were greatest for the endotoxin group compared to the control, dexamethasone and

endotoxin-dexamethasone groups (Tables 3.7 and 3.8; Figures 3.13 and 3.14). Again,

there was a significant interaction at 72 h between endotoxin and dexamethasone in the

enrofloxacin/creatinine ratios (Figure 3.13). Presumably, the interaction may have

occurred for the same reasons as in the 48 h urine enrofloxacin/creatinine ratios. The

prolonged enrofloxacin/creatinine and ciprofloxacin/creatinine ratios were most likely

due to physiological mechanisms rather than reduced metabolism of ciprofloxacin.

It is also possible that dexamethasone could induce cytochrome P450

metabolism of enrofloxacin to ciprofloxacin and contribute to the clearance of

enrofloxacin. Endotoxin could suppress P450's and decrease enrofloxacin clearance,

but endotoxin did not have an effect on clearance. Endotoxin decreased clearance of

enrofloxacin compared to control, dexamethasone and endotoxin-dexamethasone groups

(One-way analysis of variance) (Figure 3.8).

While the enrofloxacin clearance was not decreased and the volume of

distribution was not increased for the endotoxin group, the mean residence time was

significantly increased for the endotoxin group compared to the control, dexamethasone

and endotoxin-dexamethasone groups with no effect on half-life ( Table 4.5; Figure

3.11). This verifies that the transit time for enrofloxacin is increased by endotoxin.

Assuming that biliary excretion or transenteric secretion remains the same as healthy

225

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. animals, the most likely cause for the increased transit time is renal vasoconstriction.

Dexamethasone did not inhibit enrofloxacin clearance in both the infection and

endotoxin studies. Therefore, a direct effect of dexamethasone on enrofloxacin

metabolism is doubtful. Liver injury was transient at 24 h after endotoxin

administration and returned to baseline by 96 h. as measured by plasma AST. This was

not long enough to promote suppression of cytochrome P450 by systemic cytokine

release and cause a subsequent reduction of enrofloxacin biotransformation to

ciprofloxacin or oxo-enrofloxacin. Then, the direct effect of endotoxin on enrofloxacin

metabolism is also doubtful. In female rats. IL-1 and dexamethasone significantly

depressed total P450. and P450IIC12 apoenzyme. and mRNA (Wright & Morgan.

1991). Induction of IL-1 and IL-6 was inhibited by dexamethasone which would

prevent the cytokine induced suppression of cytochrome P450's (Menzozzi et al..

1994). In this study, dexamethasone did suppress the endotoxin induced release of IL-6

(Figures 3.3 and 3.4) but did not increase urine enrofloxacin/creatinine ratio with a

concomitant decrease of the ciprofloxacin/creatinine ratio in the endotoxin and

dexamethasone pigs. This strongly suggests that metabolism of enrofloxacin was not

affected by dexamethasone.

226

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. SUMMARY AND CONCLUSIONS

Previous pharmacokinetic studies have used healthy animals while diseased

animals were used in efficacy studies. The lack of pharmacokinetic studies in diseased

animals was not through scientific neglect, but the fact that the drug or antimicrobial

must be tested in healthy animals who may be screened for age. weight, medical history,

blood chemistry and receive the test article at the same time of day. with similar

conditions of fasting, food, and activity. The performance of a drug or antimicrobial or

its formulation is a tested in healthy animals by keeping all variables that might affect

pharmacokinetics parameters constant. However, many veterinary drugs are not

intended for healthy animals, but rather for those animals with one or more pathological

conditions that are manifested as disease processes. These disease processes alter the

normal function of the body. Consequently, the normal functions involved in

distribution and elimination of a drug may be altered by disease.

Because the body handles many drugs and endogenous substances similarly,

diseases that alter the pharmacodynamics of endogenous substances may also alter the

pharmacodynamics of drugs. Diseases that alter pharmacodynamics can also alter

pharmacokinetic parameters. The onset and duration of action of a drug is dependent on

the rate and extent of distribution. A change in the apparent volume of distribution may

change the drug concentration in blood or plasma and at the receptor site. Diseases can

modify a number of kinetic parameters and alter steady state concentrations and.

thereby, influence efficacy or toxicity. Unfortunately, it is not easy to standardize

disease models. Disease models are ever changing and never constant, dependent on the

227

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. state of the disease, which makes it difficult to find a population of animals who are

homogenous for the disease, disease state, therapy, age, weight, genetics, diet, housing

and stress factors.

