the isolated perfused equine distal limb and …€¦ · pharmacology and toxicology, july 8-12,...

UNIVERSITY OF VETERINARY MEDICINE HANNOVER

DEPARTMENT OF PHARMACOLOGY, TOXICOLOGY AND PHARMACY

THE ISOLATED PERFUSED EQUINE DISTAL LIMB AND

SYNOVIOCYTE CULTURES AS MODELS FOR

PHARMACOLOGICAL STUDIES

THESIS

SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE

DOCTOR OF PHILOSOPHY (PHD)

IN THE FIELD OF PHARMACOLOGY

AWARDED BY THE UNIVERSITY OF VETERINARY MEDICINE HANNOVER

BY

MAREN FRIEBE

(BIELEFELD)

HANNOVER 2013

Supervisor: Prof. Dr. Manfred Kietzmann

Supervision group: Prof. Dr. Manfred Kietzmann

Prof. Dr. Karsten Feige

PD Dr. Fritz Thorey

1st Evaluation: Prof. Dr. Manfred Kietzmann

Department of Pharmacology, Toxicology and Pharmacy

University of Veterinary Medicine Hannover

Prof. Dr. Karsten Feige

Clinic for Horses

University of Veterinary Medicine Hannover

PD Dr. Fritz Thorey

Department of Orthopedic Surgery

Hannover Medical School

2nd Evaluation: Prof. Dr. Anton Fürst

Equine Hospital, Vetsuisse Faculty

University of Zurich

Date of final exam: April 23, 2013

This project was financially supported by the German Equestrian Foundation (FN,

Warendorf, Germany).

Für meine Eltern

This project has been published in parts:

Publications

FRIEBE M., J. STAHL, M. KIETZMANN (2012)

The isolated perfused equine distal limb as an ex vivo model for pharmacokinetic studies

Journal of Veterinary Pharmacology and Therapeutics

Accepted: July 23, 2012. doi: 10.1111/jvp.12001

FRIEBE, M., S. SCHUMACHER, J. STAHL, M. KIETZMANN (2013)

Synovial distribution of “systemically” administered acetylsalicylic acid in the isolated

perfused equine distal limb

BMC Veterinary Research 9, 56

doi: 10.1186/1746-6148-9-56

Poster presentations at scientific meetings

FRIEBE, M., K. BUNTENKÖTTER, S. SCHUMACHER, M. KIETZMANN

Distribution of acetylsalicylic acid into synovia in an ex vivo model of the equine distal limb

Presented at the 12th International Congress of the European Association for Veterinary

Pharmacology and Toxicology,

July 8-12, 2012; Noordwijkerhout, the Netherlands

FRIEBE, M., J. STAHL, S. SCHUMACHER, M. DÜE, M. KIETZMANN

The isolated perfused equine distal limb as a new ex vivo model for pharmacokinetic studies

Presented at the 17th Congress on Alternatives to Animal Testing,

September 5-8, 2012; Linz, Austria

FRIEBE, M., S. SCHUMACHER, K. BUNTENKÖTTER, M. DÜE, M. KIETZMANN

Distribution of acetylsalicylic and salicylic acid into synovia in an ex vivo model of the equine

distal limb

Presented at the 19th International Conference of Racing Analysts and Veterinarians,

September 17-21, 2012; Philadelphia, PA, USA

TABLE OF CONTENT

VII

Table of content

1 INTRODUCTION ..................................................................................................... 13

2 LITERATURE REVIEW ............................................................................................. 15

2.1 Isolated perfused organs in pharmacological research ................................... 15

2.1.1 Perfusion media and systems ............................................................... 15

2.1.2 Viability parameters .............................................................................. 16

2.1.3 Applications........................................................................................... 16

2.2 Substances investigated using the isolated perfused equine distal limb ......... 19

2.2.1 Corticosteroids ...................................................................................... 19

2.2.1.1 General pharmacology ................................................................... 19

2.2.1.2 Duration of action ........................................................................... 20

2.2.1.3 Betamethasone .............................................................................. 21

2.2.1.4 Mechanism of action – genomic effects ......................................... 22

2.2.1.5 Intra-articular use of glucocorticoids ............................................... 22

2.2.1.6 Pharmacokinetics after intra-articular application ........................... 23

2.2.2 Nonsteroidal anti-inflammatory drugs (NSAIDs) ................................... 25

2.2.2.1 Role of prostaglandins in inflammation ........................................... 26

2.2.2.2 Isoforms of cyclooxygenase ........................................................... 26

2.2.2.3 NSAIDs for treating joint disease .................................................... 27

2.2.2.4 Acetylsalicylic and salicylic acid ..................................................... 30

2.2.2.5 Salicylic acid as a threshold substance .......................................... 31

2.3 Determination of an effective concentration in the joint ................................... 32

2.3.1 Cultured equine synovioctes as a model of inflammation ..................... 32

2.3.2 Role of prostaglandin E2 in joint disease ............................................... 33

TABLE OF CONTENT

VIII

3 THE ISOLATED PERFUSED EQUINE DISTAL LIMB AS AN EX VIVO MODEL FOR

PHARMACOKINETIC STUDIES .................................................................................. 34

3.1 Abstract ........................................................................................................... 35

4 SYNOVIAL DISTRIBUTION OF “SYSTEMICALLY” ADMINISTERED ACETYLSALICYLIC ACID IN

THE ISOLATED PERFUSED EQUINE DISTAL LIMB ........................................................ 36

4.1 Abstract ........................................................................................................... 37

5 PRIMARY EQUINE SYNOVIOCYTES IN A MODEL OF INFLAMMATION ............................... 39

5.1 Abstract ........................................................................................................... 39

5.2 Materials and Methods .................................................................................... 39

5.2.1 Cell culture ............................................................................................ 39

5.2.2 Verification of equine synoviocytes ....................................................... 45

5.2.3 In vitro inflammation model with equine synoviocytes ........................... 47

5.2.4 Statistical analysis ................................................................................. 49

5.3 Results ............................................................................................................ 49

5.3.1 Isolation and culture of synoviocytes .................................................... 49

5.3.2 Verification of synoviocytes with immunocytochemistry ........................ 50

5.3.3 In vitro inflammation model ................................................................... 51

6 GENERAL DISCUSSION .......................................................................................... 55

6.1 Model characteristics ...................................................................................... 55

6.2 Betamethasone ............................................................................................... 57

6.3 Acetylsalicylic and salicylic acid ...................................................................... 58

6.4 Outlook ............................................................................................................ 60

7 SUMMARY ............................................................................................................ 63

8 ZUSAMMENFASSUNG ............................................................................................ 65

9 REFERENCES ....................................................................................................... 67

TABLE OF CONTENT

IX

10 APPENDIX ............................................................................................................ 83

10.1 Cell culture experiments ................................................................................ 83

10.1.1 Cell viability ........................................................................................ 83

10.1.2 PGE2 concentrations ......................................................................... 84

10.2 Maren Friebe’s contribution to the manuscripts ............................................ 85

ABBREVIATIONS

X

List of abbreviations

° C Degree Centigrade

µg Microgram

µl Microliter

µm Micrometer

Approx. Approximately

ASA Acetylsalicylic acid

BM Betamethasone

BSA Bovine serum albumin

cm Centimeter

CO2 Carbon dioxide

COX Cyclooxygenase

DMEM Dulbecco’s modified Eagle’s medium

DMSO Dimethyl sulfoxide

E. coli Escherichia coli

EDTA Ethylendiamin tetraacetic acid

ELISA Enzyme-linked immunosorbent assay

e.g. For example

et al. et alii

FCS Fetal calf serum

FEI Fédération Equestre International

Fig. Figure

FITC Fluorescein isothiocyanate

FN Fédération Equestre Nationale, German equestrian foundation

g Gram

GCR Glucocorticoid receptor

h Hour

HPLC High performance liquid chromatography

HPAA Hypothalamic-pituitary-adrenal axis

i.a. Intra-articular

IFHA International Federation of Horseracing Authorities

ABBREVIATIONS

XI

i.m. Intramuscular

i.e. That is

i.v. Intravenous

kg Kilogram

L Liter

LDH Lactate-dehydrogenase

LOD Limit of detection

LOQ Limit of quantification

LPS Lipopolysaccharide

mg Milligram

min Minute

mL Milliliter

mm Millimeter

MP Methylprednisolone

n Number of experiments

ng Nanogram

NSAID Nonsteroidal anti-inflammatory drug

PBS Phosphate buffered saline

PG Prostaglandin

pg Picogram

PLA2 Phospholipase A2

p.o. Per os

RMTC Racing Medication and Testing Consortium

s Second

SA Salicylic acid

s.c. Subcutaneous

SD Standard deviation

SEM Standard error of the mean

Tab. Table

TM Trademark

UV Ultraviolet

FIGURES & TABLES

XII

List of figures

FIGURE 1: Generalized ring structure and numbering of corticosteroids .................... 20

FIGURE 2: The arachidonic acid cascade. ................................................................. 25

FIGURE 3: Chemical structures of acetylsalicylic and salicylic acid. ........................... 30

FIGURE 4: Verification of equine synoviocytes Type B. ............................................. 50

FIGURE 5: Viability testing using the MTS-test ........................................................... 52

FIGURE 6: PGE2 concentration in the culture medium after 24 h of stimulation ......... 54

List of tables

TABLE 1: Viability parameters determined in isolated perfused organs. .................... 18

TABLE 2: Different glucocorticoid esters and their duration of release. ...................... 21

TABLE 3: Studies investigating pharmacokinetic properties of glucocorticoids .......... 24

TABLE 4: NSAIDs currently available in Germany ..................................................... 29

TABLE 5: Ingredients of the DMEM basic medium ..................................................... 40

TABLE 6: Protocol for immunostaining of PGP 9.5..................................................... 46

TABLE 7: Protocol for stimulation and treatment of equine synoviocytes. .................. 48

TABLE 8: Results of the modified MTS-test ............................................................... 83

TABLE 9: PGE2 concentration (ng/mL) in the culture supernatant ............................. 84

INTRODUCTION

13

1 Introduction

Lameness caused by inflammatory or degenerative joint conditions is the most

important source of reduced performance in equine athletes (CARON 2005;

GOODRICH and NIXON 2006). Treatment options include administration of

substances directly into the joint or systemically via the blood circulation

(GOODRICH and NIXON 2006).

In order to investigate how long a drug remains in the joint as well as its duration of

action within the joint, pharmacokinetic and pharmacodynamic studies are needed.

In vivo studies that involve living horses, however, are expensive, elaborate and

ethically questionable. An alternative might be ex vivo models of the organs and

tissues of interest, as they have already been successfully established in other fields

of pharmacological research.

It was the aim of this PhD-project to set up appropriate ex vivo alternatives for

pharmacokinetic as well as pharmacodynamic investigations concerning treatment of

the equine joint. The first two studies in this thesis deal with the ex vivo model of the

isolated perfused equine distal limb that was established to investigate the

distribution of substances administered to a horse’s joint either locally (i.e. via intra-

articular application) or systemically. In the third study, a cell culture model of primary

equine synoviocytes was established as a tool for assessing anti-inflammatory

potency of drugs within a joint. As a first application, those concentrations measured

in the isolated perfused equine distal limb were applied to the cells and examined for

their anti-inflammatory properties.

LITERATURE REVIEW

15

2 Literature review

2.1 Isolated perfused organs in pharmacological research

Ever since RUSSELL and BURCH (1959) introduced their concept of the 3Rs -

replacement, reduction, and refinement - for the use of animals in science, various

kinds of ex vivo or in vitro models have been established in many areas of

pharmacological research. Isolated perfused organs have been introduced because

they hold the advantage of resembling the in vivo situation as closely as possible,

including the preservation of anatomic structures (GRONEBERG et al. 2002). While

isolated perfused organs of laboratory animals have been employed in

pharmacological research for many decades (LANGENDORFF 1895; MILLER et al.

1951; WEISS et al. 1959), organs of larger animals have been introduced in the last

30 years as a useful tool mainly in toxicity testing and absorption studies (RIVIERE

and MONTEIRO-RIVIERE 1991). Since they mostly employ organs from animals

killed for unrelated purposes, a reduction of the number of animals used for research

could be achieved (KIETZMANN et al. 1993; VON BAEYER et al. 1997; BÄUMER et

al. 2002; GROSSE-SIESTRUP et al. 2002). Examples include the isolated perfused

porcine skin flap (RIVIERE et al. 1986), equine skin flap (BRISTOL et al. 1991),

bovine udder (KIETZMANN et al. 1993), bovine uterus (BÄUMER et al. 2002), and

porcine forelimb (WAGNER et al. 2003). PATAN et al. (2009) have investigated

effects of long-term extracorporeal blood perfusion on an isolated equine forelimb

with regard to changes in the laminar tissue of the hoof and used this setup as a

model for studying laminitis induction with endotoxin (PATAN-ZUGAJ et al., 2012).

Apart from ethical reasons for the replacement of animal experiments, it also entails

reduction of costs and labor associated with keeping large animals such as horses.

2.1.1 Perfusion media and systems

Generally, perfusion media can be categorized in two major groups: (diluted) blood

and acellular fluids such as Tyrode or Krebs-Ringer solution. The latter group is

easier to supply but frequently needs substitution with components to increase the

LITERATURE REVIEW

16

colloid osmotic pressure within the vessels, e.g. albumin (RIVIERE et al. 1986),

bovine serum albumin (BRISTOL et al. 1991) or dextran (KIETZMANN et al. 1993).

Blood is diluted and needs anticoagulation in order to be usable (BÄUMER et al.

2002; WAGNER et al. 2003; PATAN et al. 2009). It holds the advantage of

resembling the in vivo situation even more closely but is often hard to obtain,

especially if autologous blood is desired. Furthermore, it is more demanding to

handle with regard to oxygen supply and because of blood clotting and hemolysis.

Perfusion systems are either closed, with the venous perfusate being recirculated, or

open, meaning that the venous perfusate is disposed of. The former type consumes

less perfusion medium while it is more challenging to restore the perfusion fluid for

recirculation.

2.1.2 Viability parameters

The monitoring of vitality is essential when working with isolated perfused organs

(WARD and BUTTERY 1979) to ensure sufficient tissue viability and to determine the

maximum timespan for which the organ can be used. To do so, various biochemical,

physiological and morphological endpoints are usually looked at (RIVIERE and

MONTEIRO-RIVIERE 1991). TABLE 1 summarizes viability parameters analyzed in

several studies implementing isolated perfused organs.

2.1.3 Applications

As indicated above, isolated perfused organs have mainly been used for toxicity

testing and absorption studies. Examples include percutaneous absorption studies of

organophosphates, steroids, benzoic acid, caffeine, dexamethasone, benzoyl

peroxide, etofenamate, nitroglycerin, and estradiol as well as mucous membrane

irritation and - inflammation studies (CARVER et al. 1989; WILLIAMS et al. 1990;

KIETZMANN et al. 1993; BÄUMER et al. 2002; WAGNER et al. 2003). It has to be

emphasized that these ex vivo models were not designed to completely replace

animal experiments but to reduce them.

LITERATURE REVIEW

17

Likewise, pathophysiological mechanisms can be studied using isolated perfused

organs: PATAN-ZUGAJ et al. (2012) demonstrated that an exposure to LPS led to

significant laminitis-like changes in the laminar tissue as well as to metabolic

changes in their model of the isolated perfused equine distal limb.

