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THE DISPOSITION OF METRONIDAZOLE IN GOATS AND ITS RELEVANCE TO THE TREATMENT OF
ANAEROBIC INFECTIONS
A THESIS
PR ESENTED IN PARTIAL FULFIL MENT OF THE R EQUIRE MENTS F O R THE DEGREE OF
MASTER OF VETERINARY SCIENC E AT
MASSEY UNIVERSITY
Elizabeth Amanda Dillon 1988
ABSTRACT
The recent commercial developments in goat farming in New Zealand, have led to an increase in the value of individual goats and to a growing interest in caprine diseases.
The importance of anaerobic bacteria other than the clostridia, as potential pathogens in humans and animals, has also only recently been recognised, even though anaerobic bacteria have been identified since 1 8 6 1 . Various members of this bacterial group are known to be involved in different conditions of goats, particularly in wound and foot infections.
Metronidazole (Flagyl1 ) is a bactericidal agent which has a specific action against anaerobic micro-organisms. This drug is already widely used in the treatment of selected diseases in dogs, cats and humans, but there was little information available on its use in goats.
The study which forms the basis of this thesis, was to investigate the disposition of metronidazole in eight goats. Both IV and IM routes of administration w ere studied in the form of a cross-over experiment. Silicone tubing "cages" w ere implanted subcutaneously, so that the metronidazole concentration versus time profile could be determined, both in serum and in interstitial fluid.
The analysis of serum and tissue cage fluid samples was undertaken using a high pressure liquid chromatography unit, w hich proved to be reliable over the range of concentrations tested. The system consisted of a Waters Model 6000 A solvent delivery system, a U6 K injector, a Z-module radial compression separation system and a Waters programmable automa tor, Model 7 1 0 . The mobile phase used was a 75:25 mixture of aqueous potassiu m hydrogen phosphate and methyl alcohol; this was adjusted to a running speed of 1 .5 mls per min.
A 450 variable wavelength detector was set at either 0 . 0 1 or 0 .04 absorbance units, and a constant wavelength of 3 1 2 nm.
Given these concentration profiles, a full pharmacokinetic analysis was carried out using standard statistical procedures. The following values were determined:
1 . 'Flagyl', May &. Baker (NZ) Ltd.
maximum serum and tissue cage fluid concentrations (C maxiV, CmaxiM, C maxiV tc>, time to maximum serum and tissue cage concentration (T maxiV, T maxiM, C maxiMtc' T maxiMtc>, serum concentration (extrapolated) at zero time (B, B'), half-life (q), elimination rate constant (8,S'), volume of distribution (V d(area)), area under the concentration curve (A UC), total body clearance (ClB), absorption rate constant (kab), percentage penetration of metronidazole into tissue cage fluid, percentage of drug absorbed into the systemic circulation followi ng IM administration (F), and the total amount of drug which was absorbed into the systemic circ ulation (in mg/kg).
Follow ing IV administration of a 0 .5% w /v solution of metronidazole at a dose rate of 2 0 mg/kg BWgt, the d at 0.94 ± 0 . 0 8 per hour (n=8) was rapid and consistent w i th a high figure for the eli mination rate constant at 0. 7 9 ± 0.09 per hour (n=8 ). The total body clearance, a more sensitive indicator of the biotransformation and excretion processes than d, was also rapid (0.32 ± 0.06 1/kg/hr) which is in keeping with the efficient drug metabolism of the goat.
This m ay account for the low V d(area) which was unexpected for a basic drug of this nature in the ruminant.
Critical parameters for drug concentrations and durations of effect are summarized in Table I.
Metronidazole was rapidly detected in both sera and interstitial fluid (within 0.25 hrs) following the intramuscular administration of a 40% suspension of metronidazole at a dose rate of 20 mg/kg BWgt. The uptake of metronidazole from the injection sites differed markedly betw een individual goats, resulting in a mean absorption percentage of 42.4% ± 8.8% (n = 8), equivalent to 8.4 mg/kg BWgt. Maximum serum levels w ere achieved w i thin approximately one hour of IM administration, but the peak w as more than ten-fold lower than the corresponding concentration found in serum following IV administration. Peak tissue cage drug concentrations w ere not achieved until four hours after IM administration.