One of the objectives of this investigation was to determine whether a

Actinobacillus pleuropneumoniae pulmonary infection model could be standardized.

This goal was accomplished as demonstrated by the pigs becoming ill within 2 hours of

inoculation with APP. where signs of labored breathing and lethargy were observed.

Systemic interleukin-6 concentrations were highly elevated in the infected pigs. This

was another indication that APP infection was accomplished. Infection was verified by

gross pathology observations, increased lung weights and microscopic pathology results

of necrotizing, fibrinopurulent pneumonia.

The second objective was to examine the effect of infection and dexamethasone

on pharmacokinetic parameters in swine after administering enrofloxacin. Clearance

was reduced for the dexamethasone treated pigs, but clearance was not altered in

infected pigs. This change was attributed to increased glomerular filtration rate and

filtration fraction. The volume of distribution based on the elimination phase was

decreased in the infected pigs, and was influenced by the elimination rate constant. The

volume of distribution at steady state was also decreased in the infected pigs and is

independent of the elimination rate constant. This means that decreased volumes of

distribution may be due to not only an increased elimination rate constant but could also

be to decreased protein binding in plasma and tissues and/or changes in blood pH or

flow. Mean residence time and half-life were decreased in the dexamethasone treated

228

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. pigs. The changes in mean residence time and half-life reflected the change of

increased clearance in the dexamethasone treated pigs. The urine

enrofloxacin/creatinine ratio was decreased for the dexamethasone treated pigs which

demonstrated a "washout” effect of dexamethasone by increasing the glomerular

filtration rate and filtration fraction. The ciprofloxacin/creatinine ratio and clearance in

the infected pigs were not changed which means that enrofloxacin metabolism was not

altered by infection. Finding the suspect oxo-enrofloxacin peak in the urine samples of

the control, dexamethasone. infected and non-infected dexamethasone pigs was

completely unexpected. Other investigators not did not find metabolites or conjugates

after ciprofloxacin administration (Nouws et al., 1988). The presence of oxo-

enrofloxacin should be confirmed by mass-spectroscopy before HPLC analysis of the

urine peaks. Dexamethasone could have depressed metabolism by cytochrome P450's

but this would not be obvious from the data because excretion and metabolism are

components of clearance. Infection may change the appropriate dosage of enrofloxacin

because the volume of distribution is changed. Decreased volume of distribution could

mean less drug at the receptor site. Withdrawal times should not be altered because

infection did not change enrofloxacin clearance.

The third objective was to compare the results of the infection study to an

endotoxin study. It was postulated that the use of endotoxin would mimic the

Actinobacillus pleuropneumoniae infection and induce similar pharmacokinetic

changes. The endotoxin treated pigs became ill within 30 minutes as shown by very

elevated interleukin-6 plasma concentrations, which proves that systemic signs were

229

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. initiated. As observed in the infection study, clearance o f enrofloxacin was increased in

the dexamethasone treated pigs. The volumes of distribution of the elimination phase

and at steady state were also increased in the dexamethasone treated pigs which was not

observed in the infection study. The combined effect of volume of distribution increase

and clearance increase resulted in no change in half-life in the dexamethasone treated

pigs. Mean residence time was increased for the endotoxin group. Clearance was

decreased for the endotoxin group which was further manifested as increased urine

enrofloxacin/creatinine and ciprofloxacin/creatinine ratios. In the endotoxin study, the

washout of enrofloxacin and ciprofloxacin was evident in the urine samples of

dexamethasone treated pigs as in the infection study. If the clearance of enrofloxacin

was decreased by reduced metabolism of enrofloxacin. it is assumed that renal function,

hepatic function and blood flow were not altered. However, it is more probable that

renal blood flow was decreased as a response to endotoxic shock and hypotension.

Dexamethasone enhanced enrofloxacin clearance and suppressed IL-6 plasma

concentrations which suggests that cytochrome P450 activity was not decreased in

endotoxin treated pigs. Therefore, dexamethasone had a positive influence on

enrofloxacin clearance, rather than producing a blockade of cytochrome P450

metabolism and decreased clearance, along with its anti-inflammatory role.