LITERATURE REVIEW

18

TABLE 1: Viability parameters determined in isolated perfused organs.

Viability parameter Indicative of Author

Glucose consumption Glucose metabolism (RIVIERE et al. 1986; BRISTOL et al.

1991; KIETZMANN et al. 1993;

BÄUMER et al. 2002; WAGNER et al.

2003; PATAN et al. 2009)

Lactate production Anaerobic conditions (RIVIERE et al. 1986; BRISTOL et al.


BÄUMER et al. 2002; WAGNER et al.

2003; PATAN et al. 2009)

LDH activity Cell destruction (RIVIERE et al. 1986; KIETZMANN et

al. 1993; BÄUMER et al. 2002;

PATAN et al. 2009)

Blood gas analysis Oxygen supply (PATAN et al. 2009)

Free hemoglobin Hemolysis (WAGNER et al. 2003; PATAN et al.

2009)

Vascular resistance Functionality (RIVIERE et al. 1986; BRISTOL et al.

1991; WAGNER et al. 2003)

Venous perfusate pH Anaerobic conditions (RIVIERE et al. 1986; KIETZMANN et

al. 1993; PATAN et al. 2009)

Skin surface temperature Peripheral blood

supply

(KIETZMANN et al. 1993)

Weight increase Edema formation (BRISTOL et al. 1991; KIETZMANN et

al. 1993; BÄUMER et al. 2002;

PATAN et al. 2009)

Skin fold thickness Edema formation (KIETZMANN et al. 1993)

Histological examination

Morphological

alterations

(RIVIERE et al. 1986; BRISTOL et al.


WAGNER et al. 2003; PATAN et al.

2009)

LDH = Lactate dehydrogenase, Italics: Hemoperfused organs

LITERATURE REVIEW

19

2.2 Substances investigated using the isolated perfused equine distal limb

In the study presented, we chose to investigate the distribution of two representatives

of those substance classes that are commonly used for the treatment of inflammatory

joint disease in horses, namely glucocorticoids and nonsteroidal anti-inflammatory

drugs (LEES 2003). These representatives were betamethasone and (acetyl-)

salicylic acid.

2.2.1 Corticosteroids

2.2.1.1 General pharmacology

Corticosteroids are 21-carbon steroid hormones composed of four rings that are

synthesized in the adrenal cortex from cholesterol. They can be further divided into

two groups: mineralocorticoids, mainly responsible for regulating water and

electrolyte balance, and glucocorticoids, which have an effect on various endocrine

systems in the body.

Glucocorticoids influence the intermediary metabolism by increasing liver glycogen

synthesis and storage, gluconeogenesis, lipolysis and redistribution of lipids. They

may also stimulate the central nervous system and have various effects on the

cardiovascular system, such as increasing myocardial contractions and the

expression of α-adrenergic receptors in the vascular smooth muscle and β-

adrenergic receptors in the myocardium. Glucocorticoids also decrease capillary

permeability and affect the respiratory system by increasing expression of β2–

receptors, which in turn leads to bronchodilatation. Effects on blood cells and

lymphoid tissues include an increase in the number of circulatory erythrocytes,

neutrophils, monocytes and platelets whereas the number of circulatory lymphocytes,

eosinophils and basophils is decreased (HSU 2008).

In therapeutic doses, glucocorticoids exhibit anti-inflammatory and anti-allergic

properties which are linked to the suppression of immune responses: leukocyte

migration and function are suppressed, and plasma and lysosomal membranes are

stabilized, attributable to a decrease in phospholipase A2 (PLA2) activity. As depicted

in FIGURE 2, PLA2 converts phospholipids into arachidonic acid, a precursor of

LITERATURE REVIEW

20

eicosanoids and therefore, synthesis of proinflammatory derivatives such as

prostaglandins, bradykinins, histamines and leukotrienes is suppressed (CREAMER

1999). Since bradykinin and histamine can cause pain by directly stimulating primary

afferent nociceptive fibers, and prostaglandins and leukotrienes have been shown to

sensitize nociceptors, reduction of these mediators also explains the analgesic

effects of glucocorticoids (HAMEED and IHM 2012).

2.2.1.2 Duration of action

Glucocorticoids are traditionally categorized according to the duration of their

hypothalamic-pituitary-adrenal axis - suppression. Hydrocortisone, cortisone, and

prednisolone are representatives of the short-acting (< 24 h) glucocorticoids,

triamcinolone belongs to the intermediate-acting (24 - 48 h) group, and

flumethasone, dexamethasone as well as BM are examples of long-acting (> 48 h)

glucocorticoids (UNGEMACH 2006). However, some studies suggest that the HPAA-

suppression should not be taken as an indicator for duration of pharmacological

effect (KLAUS and HAPKE 1996). Apart from the chemical structure, the formulation

FIGURE 1: Generalized ring structure and numbering of corticosteroids (left). Structure

of cortisol (top right) and betamethasone (bottom right).

LITERATURE REVIEW

21

influences the duration of action: steroid esters are considered prodrugs because the

active moiety is the free alcohol or steroid base resulting from hydrolysis

(AUTEFAGE et al. 1986; WRIGHT et al. 1986). Esterification of the alcohol at C21

significantly determines the drug’s characteristics such as the water/lipid solubility

ratio and the duration of action since tissue esterases cleave the ester to release the

free and therapeutically active base (FERGUSON et al. 2009). Examples for duration

of release are given in TABLE 2.

TABLE 2: Different glucocorticoid esters and their duration of release. Modified from

UNGEMACH (2006) .

Prodrug / Ester Duration of release

Phosphate Rapid

Hemisuccinate

Acetate 2-14 days

Diacetate

Pivalate Weeks to months

2.2.1.3 Betamethasone

The glucocorticoid betamethasone was synthesized through selective modification of

the endogenous glucocorticoid cortisol: an extra double bond was included between

C1 and C2, a methyl group was added at position C16, and a 9-α-fluoro group was

added to C9 (see FIGURE 1). These changes have increased the glucocorticoid

potency in comparison with hydrocortisone 30-fold whereas the mineralocorticoid

potency was reduced to virtually zero (FERGUSON et al. 2009).

BM belongs to the group of long-acting glucocorticoids, with a HPAA - suppression

between 36 – 72 h (UNGEMACH 2006). In Germany, BM is currently available as

Celestovet® (12.0 mg BM-21-acetate and 3.9 mg BM-21-disodiumphosphate) for

therapeutic use in horses and dogs (VETIDATA 2012). However, the preparation

used in this study was Celestan® solubile (5.3 mg BM-21-disodiumphosphate)

LITERATURE REVIEW

22

because it is known that its active agent BM-21-disodiumphosphate is used as a

rapid-acting glucocorticoid in sport horses.

2.2.1.4 Mechanism of action – genomic effects

Like other steroid hormones, glucocorticoids act by altering mRNA synthesis. After

diffusion through the cell membrane, they bind to cell receptors (GCRs), which enter

the cell nucleus and modulate the expression of target genes. One important

mechanism for anti-inflammatory and immunosuppressive effects is the

aforementioned inhibition of PLA2 via the steroid-inducible group of proteins called

lipocortin. PLA2 is the enzyme responsible for the release of arachidonic acid from

cell membranes before its further metabolization by the cyclooxygenase (COX) and

lipoxygenase (LOX) pathways (FERGUSON et al. 2009). Together with their potent

inhibition of the protein NF-κB, which is essential for enhancing inflammatory

cytokine production (BOUMPAS and WILDER 2001), the formation of prostaglandins,

leukotrienes, and platelet activating factor is inhibited. Genomic effects are generally

observed after at least 30 min because of the time needed for GCR activation,

transcription, and translation (FERGUSON et al. 2009). GCRs are present in

neutrophils, lymphocytes, monocytes, and eosinophils (AXELROD 1993).

2.2.1.5 Intra-articular use of glucocorticoids

Glucocorticoids are commonly injected intra-articularly in horses to minimize pain and

inflammation with joint disease (TROTTER et al. 1991). The use of hydrocortisone for

treatment of musculoskeletal conditions in cattle and horses was first described by

WHEAT (1955). Local, i.e. intra-articular, glucocorticoid therapy has the advantage of

directly targeting the inflamed area even with doses much lower compared with

systemic administration (DERENDORF et al. 1986; HARKINS et al. 1993;

FERGUSON et al. 2009). Glucocorticoids frequently used for intra-articular treatment

include different esters of BM, dexamethasone, methylprednisolone, and

triamcinolone (YARBROUGH 2004). Glucocorticoid activity is dependent upon the

presence of a hydroxyl group at C11, therefore, cortisone and prednisone have to be

LITERATURE REVIEW

23

converted to cortisol and prednisolone in the liver in order to be biologically active

(TROTTER 1996). Hence, glucocorticoid preparations for intra-articular use are 11-β-

hydroxyl compounds that do not require biotransformation. Traditionally, the duration

of action after intra-articular injection has been assumed to be inversely correlated

with the glucocorticoid preparation’s solubility in water: water-soluble preparations

such as phosphate and succinate esters are thought to be short-acting, whereas

more lipid-soluble esters such as acetate and acetonide have a longer duration of

action because they are absorbed with a delay (GRAY and GOTTLIEB 1983;

AUTEFAGE et al. 1986). Other determinants for the duration of action include the

rate of hydrolysis of the drug by synovial tissue esterases and the binding affinity of

the glucocorticoid to the steroid receptor in the cytoplasm of target cells (WRIGHT et

al. 1986).

2.2.1.6 Pharmacokinetics after intra-articular application

Results of studies that have investigated the distribution of glucocorticoids after intra-

articular injection to horses are listed in TABLE 3. Some preparations used consist of a

mixture of a rapid-acting solution and a more long acting suspension. However,

because of the high solubility associated with some of the short-acting solutions, it

has been suggested that they might be cleared from the joint very quickly and may

therefore be an unnecessary component (AUTEFAGE et al. 1986; WRIGHT et al.

1986).

LIT

ER

AT

UR

E R

EV

IEW

TABLE 3: Studies investigating pharmacokinetic properties of glucocorticoids after intra-articular administration to horses.

Authors Number

of horses

Joint

injected

Glucocorticoid

and dose

administered

Ester used Detection

time in

plasma

Duration of

cortisol

suppression [h]

Detection

time in urine

(AUTEFAGE

et al. 1986)

5 Tibiotarsal

joint

100 mg MP MP acetate 24 h 3 d n.i.

(LILLICH et al.

1996)

4

4

Tarsocrural

joint

100 mg

100 mg

MP acetate

Isoflupredone acetate

12 h

12 h

n.i. 72 h

22 h

(POPOT et al.

2002)

2 8 mg BM BM phosphate n.i. 36 48 h

(VINE 2006) 2 Carpal joint 11.4 mg BM BM phosphate + acetate 24 h 72 72 h

(SOMA et al.

2006)

6 Carpal joint 200 mg MP acetate > 144 h 144 n.i.

(MACHNIK et

al. 2007)

10 27 - 70 mg BM BM phosphate

BM acetate

n.i. n.i. 35 - 45 d

(SOMA et al.

2011)

6 Carpal joint 0.04 mg/kg Triamcinolone acetonide > 96 h > 96 n.i.

(LÖVENICH

2012)

6

4

Fetlock joint 4 mg BM

34.6 mg BM

BM phosphate

BM phosphate + acetate

48 - 168 h

4 d

48

48 - 384

71 - 145 h

10 d

24

BM Betamethasone, MP Methylprednisolone, n.i. not investigated

LITERATURE REVIEW

25

GLUCOCORTICOID

2.2.2 Nonsteroidal anti-inflammatory drugs (NSAIDs)

The term NSAID describes a rather broadly defined group of anti-inflammatory

substances that inhibit some component of the enzyme system that converts

arachidonic acid into prostaglandins and thromboxanes (HIGGINS and LEES 1984).

In contrast to steroidal anti-inflammatory drugs, they intervene at a later step by

inhibiting the two isoforms of the enzyme cyclooxygenase (COX) as depicted in

FIGURE 2.

PGG2

Unstable cyclic

endoperoxides

Prostacyclin

synthase LTB4 LTC

4

LTD4

LTE4

PG = Prostaglandin TX = Thromboxane LT = Leukotriene HPETE = Hydroperoxyeicosatetraenoic acid HETE = Hydroxyeicosatetraenoic acid = Inhibition

Cysteinyl

glycanase

δ-glutamyl

transpeptidase

LTA4

Hydrolase

Dehydrase

Glutathione

transferase

5-HETE 5-HPETE

Glutathione

peroxidase

PGH2

PGE2 PGI

2 TXA

2

Endoperoxide

isomerase

Thromboxane

synthase

Cell membrane phospholipid

Arachidonic acid

Phospholipase A2

Cyclooxygenase 5-Lipoxygenase

FIGURE 2: The arachidonic acid cascade. Modified from MAY and LEES (1999).

NSAID

LITERATURE REVIEW

26

2.2.2.1 Role of prostaglandins in inflammation

Arachidonic acid plays a pivotal role in inflammation as the precursor of the

eicosanoid group of mediators (see FIGURE 2). It is a component of cell membrane

phospholipid, which is released in tissue damage following activation of

phospholipase A2. After its release from the cell membrane phospholipid, arachidonic

acid serves as a substrate not only for cyclooxygenase (COX), of which there are the

two isoforms COX-1 and COX-2, but also for several lipoxygenases. Each enzyme is

part of a cascade, in which the action of further enzymes leads to the formation of

many inflammatory mediators of the eicosanoid family. The mediators are short-lived,

so that continued effect depends on maintained synthesis and release. COX

catalyzes both the formation of PGG2 and then PGG2 conversion to PGH2 via a

peroxidase function. Prostaglandins play an important role as inflammatory mediators

but also participate in synergisms: primary inflammatory mediators such as histamine

and bradykinine stimulate nociceptors to increase the discharge in afferent nerves so

that pain is sensed in spinal and brain centers (LEES 2009). Prostaglandins, e.g.

PGE2, increase intensity and duration of this afferent discharge, causing hyperalgesia

and allodynia (HSU and KANTHASAMY 2008; LEES 2009). PGE2 is an endogenous

pyrogen that leads to upward resetting of the temperature regulating center in the

anterior hypothalamus. Furthermore, prostaglandins enhance edema formation

mainly caused by primary mediators such as histamine and bradykinin by dilating

small arterioles (LEES 2009).