The maximum drug concentration in tissue cage fluid w as greater than the !VIIC upper threshold for a variety of anaerobic bacteria (1 2.5 mcg/ml), and this was maintained for 5.5 hrs. The lower limit of the MIC of 3.0 mcg/ml was exceeded for a correspondingly longer period.
iii
TABLE I Serum and tissue cage fluid drug concentrations and <l.lrations of effect, following aministr ation of metronidazole solutions
Period that nominated serum cone.
was exceeded (hr) Metronidazole
(dose rate : Cmax
mcg/ml
Tmax (hr)
50 mcg/ml
12.5 mcg/ml
3.0 mcg/ml 20 mg/kg BWgt)
0.5% w /v solution
IV
serum
t.c.f
6 3.9 ± 1 2.2 (n=5)
23.67 ± 4.46 (n=8)
<0.25
(n=5) 0.3 ± 0.05 (n=5)
1 . 4 1 ± 0.58
(n=8) 0
1 .4 ± 0.2 3 . 1 ± 0.2 (n=8) (n=8)
1 .5 ± 0.6
(n=6) 7.9 ± 1.0 (n=8)
serum 5.5 ± 0.8 1 . 0 6 ± 0.6 0 0 4.1 ± 0. 7
(n=8) (n=8) (n=8)
40% w/v suspen- --- si on
IM t.c.f 1 3.2 ± 3.9 (n=8) t.c.f Tissue cage fluid
4.1 ± 0. 7
(n=8) 0 5.0 ± 1 .5
(n=5) 8.6 ± 1 .6 (n=7)
Further pharmacokinetic analysis of the experimental data made it possible to calculate specific medication schedules for the goat. These w ere established on the basis that serum metronidazole concentrations should be maintained at a level w hich w as bactericidal for the majority of anaerobic bacteria, w hich included Bacteroides spp., Fusibacterium spp., and Clostridia spp. The recommendation given was that 0.5% w /v metronidazole solution should be administ ered at a dose rate of 20 mg/kg BWgt and repeated every 4-6 hrs.
Using the 4 0 % w /v metronidazole suspension, the dose rate should be 45 mg/kg BWgt and the medication should be repeated every 1 0-1 2 hrs. In each case the loading dose w as only fractionally greater at 2 0 . 3 mg/kg BWgt and 48.5 mg/kg B Wgt respectively.
The drug concentration in interstitial fluid (tissue cage fluid), gave some indication of the anti microbial activity in extravascular tissues, a feature which can not be extrapolated from a profile of serum concentrations.
ACKNOWLEDGEMENTS
I wish to thank my supervisors for their encouragement and assistance during this study. In particular, I thank Dr Brian Cooper for his guidance in pharmacokinetics and in the preparation of this manuscript, and Professor Neil Bruere for his interest and advice.
I gratefully acknowledge t he assistance of the following people:
Mrs Linda Dickson and Ms Hilary Burbidge for their help during the surgical procedure for tissue cage i mplantation,
Miss D ebbie Lovelock, for restraint of the animals during the sample collection periods,
Mrs Elizabeth Davies, for tests performed on serum and tissue cage fluid,
Mr Bryan McKay, Professor Roger Morris, Dr Will Marsh and !VIr Barry Butler for their advice on the use of the computer for the linear regression analysis and graphical display,
Dr D Palmer and Professor R D Jolly for their valued assistance w ith the HPLC analysis of samples.
Dr Lyndon Badcoe for the microscopic examination of the cage tissue,
and May & Baker (NZ) Ltd., for kindly supplying the metronidazole preparations, and relevant literature.
an d grat eful acknowledgment t o Mr Tom L avv ln pro duci n g · t he pho t ographic mat erial .