Additionally, the transient liver injury caused by endotoxin may not have been

substantial enough to depress hepatic metabolism of enrofloxacin as indicated by

aspartate aminotransferase plasma concentrations. It is not likely that cytochrome

P450’s are involved in the decreased clearance and increased mean residence time of

230

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. enrofloxacin in the endotoxin group for the following reasons: 1) urine

ciprofloxacin/creatinine ratios were not elevated; 2) liver injury was transient as

indicated by plasma aspartate aminotransferase concentrations. However, the presence

of a suspect oxo-enrofloxacin peak in the endotoxin study may prove interesting after

mass-spectroscopy confirmation and HPLC analysis as already mentioned for the

infection study. If the oxo-enrofloxacin ratios are not decreased for the endotoxin group

compared to the control, dexamethasone and endotoxin-dexamethasone groups, this

would support the physiological explanation for decreased enrofloxacin clearance as

opposed to reduced metabolism.

The infection and endotoxin studies confirm that the use of dexamethasone in

conjunction with enrofloxacin may result in reduced efficacy because of increased

clearance. Furthermore, the infected pigs had a decreased volume of distribution which

may reduce the concentration of enrofloxacin at the receptor site and decrease efficacy.

The contradiction of no effect of dexamethasone on volumes of distribution in the

infection study but increased volumes of distribution in the endotoxin study can not be

readily explained, except that the genetic make up of the pigs was different between the

two studies; Landrace x Yorkshire in the infection and Hampshire x Yorkshire in the

endotoxin study.

The use of model drugs, such as antipyrine, to investigate pharmacokinetic

parameters with diseased animals is a useful screening tool but can only viewed as

preliminary information prior to testing therapeutic agents. However, the variables

involved in the disease process, compared to healthy animals, make interpretation of the

231

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. results with model drugs difficult and the prediction of efficacy and toxicity impractical.

Thus, the best way to assess the influence of disease on pharmacokinetic parameters is

to use a specific therapeutic drug or antimicrobial instead of a model drug in the

presence of a disease model with a particular gene pool of domestic animals. This study

showed that Actinobacillus pleuropneumoniae. serotype 1. could be used as a disease

model in United States domestic pigs (Yorkshire crosses) to test the effects of infection

on pharmacokinetic parameters of enrofloxacin. a therapeutic fluoroquinolone.

232

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Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. VITA

Lynn O. Post was bom on June 9, 1948 in Jamestown. New York. He graduated

from Randolph Central School. Randolph. New York in 1966. In the Fall of 1966. he

entered Cornell University. Ithaca, New York. In the summer of 1968. he took a leave

of absence from Cornell University and served a three year tour of duty in the United

State Army. Two years of his military tour of duty were spent in the Republic of South

Vietnam as a veterinary technician. He resumed his studies at Cornell University and

obtained a bachelor of science degree in animal science in January of 1974. He married

Marcia L. Frey Post in 1974. He was employed by Albany Medical College. Albany.

New York from 1973 to 1974. and by the New York State Department of Mental

Hygiene from 1974 to 1977. He moved to Texas in 1977 to pursue a master of science

degree in veterinary toxicology while employed by the Texas Veterinary Medical

Laboratory. College Station. Texas. After receiving his master of science degree in

1982. he was accepted to the Texas A&M. School of Veterinary Medicine. College

Station. Texas. Upon graduation from veterinary school in 1986. he engaged in private

veterinary practice as a sole owner in Huntsville, Texas. He left private practice in 1989

and was employed by the United States Department of Agriculture until the Fall of

1990. He returned to graduate school in 1990 to pursue a doctor of philosophy degree

in the Department of Veterinary Physiology, Pharmacology and Toxicology at the

Louisiana State University, School of Veterinary Medicine. Baton Rouge. Louisiana. In

the spring of 1994 he was employed by the Food and Drug Administration, Center for

Veterinary Medicine and completed his research at the Center for Veterinary Medicine's

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Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. research facilities in Laurel. Maryland. In 1996 he became a diplomat of the American

Board of Veterinary Toxicology. Dr. Post currently resides in Keameysville. West

Virginia.

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Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. DOCTORAL EXAMINATION AND DISSERTATION REPORT

Candidat e: Lynn O. Post

Major Field: veterinary Medical Sciences

Title of Dissertation: T h e Effect of Pulmonary Infection (Actinobacillus Pleuropneumoniae), Endotoxin and Dexamethasone on Enrofloxacin Pharmacokinetic Parameters in Swine

Approved:

Major Profptesor ^md

Dean of !e graduate School

EXAMINING COMMITTEE:

Date of Examination:

1 -an-qa

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