2.2.2.2 Isoforms of cyclooxygenase

COX-1 is a membrane-bound enzyme. It first cyclizes arachidonic acid to form PGG2

and adds a 15-hydroperoxy group to convert PGG2 to PGH2. COX-1 is expressed

constitutively in many tissues and in blood platelets. It is involved in “housekeeping”

functions such as blood clotting, regulation of vascular homeostasis, renoprotection,

gastroprotection, and coordination of the actions of circulating hormones (LEES

2009). COX-2 is both an inducible and a constitutive isoform that is encoded by a

different gene than COX-1, but it has a 60 % structure homology with COX-1 at the

LITERATURE REVIEW

27

amino acid level (HSU and KANTHASAMY 2008). Induction of COX-2 synthesis is

stimulated by proinflammatory cytokines, growth factors, and lipopolysaccharide as

well as mitogens, but it is also constitutively expressed in monocytes, macrophages,

endothelial cells, brain, spinal cord, ovary, uterus, and ciliary body in the eye (LEES

2009). COX-2 is a better competitor than COX-1 for arachidonic acid released within

the cell and produces both pro- and anti-inflammatory prostaglandins at sites of

inflammation (GILROY et al. 1999; LEES 2009). While most data support COX-2 as

the isoform which generates prostaglandins at sites of inflammation, some findings

also indicate a role for COX-1 (SMITH et al. 1998). Many NSAIDs, including

acetylsalicylic acid and indomethacin, are more effective at inhibiting COX-1 than

COX-2 while others, such as ibuprofen and meclofenamic acid, are equipotent at

inhibiting both isoforms (MEADE et al. 1993; MITCHELL et al. 1993). It has been

proposed that this inhibition of the constitutively produced COX-1 is responsible for

many adverse effects of NSAIDs, including gastric ulceration, renal function

impairment and platelet dysfunction (VANE and BOTTING 1995). In an attempt to

decrease these side-effects, new compounds that more selectively inhibit the COX-2

isoform have been developed (LEES 2009). Although numerous COX-2 selective

products are available for use in humans, few of these compounds have been

studied in veterinary species. Further complicating the issue, it appears that enzyme

selectivity is very species dependent (KOLLIAS-BAKER and COX 2004). For

example, carprofen has been reported to be a selective COX-2 inhibitor in the dog

but this selectivity has not been confirmed in the horse (VANE and BOTTING 1995;

RICKETTS et al. 1998).

2.2.2.3 NSAIDs for treating joint disease

The administration of NSAIDs began over 100 years ago with the introduction of

salicylic acid for the treatment of rheumatic disease (BRUNE and BECK 1991).

Nowadays, they are the most commonly prescribed agents for use in horses, with

musculoskeletal pain and inflammation being the most widespread indications for

chronic administration (KOLLIAS-BAKER and COX 2004). Their ability to inhibit the

arachidonic acid cascade has been a mainstay for the treatment of joint disease for

LITERATURE REVIEW

28

many years (FRISBIE 2011). Analgesic and antiphlogistic properties are mediated

through the inhibition of prostaglandin and thromboxane production, which are

intimately involved in pain, altered cartilage metabolism and ongoing inflammation in

damaged joints. It has also been suggested that NSAIDs may play a role in

mediating joint pain at the level of the spinal cord (MALMBERG and YAKSH 1992),

even though this has not yet been investigated in the horse.

Clinically recommended routes of administration include the oral, intravenous,

intramuscular, and subcutaneous injection. Substances used for the treatment of

equine joint disease are listed in TABLE 4.

LITERATURE REVIEW

29

TABLE 4: NSAIDs currently available in Germany for treating musculoskeletal pain in

horses. Note that drugs in italics are not available for food-producing horses.

Drug Route of

administration

Recommended dosage

Firocoxib i.v.

p.o.

0.09 mg/kg as initial treatment

0.1 mg/kg s.i.d. up to 14 d (PLUMB 2008)

Flunixin

meglumine

i.m., i.v.

p.o.

1.1 mg/kg (SOMA et al. 1988; LANDONI and LEES 1995);

0.5 - 2.0 mg/kg (TOUTAIN et al. 1994)

1.1 mg/kg (LANDONI and LEES 1995)

Ketoprofen i.m., i.v. 2.2 mg/kg s.i.d. for 5 days (OWENS et al. 1995)

Meloxicam i.m., i.v., s.c.

p.o.

0.6 mg/kg as initial treatment

0.6 mg/kg s.i.d. up to 14 d (LÖSCHER et al. 2006)

Metamizol i.m., i.v. 5.0 - 22.0 mg/kg (J.J. BERTONE and HORSPOOL 2004)

Phenylbutazone i.v.

p.o.

4.4 mg/kg (LEES et al. 1987b; TOUTAIN et al. 1994; OWENS et

al. 1996); 4.4 mg/kg then 2.2 mg/kg (RAEKALLIO et al. 1997)

4.4 mg/kg b.i.d. for 1 day, 2.2 mg/kg b.i.d. for 4 days; 2.2 mg/kg

s.i.d. for 7 days (TAYLOR et al. 1983; LEES et al. 1986)

Suxibuzone p.o. 6.25 mg/kg b.i.d. for 2 d, then 3.1 mg/kg b.i.d. for 3 d, then 3.1

mg/kg q48h (BISHOP 2005)

Vedaprofen p.o. 2 mg/kg initially, then 1 mg/kg b.i.d. for up to 14 d

(J.J. BERTONE and HORSPOOL 2004)

i.m. intramuscular, i.v. intravenous, s.c. subcutaneous, p.o. orally, s.i.d. once daily, b.i.d.

twice daily, q48h every 48 h

LITERATURE REVIEW

30

2.2.2.4 Acetylsalicylic and salicylic acid

Acetylsalicylic acid is one of the oldest known NSAIDs and the prototype of the

salicylate drugs (COLLIER 1971). It elicits its effect via acetylation and irreversible

inhibition of COX-1, resulting in decreased prostaglandin synthesis and anticoagulant

effects due to the blockade of thromboxane A2 production (HSU and KANTHASAMY

2008). In higher concentrations, it also irreversibly inhibits COX-2 (LEES 2009). ASA

is rapidly deacetylated to SA in virtually all body tissues and blood by endogenous

esterases (KIETZMANN et al. 2002; BROOME et al. 2003).

In horses, the half-life of ASA after intravenous administration is reported to be as

short as 5-10 min (LEES et al. 1987a). Despite this rapid clearance, ASA is very

effective as an antithrombotic agent: relatively small doses can significantly prolong

bleeding times. This prolongation is due to the irreversible inhibition thromboxane A2

in platelets. Since platelets do not contain nuclei, this anticoagulant effect cannot be

reversed until new platelets are formed (KIETZMANN et al. 2002; LEES 2009). As an

analgesic or anti-inflammatory agent, large amounts of ASA have to be administered

because of its very short half-life, rendering its oral application rather impractical

(MAY and LEES 1996). Therefore, ASA is almost exclusively used for the treatment

and prevention of diseases that have arterial thrombosis as a part of their

pathogenesis, e.g. thromboembolic colic or disseminated intravascular coagulation

(BROOME et al. 2003).

FIGURE 3: Chemical structures of acetylsalicylic and salicylic acid.

LITERATURE REVIEW

31

2.2.2.5 Salicylic acid as a threshold substance

Formulating threshold levels is necessary for those substances that a horse is

frequently, but inadvertently exposed to and that may e.g. be a natural part of the

horse’s diet. Without a threshold level, any detection of the substance would have to

be reported as a violation of medication rules. On the other hand, there are also

threshold levels for endogenous substances, an example would be testosterone, for

which different threshold levels exist for geldings and mares (TOUTAIN 2010b). It is

important that the concentration of the substance below the threshold level is not a

threat to the horse’s welfare, does not alter its performance nor compromise the

integrity of the sport (TOUTAIN 2010a).

There are differences between the equestrian federations concerning not only the

concentrations at which threshold levels are fixed but also the threshold substances

as such. An example is dimethyl sulfoxide (DMSO), for which the international

federation for equestrian sports (FEI) and the international federation of horseracing

authorities (IFHA) have established a threshold, whereas this is not the case in the

regulations of the racing medication and testing consortium (RMTC), the national

horseracing authority of the United States (TOUTAIN 2010b).

Since SA naturally occurs in certain plants used as horse feed (such as alfalfa hay), it

may be a component of horse urine (BEAUMIER et al. 1983). Therefore, the rule in

equine sports, according to which neither a drug nor its metabolite may be detectable

in blood or urine during competition, cannot be applied (ANONYMUS 2012). In order

to investigate the normal concentration of SA in horse urine and to formulate

threshold levels, two research groups independently conducted feeding experiments

and analyzed urine samples (BEAUMIER et al. 1983; LAKHANI et al. 2004).

BEAUMIER et al. (1983) postulated a urine threshold concentration of 750 µg/mL,

whereas LAKHANI et al. (2004) suggested the limit of 625 µg/mL. The latter

concentration was adapted by the FEI and the German equestrian federation (FN).

Later, in an attempt to harmonize SA threshold levels between different horse sport

organizations, the FEI has raised the original threshold levels of 625 µg/mL (urine)

and 5.4 µg/mL (plasma) to 750 µg/mL and 6.5 µg/mL, respectively (ANONYMUS

2010). Threshold levels are now aligned with those of the IFHA, whereas the FN has

LITERATURE REVIEW

32

maintained its original concentrations of 625 µg/mL (urine) and 5.4 µg/mL (plasma)

(ANONYMUS 2011).

2.3 Determination of an effective concentration in the joint

Drug effects can be readily assessed using in vitro or in vivo pharmacodynamic

models (KAMERLING and OWENS 1994). Classically, the effective dose of an

antiphlogistic drug is determined from a dose-titration study by use of an appropriate

experimental method of inducing inflammation.

2.3.1 Cultured equine synovioctes as a model of inflammation

In living horses, intra-articular lipopolysaccharide (LPS) injection is an established

model for induction of transient sterile inflammation. It has been used to study e.g.

clinical symptoms, drug pharmacokinetics and the effect of therapeutic intervention

(PALMER and BERTONE 1994a; MORTON et al. 2005; DE GRAUW et al. 2009;

MEULYZER et al. 2009). Also in vitro, cultured equine synoviocytes synthesize large

amounts of PGE2 after stimulation with LPS (LANDONI et al. 1996; MOSES et al.

2001; BRISTON et al. 2009), which is regarded as a surrogate effect of inflammation

(TOUTAIN and CESTER 2004). Because of their importance in the pathogenesis of

osteoarthritis, synoviocytes are considered to be a target for therapeutic activity in

horses (BYRON et al. 2008). This has led to numerous studies investigating anti-

inflammatory properties of substances in the joint, using cultured synoviocytes as an

in vitro model for articular inflammation (LANDONI et al. 1996; FREAN and LEES

2000; BYRON et al. 2008).

Synoviocytes can be divided into two groups: type A cells, or macrophagic cells, and

type B cells, or fibroblast-like cells. The former cells are non-fixed and can

phagocytose cell debris and wastes in the joint cavity; furthermore, they possess the

ability to present antigen. Type B synoviocytes are regarded as proper synoviocytes;

their dendritic processes form a regular network on the luminal surface of the

synovial membrane (IWANAGA et al., 2000). They are responsible for production of

LITERATURE REVIEW

33

both synovial fluid and the extracellular matrix in the synovial intima (KITAMURA et

al. 1999).

2.3.2 Role of prostaglandin E2 in joint disease

Joint disease in the horse is commonly characterized by inflammation of the

synovium (OWENS et al. 1996), involving synoviocytes producing pro-inflammatory

cytokines (DINGLE et al. 1979; SAKLATVALA and DINGLE 1980),

metalloproteinases (OKADA et al. 1992), and eicosanoids (MAY et al. 1989;

LINDSLEY and SMITH 1990; HULKOWER et al. 1993). Of the latter group,

prostaglandin E2 (PGE2) has been identified as an important mediator of

inflammation and hyperalgesia through its enhancement of vascular permeability,

vasodilatory properties, and sensitization of joint nociceptors (HIGGINS et al. 1987;

PALMER and BERTONE 1994b; MAY and LEES 1996). In the joint, synovial tissue

is believed to be the major source of PGE2 in synovial fluid (WITTENBERG et al.

1993). In horses, increased PGE2 concentrations are present in synovial fluid with a

range of joint pathologies (MAY et al. 1994; KIRKER-HEAD et al. 2000). BERTONE

et al. (2001) reported PGE2 to be a good to excellent marker of acute and chronic

joint disease in horses.

THE ISOLATED PERFUSED EQUINE DISTAL LIMB

34

3 The isolated perfused equine distal limb as an ex vivo model for

pharmacokinetic studies

Maren Friebe, Jessica Stahl, Manfred Kietzmann

Department of Pharmacology, Toxicology and Pharmacy; University of Veterinary

Medicine Hannover, Foundation, Hannover, Germany

Accepted for publication in: Journal of Veterinary Pharmacology and Therapeutics

Accepted: July 23, 2012. doi: 10.1111/jvp.12001

Published online: August 23, 2012

THE ISOLATED PERFUSED EQUINE DISTAL LIMB

35

3.1 Abstract

Even though intra-articular treatment plays an important role for the treatment of

joint-related lameness in horses, little is known about pharmacokinetic properties of

substances used. Therefore, an ex vivo model for pharmacokinetic studies was

developed using distal forelimbs of slaughtered horses. The extremity was perfused

with gassed tyrode solution for up to 8 h; tissue viability was confirmed by

measurements of glucose consumption, lactate production and lactate

dehydrogenase activity in the perfusate. Standard criteria for tissue viability had been

determined in preliminary experiments (n=11) which also included histological

examinations of the joint capsule. As the model’s first implementation, the articular

efflux rate of betamethasone (BM), administered as betamethasone disodium

phosphate intra-articularly to the fetlock joint (4 mg BM / joint), was investigated. The

concentration of BM in the venous perfusate of the radial vein was measured by

means of high performance liquid chromatography. The average BM efflux rate per

minute was calculated to be 5.1 µg/min with values ranging from 9 µg/min to

2.9 µg/min. 7.5 h after i.a.-application, 2.3 mg BM had left the joint via the radial vein.

Using this inexpensive setup, the model presented allows studying a variety of

pharmacological topics without the ethical limitations of animal studies.

KEYWORDS: Isolated perfused equine distal limb, betamethasone, intra-articular

injection, articular efflux

SYNOVIAL DISTRIBUTION OF “SYSTEMICALLY” ADMINISTERED ACETYLSALICYLIC ACID

36

4 Synovial distribution of “systemically” administered

acetylsalicylic acid in the isolated perfused equine distal limb

Maren Friebe, Stephan Schumacher , Jessica Stahl, Manfred Kietzmann (2013)

Department of Pharmacology, Toxicology and Pharmacy; University of Veterinary

Medicine Hannover, Foundation, Bünteweg 17, 30559 Hannover, Germany

BMC Veterinary Research 9, 56

doi: 10.1186/1746-6148-9-56

SYNOVIAL DISTRIBUTION OF “SYSTEMICALLY” ADMINISTERED ACETYLSALICYLIC ACID

37

4.1 Abstract

Background

This study investigated synovial concentrations of acetylsalicylic acid (ASA) and its

metabolite salicylic acid (SA) in the equine fetlock joint following systemic

administration of ASA. Salicylates were chosen because SA is the only nonsteroidal

anti-inflammatory drug for which threshold levels exist for plasma and urine in equine

sports. To avoid animal experiments, the study was conducted using an ex vivo

model of the isolated perfused equine distal limb in combination with plasma

concentrations obtained from literature. Salicylate concentrations in the joint were

determined using microdialysis and high performance liquid chromatography (HPLC).

Any anti-inflammatory effect of synovial ASA concentrations was assessed using an

ASA EC50 (half maximal effective concentration) determined in equine whole blood.

Results

The ASA concentration in the synovial fluid (n=6) reached a maximum of 4 µg/mL,

the mean concentration over the entire perfusion period was 2 µg/mL. Maximum SA

concentration was 17 µg/mL, the average was 14 µg/mL. ASA and SA concentration

in the synovial fluid exceeded systemic concentrations 2 h and 3.5 h after “systemic”

administration, respectively.