V
TABLE OF CONTENTS
Abstract
Acknowledgements
List of tables List of figures List of plates
Chapter 1
Chapter 2
Chapter 3 Chapter 4
Chapter 5
Chapter 6 References Appendix I Appendix 11
Appendix Ill
General introduction
Anaerobic bacteria and associated infections : a review of the literature Metronidazole a review of the literature
Methods of measuring the concentration of drugs outside the vascular compartment
Determination of the disposition of metronidazole in goats
General discussion and summary
Materials and methods
Plots of metronidazole concentration in serum and tissue cage fluid of seven goats, periods up to 24 hours after IV administra
tion of 0.5% w /v metronidazole solution
Plots of metronidazole concentration in serum and tissue cage fluid of seven goats, at periods up to 24 hours after IM adminis
tration of 40% w /v metronidazole suspension
Page ii
V
vii
viii ix
1
3 12 25
28
56 64 75 88
89
Table 3 : 1
4 : I
5 : I 5 : n
5 : m
5 : IV
LIST OF TABLES
D isposition values in lactating cattle and sheep following intraperitoneal or intrauterine administra
tion of 0.5% w/v metronidazole solution (expressed as mean ± standard deviation)
Materials, procedures and anatomical sites involved in the determination of "interstitial fluid" drug concentrations
Mean total protein in sera and tissue cage fluid Mean differential cell counts from pooled tissue cage fluid
Least squares linear regression analysis follow ing intravenous administration of 0.5% w /v metronidazole solution (Dose 20 mg/kg BWgt)
Pharmacokinetic values for the disposition of metronidazole in goat tissue cage fluid, following t he intravenous administration of 0.5% w /v solution (Dose 20 mg/kg BWgt).
Page
19
26
40 41 45
48
5 V Phar macokinetic values for the disposition of 49 metronidazole in goat sera, following the
intravenous administration of 0.5% w/v solution (Dose 20 mg/kg BWgt)
5 VI Phar macokinetic values for the disposition of 50 metronidazole in goat sera fluid following the
intramuscular administration of 40% w /v metronidazole suspension (Dose 20 mg/kg BWgt)
5 Vll P harmacokinetic values for the disposition of 51 metronidazole in tissue cage fluid following the
intramuscular administration of 40% w /v metronidazole suspension (Dose 20 mg/kg BWgt)
5 vm Least squares linear regression analysis following 5 2 intramuscular administration of 40% w /v metronidazole
solution (Dose 20 mg/kg B Wgt)
5 : IX Recommendations for metronidazole administration 5 5
schedules in the goat
App. I:1 Time intervals between the taking of blood or tissue cage 78 fluid samples in goats following IV or IM injection of a
0.5 w /v solution or a 40 w /v suspension respectively of metronidazole
vii
Figure 3 : 1
3 : 2
3 : 3
3 4
5 1
5 2
5 3
LIST OF FIGURES
Chemical structure of enteromycin
Chemical structure of azomycin (2-nitroimidazole)
Chemical structure of metronidazole
(1-hydroxyethyl-2-methyl-5-nitroimidazole)
Chemical structure of dimetridazole (1, 2-dimethyl-5-nitroimidazole)
Diagrams of the right (a) and left (b) sides of a goat, showing the anatomical sites where the tissue cages were implanted subcutaneously. The numbers indicate the random order of tissue cages from which the fluid samples were collected
Sequence of events involved in the determination of metronidazole disposition kinestics in goats
Metronidazole concentration in serum and tissue cage fluid of goat Dopey (Do), following intravenous
administration of 0.5% w /v metronidazole solution (serum metronidazole concentration = 61.52 + 0.26 time + e)
Page 1 4 14 15
15
32
38
46
5 : 4 Metronidazole concentration in serum and tissue cage 53
fluid for goat Happy (Ha), following intramuscular administration of 40% w/v metronidazole solution (serum metronidazole concentration = 3.89 + 0.06 time + e)
App. 1:1 Semilogarithmic plot of plasma drug concentration 83 versus time following administration of a single
intravenous dose. Graphical technique for estimating the half-life of a drug is illustrated (from Baggot, J .D. [1977]): Principles of Drug Disposition in Domestic Animals. Published by Saunders Company, London.
App. 1:2 Diagrammatic representation of the trapezoidal 83 method used to calculate the area under the plasma
concentration time-curve (AUC). (From Baggot, J.D.
[1978]): J. vet. Pharmacal. Therap. 1: 111-118.
viii
Plate
5 1
5 2
5 3
5 4
5 5
5 6
5 7
6 1
LIST OF PLATES
Six of the animals used in the experiment
Materials used in the manufacture of tissue cages
Series of steps involved in the surgical implantation of the tissue cages
Series of steps involved in the removal of the implanted tissue cages
Straw-coloured samples of tissue cage fluid
Sectioned tissue cage after removal, showing the tissue mass within the cage and fibrous attachments extending through the perforations of the tube
Necrotic core within an infected tissue cage
Exposure of an implanted tissue cage, to show the thick fibrous capsule
Page
30
31
33
36
39
42
44 59