Conclusions

ASA and SA accumulated in the in the synovial fluid of the ex vivo model despite

decreasing systemic concentrations. This suggests a prolonged anti-inflammatory

effect within the joint that remains to be further elucidated.

Keywords

Acetylsalicylic acid, salicylic acid, isolated perfused equine distal limb, synovial fluid,

horse, microdialysis

PRIMARY EQUINE SYNOVIOCYTES IN A MODEL OF INFLAMMATION

39

5 Primary equine synoviocytes in a model of inflammation

5.1 Abstract

In order to test anti-inflammatory effects of substances, cultured synoviocytes in vitro

are a common approach (LANDONI et al. 1996; ARMSTRONG and LEES 2002).

Prostaglandin E2 (PGE2) has been reported to be an important mediator of

inflammation and hyperalgesia (MAY et al. 1994; PALMER and BERTONE 1994b).

Also, it has been demonstrated that cultured equine synoviocytes synthesize large

amounts of PGE2 when stimulated with LPS in vitro (LANDONI et al. 1996). To

assess the anti-inflammatory potency of substances, cells were stimulated with LPS

and either treated with decreasing concentrations of betamethasone, acetylsalicylic

acid, or salicylic acid. The resulting PGE2 production was quantified and compared

with the untreated vehicle group. The lowest concentrations capable of significantly

suppressing PGE2 production were 2.55x10-9 mol/L for BM and 7.77x10-6 for ASA.

For SA, a decrease in PGE2 production was present with increasing SA

concentration, but this decrease was not statistically significant. Results of this study

help to range in the anti-inflammatory effects of intra-articular drug concentrations

measured in the ex vivo model of the isolated perfused equine distal limb.

5.2 Materials and Methods

5.2.1 Cell culture

Culture media

The basic culture medium used for the primary equine synoviocytes was Dulbecco’s

modified Eagle’s medium (DMEM; PAA, Pasching, Germany). All ingredients of this

basic medium are listed in TABLE 5.


40

TABLE 5: Ingredients of the DMEM basic medium [mg/mL].

Calcium Chloride anhydrous

Ferric(III)-Nitrate • 9H2O

Potassium Chloride

Magnesium Sulphate anhydrous

Sodium Chloride

Sodium Dihydrogen Phosphate • 2 H2O

Sodium Hydrogen Carbonate

L-Arginine • HCl

L-Cystine

L-Glutamine

Glycine

L-Histidine • HCl • H2O

L-Isoleucine

L-Leucine

L-Lysine • HCl

L-Methionine

L-Phenylalanine

200.00

0.10

400.00

97.70

6400.00

141.31

3700.00

84.00

48.00

584.00

30.00

42.00

105.00

105.00

146.00

30.00

66.00

L-Serine

L-Threonine

L-Tryptophan

L-Tyrosine

L-Valine

D-Calcium-Pantothenate

Choline Chloride

Folic Acid

Myo-Inositol

Nicotinamide

Pyridoxal • HCl

Riboflavin

Thiamine • HCl

D-Glucose anhydrous

Phenol Red

Sodium Pyruvate

42.00

95.00

16.00

89.47

94.00

4.00

4.00

4.00

7.20

4.00

4.00

0.40

4.00

1000.0

15.00

110.00

The basic medium was substituted for different purposes as follows:

1. DMEM regular

DMEM basic culture medium

10 % heat inactivated fetal calf serum

50 IU/mL penicillin

50 µg/mL streptomycin

2. Cryomedium

DMEM basic culture medium

20 % heat inactivated fetal calf serum

50 IU/mL penicillin

50 µg/mL streptomycin

10 % glycerol


41

Substances

Heat inactivated fetal calf serum

EDTA (Versen)

Trypsin

Penicillin

Streptomycin

Glycerol

DMSO

Betamethasone

LPS (E.coli, O111:B4)

Collagenase, type VIII from

Clostridium histolyticum

Trypan blue

PAA Laboratories, Pasching, Germany

Biochrom AG, Berlin, Germany

Biochrom AG, Berlin, Germany



Merck, Darmstadt, Germany

Sigma-Aldrich, Steinheim, Germany





Disposable materials

6-mm biopsy punch

96-well tissue culture plate

6-well tissue culture plate

25 cm2 cell culture flask, 50 mL

Sterile cell scraper

Scalpel blade

Minisart® filter unit

Cryovials, 1 mL

kai Europe, Solingen, Germany

Greiner BIO-ONE, Frickenhausen, Germany



TPP, Omnilab, Mettmenstetten, Germany

Bayha, Tuttlingen, Germany

Millipore, Carrigtwohill, Ireland


Cell culture equipment

Incubator CO2 -auto-zero

Centrifuge

Sterile work bench

Phase contrast microscope

Freezing container

Neubauer counting chamber

Heraeus-Kulzer, Hanau, Germany

5804 R, Eppendorf, Hamburg, Germany

LaminAir HLP 2472, Heraeus-Kulzer, Hanau, Germany

Axiovert 25, Zeiss, Oberkochen, Germany

"Mr. Frosty", Sigma-Aldrich, Steinheim, Germany

VWR International, Darmstadt, Germany


42

Prostaglandin measurement

Prostaglandin E2 Express EIA Kit -

Monoclonal

Cayman Chemical, Ann Arbor, MI, USA

Substances and materials for immunocytochemistry

Methanol

Acetone

Bisbenzimide

Primary antibody

Secondary antibody

Microscope slides

Mounting medium

Cover slips

Fluorescence microscope

AppliChem, Darmstadt, Germany



Mouse-anti-human protein gene product 9.5 IgG;

Biozol, Eching, Germany

F(ab)2 Goat anti-mouse IgG:FITC;

Serotec, Düsseldorf, Germany

Roth, Karlsruhe, Germany

Fluoromount, Sigma-Aldrich, Steinheim, Germany

VWR International, Darmstadt, Germany

Leica DM LB, Wetzlar, Germany

Equipment

Microplate Reader

Orbital shaker

Precision scale

MRX , Dynatech Laboratories, Denkendorf, Germany

Reax 2000, Heidolph, Kehlheim, Germany

AKS 120-4, Kern & Sohn, Balingen, Germany

Solutions

PBS (0.01 mol/L; pH 7.4)

NaCl

KCl

Na2HPO4 2 • H2O

KH2PO4

Blocking buffer

Triton X-100

BSA

in 1 mL PBS

137 mmol/L

2.7 mmol/L

6.5 mmol/L

1.5 mmol/L

2.5 µL

10 mg








43

Animals

All equine distal limbs used for the isolation of synoviocytes were obtained from

warmblood horses of various sex and age slaughtered for meat production at a local

abattoir. Cells were isolated within 3 h after death of the respective horse. Only

synoviocytes from macroscopically healthy joints were included in this study, criteria

for selection were as follows: synovial membrane did not show signs of acute or

chronic inflammation; articular cartilage was free of erosion or wear lines,

periarticular osteophytes were not observed; synovial fluid appeared normal in

quantity, viscosity, and color (FREAN and LEES, 2000).

Isolation of equine synoviocytes

Synoviocytes were isolated based on a protocol by FREAN and LEES (2000).

Synovial membrane was harvested aseptically from the cranial joint recess of the

metacarpophalangeal (fetlock) joint using 6-mm-biopsy punches. The synovial

membrane was dissected from the underlying adipose subintima. Tissue pieces were

collected in 10 mL PBS containing 50 IU/mL penicillin and 50 µg/mL streptomycin

and transferred to a laminar flow hood for further processing. The intimal side of the

membrane section was subjected to enzymatic digestion in type VIII collagenase

from Clostridium histolyticum (312.5 collagen digestion units/mL) in a 6-well-plate for

3 h at 37 °C and 5 % CO2 in a humidified atmosphere. Subsequently, the

supernatant was removed and centrifuged at 4 °C at 350 x g for 10 min. Pooled from

both joints of the same horse, cells were resuspended in 5 mL DMEM regular and

seeded in 25 cm2 flasks so that synoviocytes of six different horses were available.

Culture of equine synoviocytes and cryoconservation

Cells were cultured under standard conditions (37 °C, 5 % CO2 in a humidified

atmosphere). Culture medium was exchanged every two to three days. Synoviocytes

were passaged after six to eight days. Before detachment, all medium was removed

and the cells were washed with 2 mL sterile PBS per flask. Cells were then detached

by adding 2 mL EDTA solution for 5 min at room temperature. The EDTA was


44

removed and 1.5 mL trypsin solution was added for 10 min at 37 °C, 5 % CO2 until

the cells were detached and separated. Regular DMEM medium containing 10 %

heat inactivated fetal calf serum, 50 IU/mL penicillin and 50 µg/mL streptomycin was

added to inactivate trypsin before centrifugation of the cells at 4 °C at 350 x g for

10 min. The supernatant was decanted and the cells were seeded in fresh medium at

a density of 2,000 cells/cm2 into 25 cm2 cell culture flasks.

Equine synoviocytes were cryoconserved and stored in liquid nitrogen at -196 °C in

order to have cells available at any time. The cells were detached as described

earlier. After centrifugation, the cells were resuspended in 1 mL of cryomedium and

transferred to cryovials at a density of 1,000,000 – 4,000,000 cells/mL. The vials

were stored at – 80 °C in a freezing container for 4 h and then transferred to liquid

nitrogen. For thawing, cryovials were put in a water bath at 37 °C until only a frozen

core of approx. 2 mm diameter remained. Cryovials were then placed under the

sterile bench where the cells were carefully resuspended in pre-warmed regular

DMEM, transferred to culture flasks and placed in the incubator. Medium was

exchanged 4 h after seeding and from then on every two to three days.

Cell count was routinely determined during passaging: 50 µL of a cell suspension

were diluted with 100 µL trypan blue suspension (40 mg trypan blue in 10 mL aq.

dest.) and 10 µL of this suspension placed in a Neubauer counting chamber. The

cells were counted in the four quadrants of the chamber using a 100-fold

magnification under the microscope. Since all cells incorporate the blue dye but only

viable cells are able to eliminate it, the trypan blue staining allows for a differentiation

between viable and dead cells. Under the microscope, dead cells display a blue

cytoplasm and nucleus, whereas viable cells appear unstained. The total cell count

was calculated using the following equation:

Cells counted = Number of cells in 4 counting chamber quadrants

Cells total = Cell count in 1mL of culture medium


45

Cell viability after stimulation and/or treatment was evaluated using a colorimetric test

in which the yellow MTS tetrazolium compound 3-(4,5-dimethylthiazol-2-yl)-5-(3-

carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium is reduced to a brown

formazan compound by enzymatic activity of viable cells. The resulting intracellular

formazan can be quantified by spectrophotometric means.

5.2.2 Verification of equine synoviocytes

Protein Gene Product 9.5, originally isolated from human brain as a brain-specific

protein, has also been described as a specific cytochemical marker for the

identification of equine type B synoviocytes (KITAMURA et al. 1999). In equine type

B synoviocytes, immunoreactivity for PGP 9.5 is diffusely distributed throughout the

cytoplasm. Synoviocytes of each horse were analyzed by immunocytochemical

staining for PGP 9.5 and only the positive cultures were employed in the

inflammation experiments.

Immunocytochemistry

For immunocytochemistry staining, cells were first seeded on 12-mm-cover glasses

and grown to confluence. To do so, sterilized cover glasses were placed in a 24-well-

plate and covered with 10,000 cells in 500 µL regular DMEM per well. As soon as the

cells were confluent, all medium was removed and cells were fixed with cold

methanol and acetone (1:1) for 5 min. Slides were either stained immediately

afterwards or stored in sterile PBS at 4 °C.

During the entire staining protocol, care was taken to avoid drying of the slides. The

detailed staining protocol is given in TABLE 6. Bisbenzimide binds to DNA and is a

nuclear counterstain. After staining, slides were stored at 4 °C and protected from

light for 24 h before visualizing the results using a fluorescence microscope and the

software KS 400.


46

TABLE 6: Protocol for immunostaining of PGP 9.5.

Washing of cover glasses containing the fixed cells twice with 300 µL of PBS.

Blocking of non-specific staining by adding 300 µL of blocking buffer and incubation for

30 min at room temperature.

Removal of blocking buffer without rinsing.

Dilution of the primary antibody in blocking buffer (1:500). Addition of 150 µL per cover

glass and incubation at 37 °C for 1.5 h. For the negative control, addition of blocking

buffer instead of primary antibody.

Removal of primary antibody, washing twice with 300 µL of blocking buffer.

Dilution of the secondary antibody in blocking buffer (1:200) and addition of 150 µL to

each well. Incubation at 37 °C for 30 min in the dark.

- From this step forward, protection of samples from light -

Removal of secondary antibody and rinsing twice with 300 µL of PBS.

Addition of 150 µL of the diluted bisbenzimide solution (1 µg/mL) to each well,

incubation for 10 min at room temperature.

Rinsing once with 300 µL PBS, then careful removal of the cover glasses from the wells

and blotting dry to remove any excess water.

Addition of one drop of mounting medium per cover glass onto the microscope slide.

Mounting of the coverslip with the cells facing towards the microscope slide.


47

5.2.3 In vitro inflammation model with equine synoviocytes

Pooled synoviocytes from both fetlock joints of six horses were seeded in 96-well-

plates at a density of 10,000 cells per well and grown to confluence in 1-2 d. All anti-

inflammatory substances except for BM were added simultaneously with LPS in

100 µL of regular DMEM; BM was added 4 h prior to stimulation with LPS because of

the time needed for GCR activation, transcription, and translation. Cells were

incubated at 37 °C and 5 % CO2 in a humidified atmosphere. After 24 h,

supernatants were taken and centrifuged for 5 min at 4 °C and 3000 x g. Four

hundred µL of the resulting supernatant were removed and stored at - 80 °C until

analysis.

Stimulation with LPS and treatment

Synoviocytes were stimulated with LPS (E.coli O111:B4) to trigger an inflammation-

like reaction. In preliminary experiments, different concentrations of LPS were tested

for their capability to stimulate PGE2 production in the cells. They were stimulated

with 10 µg/mL or 100 µg/mL of LPS for 24 h. The optimal concentration with regard to

cell viability and PGE2 production was 10 µg/mL LPS (data not shown). This

concentration was applied in the main experiments, where LPS was diluted in regular

DMEM.

Those three anti-inflammatory agents that had been used in experiments with the

isolated perfused equine distal limb (BM, ASA and SA) were added to the LPS-

stimulated synoviocytes. BM concentrations had been determined in preliminary

experiments (data not shown). ASA and SA concentrations were those measured in

synovial fluid at the end of the ex vivo experiments plus one order of magnitude

greater and lesser, as indicated in TABLE 7. Immediately before each experiment, all

anti-inflammatory substances were dissolved in DMSO and diluted in regular DMEM

to a final DMSO concentration of 1 ‰. Four wells received the same treatment with

LPS stimulation. The control group was stimulated with LPS and treated with vehicle

only (medium with 1 ‰ DMSO). After 24 h of incubation at 37 °C, 5 % CO2 in a

humidified atmosphere, supernatants were taken, pooled, immediately centrifuged at


48

4 °C and 3000 x g for 5 min, and frozen at -80 °C until analysis for PGE2

concentration as described below.

TABLE 7: Protocol for stimulation and treatment of equine synoviocytes.

Substance Concentration

[mol/L]

Concentration

[µg/mL]

Time of application

Betamethasone 2.55 x 10-8

2.55 x 10-9

2.55 x 10-10

0.01

0.001

0.0001

4 h before stimulation

with LPS

Acetylsalicylic acid 7.77 x 10-5

7.77 x 10-6

7.77 x 10-7

3.88 x 10-6

14

1.4

0.14

0.7

Simultaneous with LPS

Salicylic acid 1.23 x 10-3

1.23 x 10-4

1.23 x 10-5

170

17

1.7

Simultaneous with LPS

Prostaglandin E2 measurement in the culture medium

To determine the PGE2 concentration in the cell culture supernatant, a competitive

enzyme immunoassay was used. This assay is based on the competition between

PGE2 in the sample and a PGE2-acetylcholinesterase (AChE) conjugate, the so-

called PGE2-tracer, for a limited amount of PGE2 monoclonal antibody. Since the

PGE2-tracer concentration is constant and the PGE2 concentration in the sample

varies, the amount of PGE2-tracer that is able to bind to the monoclonal antibody is

inversely proportional the PGE2 concentration in the sample. The antibody - PGE2

complex binds to polyclonal goat anti-mouse-IgG with which the assay wells are

covered. After washing the plate and addition of the AChE substrate, the product of

this enzymatic reaction is yellow and absorbs strongly at 412 nm. This color can be

determined spectrophotometrically and is proportional to the amount of PGE2-tracer


49

bound to the well, which is inversely proportional to the amount of free PGE2 present

in the sample. A standard curve was performed with each assay and the percentage

of binding used to calculate PGE2 concentrations. Each assay included the following

controls: blank, total activity, non-specific binding, and maximum binding.

Viability testing

After removal of the supernatants, a colorimetric test was performed as described

above to assess cell viability after stimulation and treatment, and to ensure that the

PGE2 concentration in the culture medium was not a result of cell decay.

5.2.4 Statistical analysis

The stimulation / treatment experiments were conducted six times with synoviocytes

from six different horses. Each concentration of an anti-inflammatory drug was

applied in quadruplicate. To compare PGE2 concentrations of LPS-stimulated cells

after treatment with those of that only received vehicle treatment, PGE2

concentrations were analyzed by means of a one-way analysis of variance followed

by a Dunnett’s multiple comparison test. For viability testing, metabolic activity of the

positive control group was normalized to 100 % and results of the treated cells

expressed in relation to that. They were analyzed with a one-way analysis of

variance followed by a Kruskal-Wallis test. In both cases, a p-value < 0.05 was

considered significant. Statistical calculations were performed with GraphPad Prism

5.03 software (GraphPad Software, La Jolla, CA, USA).

5.3 Results

5.3.1 Isolation and culture of synoviocytes

Equine synoviocytes were enzymatically and mechanically isolated, and taken into

culture. Cells exhibited the typical spindle shaped morphology under the phase

contrast microscope (FIGURE 4).


50

5.3.2 Verification of synoviocytes with immunocytochemistry

Primary equine synoviocyte cultures were analyzed by staining with a primary

antibody against PGP 9.5. None of the negative controls showed any staining

whereas synoviocyte cultures from all horses exhibited a diffuse staining throughout

their cytoplasm as depicted in FIGURE 4.

FIGURE 4: Verification of equine synoviocytes Type B. 1: Spindle – like morphology

(Phase contrast microscope). 2: Positive staining for protein gene product 9.5

(Fluorescence microscope).

1 100 µm

50 µm

2


51

5.3.3 In vitro inflammation model

Viability testing after LPS-stimulation

To assess the influence of anti-inflammatory treatment on the viability of cells and to

make sure that PGE2 production was not due to cell death, a colorimetric test was

performed with all 96-well-plates used as described earlier. Results are shown in

FIGURE 5. No significant deterioration in cell viability can be seen as a result of

treatment.


52

Control (

LPS) -10

2.55x10

-9

2.55x10

-8

2.55x10

0

50

100

150

LPS + Treatment [mol/L]

% C

on

tro

l

Control (

LPS) -7

7.77x10

-6

3.88x10

-6

7.77x10

-5

7.77x10

0

50

100

150


% C

on

tro

l

Control (

LPS) -5

1.23x10

-4

1.23x10

-3

1.23x10

0

50

100

150


% C

on

tro

l

FIGURE 5: Viability testing using the MTS-test after stimulation with 10 µg/mL LPS and

treatment with different concentrations [mol/L] of three anti-inflammatory drugs: (A)

Betamethasone, (B) Acetylsalicylic acid, (C) Salicylic acid. Control: Cells stimulated

with LPS without treatment. Mean ± SD of 6 horses.

(A) Betamethasone

(B) Acetylsalicylic acid

(C) Salicylic acid


53

LPS stimulation and treatment

Results for the stimulation of equine synoviocytes with LPS (10 µg/µL) with or without

anti-inflammatory treatment are illustrated in FIGURE 6.

For BM and ASA, the PGE2 inhibition was dose-dependent: for cells treated with BM

(A), the increase in PGE2 production was significantly inhibited at 2.55x10-8 mol/L

(0.01 µg/mL) and 2.55x10-9 mol/L (0.001 µg/mL) but not at 2.55x10-10 mol/L

(0.0001 µg/mL). ASA (B) significantly reduced PGE2 production at 7.77x10-5 mol/L

(14 µg/mL) and 7.77x10-6 mol/L (1.4 µg/mL), but not at 3.88x10-6 mol/L (0.7 µg/mL)

and 7.77x10-7 mol/L (0.14 µg/mL). None of the three SA (C) concentrations tested

were able to significantly inhibit PGE2 production: 1.23x10-3 mol/L (170 µg/mL),

1.23x10-4 mol/L (17.0 µg/mL), 1.23x10-5 mol/L (1.7 µg/mL).


54

+LPS -10

2.55x10

-9

2.55x10

-8

2.55x10

0

50

100

**

*


PG

E2 (

ng

/mL

)

+LPS -7

7.77x10

-6

3.88x10

-6

7.77x10

-5

7.77x10

0

50

100

*

***


PG

E2 (

ng

/mL

)

+LPS -5

1.23x10

-4

1.23x10

-3

1.23x10

0

50

100


PG

E2 (

ng

/mL

)

FIGURE 6: PGE2 concentration in the culture medium after 24 h of stimulation with

10 µg/mL LPS and treatment with (A) Betamethasone, (B) Acetylsalicylic acid or

(C) Salicylic acid. Data are given as mean ± SD of 6 horses (Analysis of variance and

Dunett’s multiple comparison test, p < 0.05).

(A) Betamethasone

(B) Acetylsalicylic acid

(C) Salicylic acid

GENERAL DISCUSSION

55

6 General discussion

It was the aim of the study presented to establish an ex vivo model of the isolated

perfused equine distal limb in order to facilitate pharmacokinetic studies concerning

the equine foot. Pharmacokinetic data are urgently needed by veterinarians who treat

equine athletes to ensure that recovered horses do not return to competition until

they are clear of all medication. The established ex vivo model was designed to

reduce the number of living horses used, which renders these studies less labor-

intensive and less expensive. Perfusion medium and -conditions were optimized with

regard to the following viability parameters: Glucose consumption, lactate production,

LDH liberation, weight increase, skin surface temperature, and histologic changes in

the joint capsule.

Two substances were administered either locally, i.e. intra-articularly, or systemically,

i.e. via the systemic circulation, as it is done to target equine joint disease in vivo.

The ex vivo model was subsequently used to quantify the amount of drug in the joint

over the perfusion period of 8 h. The remaining anti-inflammatory effect of the

respective intra-articular concentration was assessed by examining the ability to

inhibit PGE2 production following stimulation with LPS in an in vitro inflammation

model of cultured equine synoviocytes.

6.1 Model characteristics

For the ex vivo model of the isolated perfused equine distal limb, criteria have been

formulated in order to set minimum standards for tissue viability. However, one

disadvantage of the model is the relative heterogeneity of animals at the abattoir.

Even though only warmblood horses showing no signs of lameness were included in

this study, some variables such as different age, sex, and history of joint disease

remain. It has to be considered, though, that also in vivo studies recruit horses of

different age, sex, and often breed. These horses are not as uniform as laboratory

animals such as mice or rats, which are not only genetically identical due to

inbreeding but are also kept under standardized conditions.

GENERAL DISCUSSION

56

The other limitation of all isolated perfused organs, the restricted time span in which

they are viable, could be overcome by extrapolation of data using pharmacokinetic

parameters determined elsewhere, as it was done for BM.

The synovial clearance of a substance is among others dependent on the blood flow,

whose increase e.g. with exercise also increases the transport of solutes from the

joint to the circulation (DULIN et al. 2012). These physiological changes in blood flow

were not mirrored in the ex vivo model; the perfusion rate was constantly set at the

lower limit of flow rates reported in literature as a compromise between sufficient

tissue supply and prevention of edema formation (ROBINSON et al. 1975;

WONGAUMNUAYKUL et al. 2006).

The model’s most evident advantage is the possibility to investigate the distribution of

drugs administered to the equine foot without the necessity to use living horses.

DERENDORF et al. (1986) have described the so-called “flip-flop case”, according to

which the terminal phase of the concentration-time-curve of intra-articularly

administered triamcinolone acetonide and BM is represented rather by the absorption

rate from the joint than by the elimination phase. The absorption rates can be

investigated in the model since they do not require the animal’s systemic circulation

or excretory organs. It should also be possible to compare joint clearance rates of

different substance formulations. Furthermore, the model offers sampling sites that

are not accessible in the living animal such as efferent veins, circumventing the

substance’s dilution in the entire blood volume as it would be case in vivo.

In order to get some orientation concerning the influence of plasma protein binding

on synovial distribution of systemically administered substances, two extremities

were perfused with a blood-tyrode mixture (4:1) instead of tyrode solution. The

resulting synovia concentrations of ASA and SA were 49.5 % and 47.8 %,

respectively, in comparison with synovial concentrations after perfusion with cell-free

tyrode solution. These results reflect the plasma protein binding of around 50 % for

salicylates (FRASER et al. 1986). It can therefore be speculated that synovial fluid

concentrations of substances in the tyrode-perfused model represent the

concentration without plasma protein binding. Since this characteristic is known for

most drugs, synovial concentrations in tyrode-perfused limbs would simply have to

GENERAL DISCUSSION

57

be adjusted by the plasma protein binding in order to approximate the in vivo

situation.

The model has been established to mirror conditions of the healthy limb and can

therefore not reflect possible alterations due to pathologic processes. Even though

these processes may have an influence on drug distribution, the investigation under

non-diseased conditions was priority.

6.2 Betamethasone

Glucocorticoids are one of the most commonly administered classes of intra-articular

medication in equine medicine (LEES 2003; YARBROUGH 2004). In the study

presented BM, a typical representative of this drug class, was chosen as model

substance.

In order to assess the value of data obtained from the ex vivo model of the isolated

perfused equine distal limb, an extrapolation was made to estimate the time until BM

would no longer be detectable in synovial fluid of the fetlock joint. This time span was

then compared with plasma concentrations from an in vivo study, in which an intra-

articular injection of the identical dose of BM had been performed (LÖVENICH 2012).

Pharmacokinetic parameters that could not be obtained from the ex vivo model were

adapted from a study that had investigated them in vivo (DIKKER 2009).

For calculation, a three-compartment-model was used (joint, blood, peripheral

compartment). A horse of 500 kg bodyweight and a blood volume of 35 L was

assumed. In the aforementioned in vivo study (n=6 horses), LÖVENICH (2012) found

that BM was detectable in plasma for up to 168 h (LOD 4 pg/mL). With data from the

isolated perfused equine distal limb, it was extrapolated that BM concentration in the

joint would decrease to 36 ng/mL after 168 h, possibly indicating an overestimation of

the real concentration. However, it has also been reported that after intra-articular

injection, a glucocorticoid is longer detectable in synovial fluid than in plasma (CHEN

et al. 1992). Also, the return to normal concentrations is described to be slightly

delayed in synovial fluid in comparison with plasma (VINE 2006). As indicated

before, BM phosphate is known to be a more rapid-acting ester (FERGUSON et al.

2009). Mean BM concentration in the joint 7.5 h after intra-articular injection of 4 mg

GENERAL DISCUSSION

58

BM per joint (as 5.3 mg BM disodium phosphate) was calculated to be 1.7 mg. This

finding is supported by other studies in which hydrosoluble esters such as

phosphates are cleared from the joint rather quickly (AUTEFAGE et al. 1986;

DERENDORF et al. 1986). Less soluble glucocorticoid esters such as

methylprednisolone acetate, triamcinolone acetonide or BM acetate are detectable

for longer periods of time (SOMA et al. 2006; LÖVENICH 2012).

In this context, it would be interesting to know which concentration still leads to a

pharmacologic, i.e. anti-inflammatory, effect. Results of the in vitro experiments with

cultured equine synoviocytes described in this thesis have shown that a BM

concentration of as low as 1 ng/mL (2.55x10-9 mol/L) significantly suppresses PGE2

production after LPS stimulation of the cells. A study investigating PGE2 inhibition in

human rheumatoid synovia reports dexamethasone (whose structure is virtually

identical with that of BM) concentrations of 10-8 mol/L (3.93 ng/mL) to 10-6 mol/L

(393 ng/mL) to be effective (KANTROWITZ et al. 1975).

These findings indicate that rather small BM concentrations still exert an anti-

inflammatory effect, even though its presence in the joint after intra-articular

application might not be detectable in plasma samples.

6.3 Acetylsalicylic and salicylic acid

Although not frequently used for treatment of joint disease in horses, synovial

concentrations of ASA and its metabolite SA are of special interest because SA is a

threshold substance. This means that a horse may feature SA concentrations of up

to 625 µg/mL (urine) and 5.4 µg/mL (plasma) in national events in Germany

(ANONYMUS 2011) and up to 750 µg/mL (urine) and 6.5 µg/mL (plasma)

internationally (ANONYMUS 2010). Furthermore, ASA is known to have a very short

half-life in horses and might therefore be chosen if treatment with an NSAID should

appear necessary shortly before a competition.

Synovial fluid concentrations after simulated systemic administration of ASA were

obtained from the ex vivo model of the isolated perfused equine distal limb. Systemic

plasma concentrations of ASA and SA were taken over from a study that had

investigated their pharmacokinetic properties after systemic administration of ASA

GENERAL DISCUSSION

59

(20 mg/kg bodyweight) to horses (BROOME et al. 2003). ASA and SA were detected

by means of high performance liquid chromatography and UV detection with limits of

detection (LOD) at 53 ng/mL and 84 ng/mL and limits of quantification (LOQ) at

106 ng/mL and 169 ng/mL, respectively.

None of the synovial blank samples analyzed before the onset of systemic ASA

application contained any measurable concentrations of ASA or SA. After systemic

administration, the decrease of ASA and SA concentrations in the joint did not

parallel the decrease in the perfusion fluid. This accumulation in synovial fluid is in

accordance with findings in humans (SHOLKOFF et al. 1967; SOREN 1977).

Although the exact reasons for this phenomenon have never been elucidated, it can

be speculated that the accumulation is due to at least two factors: firstly, the

continuous deacetylation of ASA to SA in plasma leads to a constant supply of ASA

and SA to the joint and secondly, the lower esterase activity in the synovial fluid

results in less ASA being converted to SA so that ASA concentrations remain

relatively constant once ASA has diffused into the synovial cavity.

Particularly those synovial fluid concentrations are of clinical relevance that were

measured when the SA plasma concentration had fallen below the FEI threshold

level. In the systemic plasma concentrations adapted from BROOME et al. (2003),

this was the case after 3.5 h. Corresponding synovial fluid concentrations of ASA and

SA until the end of the experiment were 1x10-5 to 8x10-6 mol/L and 1.15x10-4 to

1.24x10-4 mol/L, respectively. A potential anti-inflammatory effect of these

concentrations would be of special importance because at the time these

concentrations were measured within the joint, the horse’s plasma concentration

would have been below the permitted threshold.

In vitro experiments within the framework of this thesis have revealed a dose-

dependent inhibition of PGE2 production after LPS stimulation of primary equine

synoviocytes for ASA, the lowest ASA concentrations capable of significantly

inhibiting PGE2 production being 7.77x10-6 mol/L. For SA, a decrease in PGE2

production could be noted with increasing SA concentration, but this decrease was

not statistically significant. These results are in accordance with data from

BUNTENKÖTTER (2012), who has calculated the following EC50 values (which

GENERAL DISCUSSION

60

describe those plasma concentrations at which half of the maximum possible effect is

provided (TOUTAIN 2009)) for horses: for ASA, the EC50 values were 1.17x10-6 mol/L

for COX I and 1.23x10-5 mol/L for COX II. For SA, an EC50 value could not be

calculated because the inhibition at the concentrations tested was too weak. Four

other studies have determined EC50 values for COX I inhibition by ASA between

1.67x10-6 mol/L and 4.45x10-6 mol/L and for COX II inhibition between 5.35x10-6

mol/L and 1.59x10-5 mol/L. However, these values were not determined specifically

for horses (CROMLISH and KENNEDY 1996; CRYER and FELDMAN 1998;

GIULIANO and WARNER 1999; HINZ et al. 2000).

With regard to SA concentrations in urine samples, BUNTENKÖTTER (2012)

observed that after oral administration of 12.5 µg/kg, 25 µg/kg, and 50 µg/kg ASA,

urine concentrations decreased rather quickly: values fell below the FEI threshold

level of 750 µg/mL after 12 h (12.5 µg/kg) or 24 h (25 µg/kg and 50 µg/kg). However,

concentrations were broadly distributed so that three out of eight horses showed a

urine concentration below the threshold as early as 7 h after administration

(12.5 µg/kg), two out of eight 12 h after administration (25 µg/kg), and two out of

sixteen horses did not show a SA urine concentration above the threshold level at all

(one each for 12.5 µg/kg and 25 µg/kg).

Together, these data give a strong hint that the observed accumulation of ASA in

synovial fluid might lead to an anti-inflammatory effect despite plasma concentrations

below the threshold level. Likewise, it has been reported that NSAIDs accumulate in

and are cleared slowly from inflammatory exudate (LANDONI et al. 1995; LANDONI

and LEES 1995). Hence, the level of SA threshold levels might have to be

reassessed in order to avert the option of treating inflammatory joint conditions in

horses with ASA by taking advantage of its short half-life in plasma and urine coupled

with an accumulation in synovial fluid.

6.4 Outlook

The ex vivo model of the isolated perfused equine distal limb appears to be a

promising tool to investigate the distribution of substances targeting the equine joint,

either administered intra-articularly or systemically, as demonstrated with the model

GENERAL DISCUSSION

61

substances BM and ASA / SA. Further applications might include the comparison of

different formulations of e.g. glucocorticoids or NSAIDs. Also, it would be interesting

to compare synovial fluid concentrations of other therapeutic agents such as

antibiotics after intra-articular with those after systemic administration.

The in vitro equine synoviocyte model of inflammation has proven to be useful for

determining effective concentrations of anti-inflammatory substances. Measurement

of the cells’ inflammatory response might be extended to other parameters of

inflammation.

SUMMARY

63

7 Summary

Maren Friebe (2013)

The isolated perfused equine distal limb and synoviocyte cultures as models

for pharmacological studies

It was the aim of this PhD-project to establish suitable models for investigating

pharmacokinetic and pharmacodynamic properties of substances administered to the

equine joint.

Firstly, in order to assess the distribution of drugs either after intra-articular or

systemic application, the ex vivo model of the isolated perfused equine distal limb

was established. Distal forelimbs of horses obtained from a slaughterhouse were

exarticulated in the carpal joint. Within 1 h after slaughter, the median artery was

canulated and extremities were perfused with oxygenated tyrode solution containing

sodium carboxymethyl cellulose at a flow rate of approx. 65 mL/min for up to 8 h.

Tissue viability during the perfusion period was verified by determination of glucose

consumption, lactate production and LDH liberation in the venous perfusate sampled

from the radial vein. Furthermore, skin surface temperature and weight increase were

monitored as indicators of peripheral blood supply and edema formation. The

integrity of the joint capsule was assessed by histologic examination. Minimum

requirements for parameters were formulated to ensure sufficient viability of the distal

limb.

The articular efflux rate of BM in the venous perfusate of the radial vein after intra-

articular injection of BM disodium phosphate (4 mg BM) into the fetlock joint was

measured with HPLC and UV detection (n=6). The average BM efflux rate per minute

was calculated to be 5.1 µg/min with values ranging from 9 µg/min to 2.9 µg/min. 7.5

h after i.a.-application, 2.3 mg BM had left the joint.

SUMMARY

64

Also, the distribution of ASA and its metabolite SA into the fetlock joint after systemic

administration of ASA was determined in the isolated perfused equine distal limb

(n=8). Plasma concentrations of ASA and SA were adapted from an in vivo study and

added to the perfusate (BROOME et al. 2003). ASA and SA concentrations in the

synovial fluid were determined using calibrated microdialysis probes and HPLC-UV.

Two distal limbs were perfused with diluted autologous blood in order to give some

orientation concerning the influence of plasma protein binding on the synovial

distribution. For the tyrode-perfused limbs (n=6), ASA concentrations in the synovial

fluid reached a maximum of 2.1x10-5 mol/L, maximum SA concentrations were

1.27x10-4 mol/L. Synovial salicylate concentrations in the hemoperfused limbs were

about 50 % lower, presumably owing to the salicylate plasma protein binding of

50 %. The decrease of ASA and SA in synovial fluid did not parallel the decrease in

plasma concentrations, indicating an accumulation in the joint.

Secondly, in order to assess the anti-inflammatory potency of concentrations

measured in the isolated perfused equine distal limb, a cell culture model of primary

equine synoviocytes was established. Cells isolated from macroscopically healthy

fetlock joints (n=6) were stimulated with LPS and treated with decreasing doses of

BM, ASA and SA. The inhibition of prostaglandin E2 production in comparison with an

untreated control was determined as an indicator of an anti-inflammatory effect.

The lowest concentrations causing a significant inhibition of PGE2 production were

2.55x10-9 mol/L and 7.77x10-6 mol/L for BM and ASA, respectively. SA

concentrations employed did not cause a significant PGE2 inhibition. These results

may be a hint for a prolonged anti-inflammatory effect in the joint in comparison with

plasma concentrations.

In summary, the isolated perfused equine distal limb and primary equine synoviocyte

cultures were successfully established as useful tools for the investigation of

pharmacokinetic and pharmacodynamic properties of substances administered to the

equine joint without the necessity to use living horses.

ZUSAMMENFASSUNG

65

8 Zusammenfassung

Maren Friebe (2013)

Das isoliert perfundierte Pferdebein und kultivierte Synoviozyten als Modelle

für pharmakologische Studien

Ziel der vorliegenden PhD-Arbeit war es, geeignete Modelle zur Untersuchung

pharmakokinetischer und pharmakodynamischer Daten von Substanzen zu

etablieren, mit denen beim Pferd Gelenksbehandlungen durchgeführt werden.

Um zunächst die Verteilung von Stoffen nach intra-artikulärer oder systemischer

Applikation zu untersuchen, wurde das ex vivo Modell des isoliert perfundierten

distalen Pferdebeins etabliert. Vordergliedmaßen von Pferden wurden an einem

Schlachthof im Karpalgelenk exartikuliert und innerhalb von 1 h nach der

Schlachtung über die A. mediana mit oxygenierter Tyrodelösung, die Natrium-

Carboxymethylcellulose enthielt, bei einer Flussrate von ca. 65 ml/min für 8 Stunden

perfundiert. Die Gewebevitalität während der Perfusion wurde durch die Bestimmung

von Glukoseverbrauch, Laktatproduktion und LDH-Freisetzung im venösen Perfusat

der V. radialis überprüft. Des Weiteren wurden Hautoberflächentemperatur und

Gewichtszunahme als Indikatoren für periphere Blutversorgung und Ödembildung

kontrolliert. Die Unversehrtheit der Gelenkkapsel wurde mittels histologischer

Untersuchung beurteilt. Mindestanforderungen für eine ausreichende Vitalität der

distalen Gliedmaße wurden formuliert.

Die Abflutung von Betamethason über die V. radialis nach intra-artikulärer Injektion

von BMdihydrogenphosphat-Dinatrium (4 mg BM) ins Fesselgelenk wurde mittels

HPLC und UV-Detektion gemessen (n=6). Die durchschnittliche BM - Abflutung pro

Minute betrug 5.1 µg/min, mit Werten zwischen 9 µg/min und 2.9 µg/min. 7.5

Stunden nach i.a.-Applikation waren 2.3 mg BM aus dem Gelenk abgeflutet.

Die Distribution von Acetylsalicylsäure und ihres Metaboliten Salicylsäure in das

Fesselgelenk nach systemischer Applikation von ASA wurde ebenfalls am isoliert

perfundierten Pferdebein untersucht (n=8). Plasmakonzentrationen von ASA und SA

ZUSAMMENFASSUNG

66

wurden aus einer entsprechenden in vivo Studie übernommen und dem Perfusat

zugesetzt (BROOME et al. 2003). Ihre Konzentrationen in der Synovialflüssigkeit

wurden mit einer kalibrierten Mikrodialysesonde und HPLC-UV bestimmt. Zwei Beine

wurden mit verdünntem autologem Blut perfundiert, um eine Orientierung hinsichtlich

des Einflusses der Plasmaproteinbindung zu erhalten. In den Tyrode-perfundierten

Gliedmaßen (n=6) wurden maximale synoviale Konzentrationen von 2.1x10-5 mol/l

(ASA) und 1.27x10-4 mol/l (SA) erreicht. Die Salicylatkonzentration in der

Synovialflüssigkeit der hämoperfundierten Beine war ca. 50 % niedriger,

wahrscheinlich aufgrund der Plasmaproteinbindung der Salicylate von 50 %. Die

Abnahme von ASA und SA in der Synovia verlief bedeutend langsamer als im

Plasma, was für eine Akkumulation im Gelenk spricht.

Um im Anschluss die antiphlogistische Wirkung der im isoliert perfundierten

Pferdebein gemessenen Synovialkonzentrationen einzuordnen, wurde ein primäres

Zellkulturmodell aus equinen Synoviozyten etabliert. Die aus makroskopisch

gesunden Fesselgelenken (n=6) isolierten Zellen wurden mit LPS stimuliert und mit

abnehmenden Konzentrationen von BM, ASA und SA behandelt. Die Inhibition der

Prostaglandin E2-Produktion im Vergleich mit einer unbehandelten Kontrolle wurde

als Indikator für einen entzündungshemmenden Effekt verwendet. Die niedrigsten

Konzentrationen, die zu einer signifikanten Hemmung der PGE2-Produktion führten,

betrugen 2.55x10-9 mol/l für BM und 7.77x10-6 mol/l für ASA. Die eingesetzten SA-

Konzentrationen führten nicht zu einer signifikanten PGE2-Hemmung. Die Ergebnisse

bedeuten einen Hinweis darauf, dass die entzündungshemmende Wirkung im

Gelenk im Vergleich zum Plasma länger andauert.

Zusammenfassend lässt sich sagen, dass das isoliert perfundierte Pferdebein und

die Synoviozytenkultur erfolgreich etabliert werden konnten. Ohne dass

Versuchspferde eingesetzt werden, ermöglichen sie die Untersuchung

pharmakokinetischer und pharmakodynamischer Charakteristika von Substanzen,

die für die Gelenksbehandlung beim Pferd eingesetzt werden.

REFERENCES

67

9 References

ARMSTRONG, S. and P. LEES (2002):

Effects of carprofen (R and S enantiomers and racemate) on the production of IL-1, IL-6 and TNF-

alpha by equine chondrocytes and synoviocytes.

Journal of veterinary pharmacology and therapeutics 25, 145-153

AUTEFAGE, A., M. ALVINERIE and P. L. TOUTAIN (1986):

Synovial fluid and plasma kinetics of methylprednisolone and methylprednisolone acetate in horses

following intra-articular administration of methylprednisolone acetate.

Equine veterinary journal 18, 193-198

AXELROD, L. (1993):

Glucocorticoids.

In: Textbook of Rheumatology

Saunders, Philadelphia, pp. 779-796

BÄUMER, W., A. MERTENS, M. BRAUN and M. KIETZMANN (2002):

The isolated perfused bovine uterus as a model for mucous membrane irritation and inflammation.

Altex 19, 57-63

BEAUMIER, P., J. FENWICK, L. YOUNG, S. HAZEN, K. HAMA, B. LAVIOLETTE, Z. DEOCAMPO, H.

PARK, N. SIO, S. TIMMINGS and F. TODI (1983):

Salicylic Acid in the Horse: Studies Conducted for Agriculture Canada.

In: Proceedings of the 5th International Conference on the Control of the Use of Drugs in Racehorses.

Toronto, pp. 12-20

BERTONE, A. L., J. L. PALMER and J. JONES (2001):

Synovial fluid cytokines and eicosanoids as markers of joint disease in horses.

Veterinary surgery 30, 528-538

BERTONE, J. J. and L. J. HORSPOOL (2004):

Appendix.

In: Equine Clinical Pharmacology

Saunders, pp. 367 - 380

REFERENCES

68

BISHOP, Y. (2005):

In: The Veterinary Formulary.

Pharmaceutical Press, London

BOUMPAS, D. T. and R. L. WILDER (2001):

Corticosteroids.

In: Arthritis and Allied Conditions

Williams and Wilkins, Philadelphia, S. 827-847

BRISTOL, D. G., J. E. RIVIERE, N. A. MONTEIRO-RIVIERE, K. F. BOWMAN and R. A. ROGERS

(1991):

The isolated perfused equine skin flap. Preparation and metabolic parameters.


BRISTON, L., J. DUDHIA and P. LEES (2009):

Age-related differences in prostaglandin E2 synthesis by equine cartilage explants and synoviocytes.


BROOME, T. A., M. P. BROWN, R. R. GRONWALL, M. F. CASEY and K. A. MERITT (2003):

Pharmacokinetics and plasma concentrations of acetylsalicylic acid after intravenous, rectal, and

intragastric administration to horses.

Canadian journal of veterinary research 67, 297-302

BRUNE, K. and W. S. BECK (1991):

Towards safer nonsteroidal anti-inflammatory drugs.

Agents and actions 32, 13-25

BUNTENKÖTTER, K. (2012):

Untersuchung zur Pharmakokinetik von Salicylsäure nach oraler Applikation von Acetylsalicylsäure

beim Pferd.

Thesis, University of Veterinary Medicine Hannover, Department of Pharmacology, Toxicology and

Pharmacy

BYRON, C. R., M. C. STEWART, A. A. STEWART and H. C. PONDENIS (2008):

Effects of clinically relevant concentrations of glucosamine on equine chondrocytes and synoviocytes

in vitro.

American journal of veterinary research 69, 1129-1134

REFERENCES

69

CARON, J. P. (2005):

Intra-articular injections for joint disease in horses.

The Veterinary clinics of North America 21, 559-573

CARVER, M. P., P. L. WILLIAMS and J. E. RIVIERE (1989):

The isolated perfused porcine skin flap. III. Percutaneous absorption pharmacokinetics of

organophosphates, steroids, benzoic acid, and caffeine.

Toxicology and applied pharmacology 97, 324-337

CHEN, C. L., J. A. SAILOR, J. COLLIER and J. WIEGAND (1992):

Synovial and serum levels of triamcinolone following intra-articular administration of triamcinolone

acetonide in the horse.


COLLIER, H. O. (1971):

Prostaglandins and aspirin.

Nature 232, 17-19

CREAMER, P. (1999):

Intra-articular corticosteroid treatment in osteoarthritis.

Current opinion in rheumatology 11, 417-421

CROMLISH, W. A. and B. P. KENNEDY (1996):

Selective inhibition of cyclooxygenase-1 and -2 using intact insect cell assays.

Biochemical pharmacology 52, 1777-1785

CRYER, B. and M. FELDMAN (1998):

Cyclooxygenase-1 and cyclooxygenase-2 selectivity of widely used nonsteroidal anti-inflammatory

drugs.

The American journal of medicine 104, 413-421

DE GRAUW, J. C., C. H. VAN DE LEST and P. R. VAN WEEREN (2009):

Inflammatory mediators and cartilage biomarkers in synovial fluid after a single inflammatory insult: a

longitudinal experimental study.

Arthritis research & therapy 11, R 35

REFERENCES

70

DERENDORF, H., H. MOLLMANN, A. GRUNER, D. HAACK and G. GYSELBY (1986):

Pharmacokinetics and pharmacodynamics of glucocorticoid suspensions after intra-articular

administration.

Clinical pharmacology and therapeutics 39, 313-317

DIKKER, L. (2009)

Untersuchung zur Pharmakokinetik von Betamethason nach intravenöser und dermaler Applikation

hinsichtlich der Dopingrelevanz beim Pferd.


Pharmacy

DINGLE, J. T., J. SAKLATVALA, R. HEMBRY, J. TYLER, H. B. FELL and R. JUBB (1979):

A cartilage catabolic factor from synovium.

The Biochemical journal 184, 177-180

DULIN, J. A., W. T. DROST, M. A. PHELPS, E. M. SANTSCHI, M. I. MENENDEZ and A. L.

BERTONE (2012):

Influence of exercise on the distribution of technetium Tc 99m medronate following intra-articular

injection in horses.


FERGUSON, D. C., L. DIRIKOLU and M. HOENIG (2009):

Glucocorticoids, mineralocorticoids and adrenolytic drugs.

In: Veterinary Pharmacology and Therapeutics

Wiley-Blackwell, pp. 771 - 793

FRASER, C. M., A. MAYS, H. E. AMSTUTZ, J. ARCHIBALD and J. ARMOUR (1986):

In: The Merck Veterinary Manual: a Handbook of Diagnosis, Therapy and Disease Prevention and

Control for the Veterinarian.

Merck & Co Inc, Rahway (USA), p. 1677

FREAN, S. P. and P. LEES (2000):

Effects of polysulfated glycosaminoglycan and hyaluronan on prostaglandin E2 production by cultured

equine synoviocytes.


REFERENCES

71

FRISBIE, D. D. (2011):

Medical Treatment of Joint Disease In: Equine Surgery

Saunders Elsevier, pp. 1114-1117

GILROY, D. W., P. R. COLVILLE-NASH, D. WILLIS, J. CHIVERS, M. J. PAUL-CLARK and D. A.

WILLOUGHBY (1999):

Inducible cyclooxygenase may have anti-inflammatory properties.

Nature medicine 5, 698-701

GIULIANO, F. and T. D. WARNER (1999):

Ex vivo assay to determine the cyclooxygenase selectivity of non-steroidal anti-inflammatory drugs.

British journal of pharmacology 126, 1824-1830

GOODRICH, L. R. and A. J. NIXON (2006):

Medical treatment of osteoarthritis in the horse - a review.

Veterinary journal 171, 51-69

GRAY, R. G. and N. L. GOTTLIEB (1983):

Intra-articular corticosteroids. An updated assessment.

Clinical orthopaedics and related research 177, 235-263

GRONEBERG, D. A., C. GROSSE-SIESTRUP and A. FISCHER (2002):

In vitro models to study hepatotoxicity.

Toxicologic pathology 30, 394-399

GROSSE-SIESTRUP, C., C. FEHRENBERG, H. VON BAEYER and D. A. GRONEBERG (2002):

Multiple-organ harvesting for models of isolated hemoperfused organs of slaughtered pigs.

Altex 19, 9-13

HAMEED, F. and J. IHM (2012):

Injectable medications for osteoarthritis.

Physical medicine and rehabilitation 4, 75-81

HARKINS, J. D., J. M. CARNEY and T. TOBIN (1993):

Clinical use and characteristics of the corticosteroids.

The Veterinary clinics of North America 9, 543-562

REFERENCES

72

HIGGINS, A. J. and P. LEES (1984):

The acute inflammatory process, arachidonic acid metabolism and the mode of action of anti-

inflammatory drugs.


HIGGINS, A. J., P. LEES and A. D. SEDGWICK (1987):

Development of equine models of inflammation. The Ciba-Geigy Prize for Research in Animal Health.

The Veterinary record 120, 517-522

HINZ, B., V. KRAUS, A. PAHL and K. BRUNE (2000):

Salicylate metabolites inhibit cyclooxygenase-2-dependent prostaglandin E2 synthesis in murine

macrophages.

Biochemical and biophysical research communications 274, 197-202

HSU, W. H. (2008):

Endocrine Pharmacology In: Handbook of Veterinary Pharmacology

Wiley-Blackwell, pp. 264-267

HSU, W. H. and A. KANTHASAMY (2008):

Nonsteroidal Anti-inflammatory Drugs.

In: Handbook of Veterinary Pharmacology


HULKOWER, K. I., J. W. COFFEY, W. LEVIN, C. M. ANDERSON, T. CHEN, W. C. HOPE, D. R.

BOLIN and D. W. MORGAN (1993):

Interleukin-1 beta induces cytosolic PLA2 in parallel with prostaglandin E2 in rheumatoid synovial

fibroblasts.

Agents Actions 39 Spec No, C5-7

IWANAGA, T., M. SHIKICHI, H. KITAMURA, H. YANASE and K. NOZAWA-INOUE (2000):

Morphology and functional roles of synoviocytes in the joint.

Archives of Histology and Cytology 63 (1), 17-31.

KAMERLING, S. G. and J. G. OWENS (1994):

Models for assessing the relationship between drug concentration and drug effect in performance

horses.

The British veterinary journal 150, 507-525

http://www.ishc.net/contents.htm

REFERENCES

73

KANTROWITZ, F., D. R. ROBINSON, M. B. MCGUIRE and L. LEVINE (1975):

Corticosteroids inhibit prostaglandin production by rheumatiod synovia.

Nature 258, 737-739

KIETZMANN, M., W. LÖSCHER, D. ARENS, P. MAASS and D. LUBACH (1993):

The isolated perfused bovine udder as an in vitro model of percutaneous drug absorption. Skin

viability and percutaneous absorption of dexamethasone, benzoyl peroxide, and etofenamate.

Journal of pharmacological and toxicological methods 30, 75-84

KIETZMANN, M., R. SCHERKL and R. SCHULZ (2002):

Pharmakologie der Entzündung und der Allergie.

In: Lehrbuch der Pharmakologie und Toxikologie für die Veterinärmedizin

Enke, Stuttgart, pp. 318 - 328

KIRKER-HEAD, C. A., V. K. CHANDNA, R. K. AGARWAL, E. A. MORRIS, A. TIDWELL, M. W.

O'CALLAGHAN, W. RAND and M. S. KUMAR (2000):

Concentrations of substance P and prostaglandin E2 in synovial fluid of normal and abnormal joints of

horses.


KITAMURA, H. P., H. YANASE, H. KITAMURA and T. IWANAGA (1999):

Unique localization of protein gene product 9.5 in type B synoviocytes in the joints of the horse.

Journal of histochemistry and cytochemistry 47, 343-352

KLAUS, A. M. and H. J. HAPKE (1996):

[Natural and synthetic glucocorticoids in the racing horse: a review of the literature].

Deutsche tierärztliche Wochenschrift 103, 494-500

KOLLIAS-BAKER, C. and K. COX (2004):

Non-steroidal anti-inflammatory drugs.


Saunders, pp. 247-266

LAKHANI, K. H., M. LAMBERT, F. SLUYTER, R. DEVOLZ, G. MAYLIN and A. J. HIGGINS (2004):

Estimation of the critical threshold value for presence of salicylic acid in the urine of thoroughbred

horses.

In: Proceedings of the 15th International Conference of Racing Analysts and Veterinarians, Dubai, 67-

77

REFERENCES

74

LANDONI, M. F., F. M. CUNNINGHAM and P. LEES (1995):

Comparative pharmacodynamics of flunixin, ketoprofen and tolfenamic acid in calves.


LANDONI, M. F., R. FOOT, S. FREAN and P. LEES (1996):

Effects of flunixin, tolfenamic acid, R(-) and S(+) ketoprofen on the response of equine synoviocytes to

lipopolysaccharide stimulation.


LANDONI, M. F. and P. LEES (1995):

Comparison of the anti-inflammatory actions of flunixin and ketoprofen in horses applying PK/PD

modelling.


LANGENDORFF, O. (1895):

Untersuchungen am überlebenden Säugethierherzen.

Arch Ges Physiol 61, 291-332

LEES, P. (2003):

Pharmacology of drugs used to treat osteoarthritis in veterinary practice.

Inflammopharmacology 11, 385-399

LEES, P. (2009):

Analgesic, antiinflammatory, antipyretic drugs.



LEES, P., C. P. EWINS, J. B. TAYLOR and A. D. SEDGWICK (1987a):

Serum thromboxane in the horse and its inhibition by aspirin, phenylbutazone and flunixin.

The British veterinary journal 143, 462-476

LEES, P., J. B. TAYLOR, A. J. HIGGINS and S. C. SHARMA (1986):

Phenylbutazone and oxyphenbutazone distribution into tissue fluids in the horse.


LEES, P., J. B. TAYLOR, T. E. MAITHO, J. D. MILLAR and A. J. HIGGINS (1987b):

Metabolism, excretion, pharmacokinetics and tissue residues of phenylbutazone in the horse.

The Cornell veterinarian 77, 192-211

REFERENCES

75

LILLICH, J. D., A. L. BERTONE, L. M. SCHMALL, A. J. RUGGLES and R. A. SAMS (1996):

Plasma, urine, and synovial fluid disposition of methylprednisolone acetate and isoflupredone acetate

after intra-articular administration in horses.


LINDSLEY, H. B. and D. D. SMITH (1990):

Enhanced prostaglandin E2 secretion by cytokine-stimulated human synoviocytes in the presence of

subtherapeutic concentrations of nonsteroidal antiinflammatory drugs.

Arthritis and rheumatism 33, 1162-1169

LÖSCHER, W., F. R. UNGEMACH and R. KROKER (2006):

Pharmakotherapie bei Haus- und Nutztieren.

Enke Verlag, Stuttgart

LÖVENICH, B. (2012)

Pharmacokinetics of betamethasone and triamcinolone acetonide after intra-articular injection

regarding doping relevance in the horse.


Pharmacy

MACHNIK, M., W. LÖHLEIN, Y. SCHRADER, K. KÖHLER, J. FREYMARK and W. SCHÄNZER

(2007):

Behandlung von Sportpferden mit Corticosteroiden: Nachweiszeiten von Flumethason und

Betamethason beim Pferd nach i.v. Applikation von Acutol und i.a. Applikation von Celestovet.

Pferdeheilkunde 5, 476 - 480

MALMBERG, A. B. and T. L. YAKSH (1992):

Hyperalgesia mediated by spinal glutamate or substance P receptor blocked by spinal

cyclooxygenase inhibition.

Science 257, 1276-1279

MAY, S. A., R. E. HOOKE and P. LEES (1989):

Identity of the E-series prostaglandin produced by equine chondrocytes and synovial cells in response

to a variety of stimuli.

Research in veterinary science 46, 54-57

REFERENCES

76

MAY, S. A., R. E. HOOKE, K. Y. PEREMANS, F. VERSHOOTEN and P. LEES (1994):

Prostaglandin E2 in equine joint disease.

Vlaams Diergeneeskd Tijdschr 63, 187-191

MAY, S. A. and P. LEES (1996):

Nonsteroidal anti-inflammatory drugs.

In: Joint disease in the horse

Saunders, Philadelphia, pp. 223 - 237

MEADE, E. A., W. L. SMITH and D. L. DEWITT (1993):

Differential inhibition of prostaglandin endoperoxide synthase (cyclooxygenase) isozymes by aspirin

and other non-steroidal anti-inflammatory drugs.

The Journal of biological chemistry 268, 6610-6614

MEULYZER, M., P. VACHON, F. BEAUDRY, T. VINARDELL, H. RICHARD, G. BEAUCHAMP and S.

LAVERTY (2009):

Joint inflammation increases glucosamine levels attained in synovial fluid following oral administration

of glucosamine hydrochloride.

Osteoarthritis and cartilage / OARS, Osteoarthritis Research Society 17, 228-234

MILLER, L. L., C. G. BLY, M. L. WATSON and W. F. BALE (1951):

The dominant role of the liver in plasma protein synthesis; a direct study of the isolated perfused rat

liver with the aid of lysine-epsilon-C14.

The Journal of experimental medicine 94, 431-453

MITCHELL, J. A., P. AKARASEREENONT, C. THIEMERMANN, R. J. FLOWER and J. R. VANE

(1993):

Selectivity of nonsteroidal antiinflammatory drugs as inhibitors of constitutive and inducible

cyclooxygenase.

Proceedings of the National Academy of Sciences of the United States of America 90, 11693-11697

MORTON, A. J., N. B. CAMPBELL, J. M. GAYLE, W. R. REDDING and A. T. BLIKSLAGER (2005):

Preferential and non-selective cyclooxygenase inhibitors reduce inflammation during

lipopolysaccharide-induced synovitis.

Research in veterinary science 78, 189-192

REFERENCES

77

MOSES, V. S., J. HARDY, A. L. BERTONE and S. E. WEISBRODE (2001):

Effects of anti-inflammatory drugs on lipopolysaccharide-challenged and -unchallenged equine

synovial explants.


OKADA, Y., M. SHINMEI, O. TANAKA, K. NAKA, A. KIMURA, I. NAKANISHI, M. T. BAYLISS, K.

IWATA and H. NAGASE (1992):

Localization of matrix metalloproteinase 3 (stromelysin) in osteoarthritic cartilage and synovium.

Laboratory investigation 66, 680-690

OWENS, J. G., S. G. KAMERLING and S. A. BARKER (1995):

Pharmacokinetics of ketoprofen in healthy horses and horses with acute synovitis.


OWENS, J. G., S. G. KAMERLING, S. R. STANTON, M. L. KEOWEN and J. S. PRESCOTT-

MATHEWS (1996):

Effects of pretreatment with ketoprofen and phenylbutazone on experimentally induced synovitis in

horses.


PALMER, J. L. and A. L. BERTONE (1994a):

Experimentally-induced synovitis as a model for acute synovitis in the horse.


PALMER, J. L. and A. L. BERTONE (1994b):

Joint structure, biochemistry and biochemical disequilibrium in synovitis and equine joint disease.


PATAN-ZUGAJ, B., F. C. GAUFF and T. F. LICKA (2012):

Effects of the addition of endotoxin during perfusion of isolated forelimbs of equine cadavers.


PATAN, B., K. D. BUDRAS and T. F. LICKA (2009):

Effects of long-term extracorporeal blood perfusion of the distal portion of isolated equine forelimbs on

metabolic variables and morphology of laminar tissue.


REFERENCES

78

PLUMB, D. C. (2008):

In: Veterinary Drug Handbook.

Wiley-Blackwell

POPOT, M. A., P. GARCIA, Y. MOULARD and Y. BONNAIRE (2002):

Detection of triamcinolone, betamethasone and methylprednisolone in the horse: Effect on urine

cortisol and cortisol metabolite concentrations.

In: Proceedings of the 14th international conference of racing analysts and veterinarians, Florida, USA

pp. 408 - 412

RAEKALLIO, M., P. M. TAYLOR and R. C. BENNETT (1997):

Preliminary investigations of pain and analgesia assessment in horses administered phenylbutazone

or placebo after arthroscopic surgery.


RICKETTS, A. P., K. M. LUNDY and S. B. SEIBEL (1998):

Evaluation of selective inhibition of canine cyclooxygenase 1 and 2 by carprofen and other

nonsteroidal anti-inflammatory drugs.


RIVIERE, J. E., K. F. BOWMAN, N. A. MONTEIRO-RIVIERE, L. P. DIX and M. P. CARVER (1986):

The isolated perfused porcine skin flap (IPPSF). I. A novel in vitro model for percutaneous absorption

and cutaneous toxicology studies.

Fundamental and applied toxicology 7, 444-453

RIVIERE, J. E. and N. A. MONTEIRO-RIVIERE (1991):

The isolated perfused porcine skin flap as an in vitro model for percutaneous absorption and

cutaneous toxicology.

Critical reviews in toxicology 21, 329-344

ROBINSON, N. E., J. M. DABNEY, W. J. WEIDNER, G. A. JONES and J. B. SCOTT (1975):

Vascular responses in the equine digit.


RUSSELL, W. M. S. and R. L. BURCH (1959):

The Principles of Humane Experimental Technique.

Methuen, London, UK.

REFERENCES

79

SAKLATVALA, J. and J. T. DINGLE (1980):

Identification of catabolin, a protein fro synovium which induces degradation of cartilage in organ

culture.

Biochemical and biophysical research communications 96, 1225-1231

SHOLKOFF, S. D., E. J. EYRING, M. ROWLAND and S. RIEGELMAN (1967):

Plasma and synovial fluid concentrations of acetylsalicylic acid in patients with rheumatoid arthritis.


SMITH, C. J., Y. ZHANG, C. M. KOBOLDT, J. MUHAMMAD, B. S. ZWEIFEL, A. SHAFFER, J. J.

TALLEY, J. L. MASFERRER, K. SEIBERT and P. C. ISAKSON (1998):

Pharmacological analysis of cyclooxygenase-1 in inflammation.

Proceedings of the National Academy of Sciences of the United States of America 95, 13313-13318

SOMA, L. R., E. BEHREND, J. RUDY and R. W. SWEENEY (1988):

Disposition and excretion of flunixin meglumine in horses.


SOMA, L. R., C. E. UBOH, Y. LUO, F. GUAN, P. J. MOATE and R. C. BOSTON (2006):

Pharmacokinetics of methylprednisolone acetate after intra-articular administration and its effect on

endogenous hydrocortisone and cortisone secretion in horses.


SOMA, L. R., C. E. UBOH, Y. YOU, F. GUAN and R. C. BOSTON (2011):

Pharmacokinetics of intra-articular, intravenous, and intramuscular administration of triamcinolone

acetonide and its effect on endogenous plasma hydrocortisone and cortisone concentrations in

horses.


SOREN, A. (1977):

Dissociation of acetylsalicylic acid in blood and joint fluid.

Scandinavian journal of rheumatology 6, 17-22

TAYLOR, J. B., A. WALLAND, P. LEES, E. L. GERRING, T. E. MAITHO and J. D. MILLAR (1983):

Biochemical and haematological effects of a revised dosage schedule of phenylbutazone in horses.


REFERENCES

80

TOUTAIN, P. L. (2009):

Mechanisms of drug action and pharmacokinetics/pharmacodynamics integration in dosage regimen

optimization for veterinary medicine.


Wiley-Blackwell, pp. 75-97

TOUTAIN, P. L. (2010a):

How to extrapolate a withdrawal time from an EHSLC published detection time: a Monte Carlo

simulation appraisal.


TOUTAIN, P. L. (2010b):

Veterinary medicines and competition animals: the question of medication versus doping control.

Handbook of experimental pharmacology 199, 315-339

TOUTAIN, P. L., A. AUTEFAGE, C. LEGRAND and M. ALVINERIE (1994):

Plasma concentrations and therapeutic efficacy of phenylbutazone and flunixin meglumine in the

horse: pharmacokinetic/pharmacodynamic modelling.


TOUTAIN, P. L. and C. C. CESTER (2004):

Pharmacokinetic-pharmacodynamic relationships and dose response to meloxicam in horses with

induced arthritis in the right carpal joint.


TROTTER, G. W. (1996):

Intra-articular corticosteroids.

In: Joint disease in the horse

Saunders, Philadelphia, pp. 237 - 256

TROTTER, G. W., C. W. MCILWRAITH, J. V. YOVICH, R. W. NORRDIN, R. H. WRIGLEY and C. H.

LAMAR (1991):

Effects of intra-articular administration of methylprednisolone acetate on normal equine articular

cartilage.


REFERENCES

81

UNGEMACH, F. R. (2006):

Pharmaka zur Beeinflussung von Entzündungen.

In: Pharmakotherpapie bei Haus- und Nutztieren

Parey, Stuttgart, pp. 364-403

VANE, J. R. and R. M. BOTTING (1995):

New insights into the mode of action of anti-inflammatory drugs.

Inflammation research 44, 1-10

VETIDATA (2012)

Glukokortikoide

www.vetidata.de, 21.12.2012

VINE, J. S., N; GLOWACKI, L; KRSTEVSKA, D; ZAHRA, P; O'CALLAGHAN, P (2006):

The metabolism and excretion of betamethasone sodium phosphate and betamethasone acetate in

the horse after intra-articular injection.

In: Proceedings of the 16th International Conference of Racing Analysts and Veterinarians

Tokyo, Japan, pp. 167-171

VON BAEYER, H., K. STAHL, M. HAUSLER, M. MEISSLER, V. UNGER, J. FRANK, C. GROSSE-

SIESTRUP, G. KACZMARCZYK, K. AFFELD, H. J. FLAIG and B. STEINBACH (1997):

[A new method of ex vivo whole blood perfusion of isolated mammalian organs, exemplified by the

kidney of swine].

Biomedizinische Technik 42, 61-68

WAGNER, S. M., A. C. NOGUEIRA, M. PAUL, D. HEYDECK, S. KLUG and B. CHRIST (2003):

The isolated normothermic hemoperfused porcine forelimb as a test system for transdermal

absorption studies.

The international journal of artificial organs 6, 183-191

WARD, L. C. and P. J. BUTTERY (1979):

The patho-physiological basis for tests of viability in isolated perfused organs.

Biomedicine 30, 181-186

WEISS, C., H. PASSOW and A. ROTHSTEIN (1959):

Autoregulation of flow in isolated rat kidney in the absence of red cells.

The American journal of physiology 196, 1115-1118

REFERENCES

82

WHEAT, J. D. (1955):

The use of hydrocortisone in the treatment of joint and tendon disorders in large animals.

Journal of the American Veterinary Medical Association 127, 64-67

WILLIAMS, P. L., M. P. CARVER and J. E. RIVIERE (1990):

A physiologically relevant pharmacokinetic model of xenobiotic percutaneous absorption utilizing the

isolated perfused porcine skin flap.

Journal of pharmaceutical sciences 79, 305-311

WITTENBERG, R. H., R. E. WILLBURGER, K. S. KLEEMEYER and B. A. PESKAR (1993):

In vitro release of prostaglandins and leukotrienes from synovial tissue, cartilage, and bone in

degenerative joint diseases.


WONGAUMNUAYKUL, S., C. SIEDLER, H. SCHOBESBERGER and C. STANEK (2006):

Doppler sonographic evaluation of the digital blood flow in horses with laminitis or septic

pododermatitis.

Veterinary radiology & ultrasound 47, 199-205

WRIGHT, J. M., C. G. KNIGHT and I. M. HUNNEYBALL (1986):

The effect of side chain structure on the biochemical and therapeutic properties of intra-articular

dexamethasone 21-esters.

Clinical and experimental rheumatology 4, 331-339

YARBROUGH, T. (2004):

Intraarticular medication.


Saunders, pp. 121-134

APPENDIX

83

10 Appendix

10.1 Cell culture experiments

10.1.1 Cell viability

TABLE 8: Results of the modified MTS-test following synoviocyte stimulation with LPS and treatment

with BM, ASA or SA, measured as spectrophotometrical extinction. Control: Stimulation with LPS and

vehicle treatment. Each value is a mean of 4 wells. SD = Standard deviation.

Horse # 1 2 3 4 5 6 Mean SD

Control 1.065 1.024 0.726 0.858 1.156 0.782 0.935 0.171

BM (mol/L)

2.55x10-8

0.750 1.0785 0.751 0.909 1.078 0.732 0.883 0.164

2.55x10-9

0.744 0.494 0.744 0.974 1.046 0.749 0.792 0.196

2.55x10-10

0.756 0.678 0.743 0.840 1.162 0.811 0.832 0.171

ASA (mol/L)

7.77x10-5

0.710 0.941 0.771 0.866 1.126 0.761 0.863 0.153

7.77x10-6

0.692 0.939 0.707 0.831 1.054 0.762 0.831 0.142

7.77x10-7

0.800 0.991 0.718 0.893 1.060 0.756 0.870 0.136

3.88x10-6

0.730 0.926 0.756 0.917 1.058 0.757 0.857 0.130

SA (mol/L)

1.23x10-3

0.668 1.056 0.731 0.875 1.019 0.745 0.849 0.161

1.23x10-4

0.729 0.895 0.808 0.965 1.152 0.767 0.886 0.156

1.23x10-5

0.719 0.970 0.883 0.711 1.182 0.804 0.878 0.179

APPENDIX

84

10.1.2 PGE2 concentrations

TABLE 9: PGE2 concentration (ng/mL) in the culture supernatant 24 h after stimulation with LPS and

treatment with BM, ASA or SA. Control: Stimulation with LPS and vehicle treatment. Each

concentration is a mean of 4 wells. SD = Standard deviation.

Horse # 1 2 3 4 5 6 Mean SD

Control 58 82 112 68 43 73 73 23

BM (mol/L)

2.55x10-8

13 10 52 27 15 40 26 17

2.55x10-9

39 12 68 36 23 55 39 23

2.55x10-10

63 83 103 65 43 79 73 25

ASA (mol/L)

7.77x10-5

2 5 9 4 2 4 4 3

7.77x10-6

28 48 62 29 23 47 40 15

7.77x10-7

41 68 103 60 30 61 61 25

3.88x10-6

53 56 75 61 28 47 53 17

SA (mol/L)

1.23x10-3

33 60 73 60 25 55 51 18

1.23x10-4

45 80 86 65 35 60 62 20

1.23x10-5

37 90 92 68 46 61 66 22

APPENDIX

85

10.2 Maren Friebe’s contribution to the manuscripts

1. The isolated perfused equine distal limb as an ex vivo model for pharmacokinetic studies:

Participation in the study design

Performance of the experiments

Writing of the manuscript.

2. Synovial distribution of “systemically” administered acetylsalicylic acid in the isolated perfused

equine distal limb

Participation in the study design

Performance of the experiments

Writing of the manuscript

THANK YOU

Danksagung

Herrn Prof. Dr. M. Kietzmann danke ich herzlich für die Überlassung des interessanten

Themas, die gute und stets freundliche Betreuung, die fachlichen Denkanstöße und

schließlich die konstruktive Kritik bei der Anfertigung dieser Arbeit.

Herrn Prof. Dr. K. Feige und PD Dr. F. Thorey danke ich für die Ratschläge und

Anregungen im Rahmen der Betreuergruppe.

Der Deutschen Reiterlichen Vereinigung (FN), insbesondere Herrn Dr. Michael Düe,

danke ich für die finanzielle Unterstützung der Studie.

Allen Mitarbeitern der Arbeitsgruppe Toxikologie danke ich für die abwechslungsreichen

drei Jahre. Speziell möchte ich Frau Dr. Jessica Stahl für den Beistand rund um die

HPLC sowie die jederzeit hilfsbereite Art und Herrn Stephan Schumacher, PhD, für die

umfangreiche Einführung in Mikrodialyse und Statistik danken. Viktoria Garder und

Caroline Schulz danke ich für die souveräne Unterstützung in allen größeren und

kleineren Fragen und die unterhaltsame Zeit. Danken für die Hilfe im Labor möchte ich

außerdem Hans-Herbert Bohr, Theiss Wystemp und Alina Narten.

Meinen Mitdoktoranden im Laufe der Jahre (auch denen aus der 1. Etage), ganz

besonders der aktuellen Besetzung des „kleinen“ Doktorandenzimmers Isabelle Roth und

Stefanie Mielke, danke ich für die fachlichen und nicht fachlichen Gespräche und

Ratschläge, den Spaß und das Gefühl, nicht alleine zu sein.

Sonja, Maike, Hellen, Kathrin, Katha & Anne-Sophie danke ich für ihre Freundschaft seit

dem Studium und viele schöne Erinnerungen an unsere gemeinsame Zeit in Hannover.

Meinen Eltern und meinem Bruder Thomas danke ich von Herzen für die Unterstützung

während meines Studiums und meiner Doktorarbeit. Ohne Euch wäre das alles nicht

möglich gewesen.

Meinem lieben Jan danke ich für die Zuneigung und die Ablenkung und dafür, dass er

immer an mich glaubt. Ich freue mich auf die Zukunft mit Dir!

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