Antimicrobial therapy in horses:
a pharmacologist perspective
Pierre-Louis Toutain
National Veterinary School; Toulouse ,France
30th October 2014;
Department of Veterinary Disease Biology
University of Copenhagen
Steps for a rationale selection of an
antimicrobial (AM) drug
1.
2.
3.
4.
5.
6.
7.
8.
Identity of the affecting MO
In vitro AM susceptibility of the bug
Nature and site of infection
The pharmacokinetic (PK) characteristics of the
selected AM
The pharmacodynamics (PD) properties of the
selected AM
PK and PD integration (PK/PD indices)
Safety issues
Cost of the therapy
1-Why plasma concentrations are
relevant for AMD and why to
compare free plasma concentration
to MICs?
Nature and site of infection
Where are located the pathogens
Extra Cellular Fluid
Bound
Free
Most bacteria of clinical
interest
- respiratory infection
- wound infection
- digestive tract inf.
Free
±MIC
MO
Cell
(in phagocytic cell most often)
• Legionnella spp
• mycoplasma (some)
• chlamydiae
• Brucella
• Cryptosporidiosis
• Listeria monocytogene
• Salmonella
• Mycobacteria
• Rhodococcus equi
2-The right dosage regimen
to control the efficacious
plasma concentration
What are the elements of a dosage
regimen
• The dose
–A PK/PD variable
• The dosing interval
• The treatment duration
–When to start
–When to finish
A fundamental relationship
PK
PK
(0 to 1)
!
PD
X MIC
PK
(0 to 1)
A dose can be determined rationally
using a PK/PD approach
8
Question: what is the daily dose for
enrofloxacin for different possible MIC90
• What we know:
–
–
–
–
Plasma clearance: 2.5L/Kg/24h
Bioavailability by intragastric route of 80%
Extent of binding of ~ 20%
MIC90
MO
µg/mL
E. Coli ; S. aureus
Pseudomonas aeruginosa
Strept. zooepidemicus
Rhodococcus equi
0.25
0.50
1.00
2.00
– The PK/PD index for optimization: AUC/MIC=125
• Or equivalently : the average plasma concentration over the
dosing interval should be 5 folds the MIC
It has been developed surrogates indices
(predictors) of antibiotic efficacy taking into
account MIC (PD) and exposure antibiotic
metrics (PK)
Practically, 3 indices cover all situations:
•AUC/MIC
•Time>MIC
• Cmax/MIC
Recommandations thérapeutiques en fonction de la
bactéricide
Pattern de la
bactéricidie
Antibiotiques
Objectifs
therapeutiques
Paramètre
PKPD
Type I
Concentration
dépendant & effets
prolongés
Aminoglycosides
Quinolones
Optimiser les
concentrations
Cmax/MIC
24h-AUC/MIC
Type II
Temps dépendant &
pas de rémanence
Pénicillines
Céphalosporines
Optimiser la
durée
d’exposition
T>MIC
Type III
Temps dépendant &
effets rémanents
dose-dépendant
Macrolides
Tétracyclines
Optimiser les
quantités (doses)
24h-AUC/MIC
The dose for enrofloxacin
MO
MIC: µg/mL
E. Coli ; S. aureus
Pseudomonas aeruginosa
Strept. zooepidemicus
Rhodococcus equi
0.25
0.50
1.00
2.00
The dose for enrofloxacin
AUC/MIC=125
MO
MIC (µg/mL)
Dose (mg/kg)
E. Coli ; S. aureus
0.25
4.9
Pseudomonas aeruginosa
Strept. zooepidemicus
Rhodococcus equi
0.50
9.77
1.00
19.5
2.00
39.1
3-Variability of plasma clearance
in horses
Drugs, age
15
AMD: plasma clearances
Low or high?
Drug
Sulphadoxine
Gentamicin
Sulphamethoxazole
Amikacin
Oxytetracycline
Rifampin
Sulphadiazine
Cefoxitin
Metronidazole
Enrofloxacin
Ampicillin
Ticarcillin
Amoxicillin
ClB
(mL/kg/min)
0.32
1.2
1.2
1.23
1.25
1.34
1.45
1.72
1.97
2.33
2.89
3.1
4.55
Drug
Trimethoprim
Ceftriaxone
Cafazolin
Cefadroxil
Penicillin
ClB
(mL/kg/min)
5.03
5.22
5.27
6.95
8.5
Chloramphenicol
Ciprofloxacin
Clarithromycin
Erythromycin
8.8
9.7; 18
21.1
26.6
16
AMD: plasma clearances
Effect of age
Effect of breed, fever, sex, ….
chloramphenicol
Age (days) clearance (ml/kg/min)
1
2.25
3
6.23
7
8.86
4
9.63
42
9.68
A foal is not only a
small horse
17
AMD: protein binding
• MIC are free concentrations
• Only the free concentration is active
• No example of drug/drug interaction
leading to increase the free drug
concentration by displacement (eg with
NSAID)
Low or high?
drug
Ampicillin
Gentamicin
Cefazolin
Enrofloxacin
Amoxicillin
Penicillin
doxycycline
%
8
8
8
22
37
52
82
18
AMD: bioavailability
Low or high?
Large influence of the route of administration
and of the formulations
19
Bioavailability
• Bioavailability quantifies the proportion
of a drug that is absorbed and
available to produce its systemic effect
– Extent (overall exposure)
– Rate (T>MIC)
Bioavailability
Definition
• Absolute
– amount of administered drug which enters the
systemic (arterial) circulation and the rate at
which the drug appears in the blood stream
• Relative
– to compare formulations (bioequivalence)
– to compare routes of administration
IV route of administration
by definition F=100%
Not always the case for AMD administered as prodrug such as esters as
erythromycin estolate
22
Oral route of administration
24
Oral route: several possible
modalities
Mash
Intragastric
Perlingual
Fed vs unfed (food withheld for 12h )
25
Oral enrofloxacin : no food effect
AUC
(µg.h/ml)
5 mg/kg
Steinman et al JPT 2006
T1/2 (h)
Cmax
(µg/ml)
Fasted
Hay
concentrate
18.5
12.5
13.9
8.1
7.6
7.9
1.7
1
1.3
Rifampin administration before
and after feeding
The Royal Veterinary College
Peter Lees
July 2003
Bioavailability: 68% (fasted) vs 26% (fed)
28
Influence of food on the F% of
erythromycin (base)
Food withheld=26% (6-44%)
Fed =7.7% (1-18%)
Lakritz et al AJVR, Vol 61, No. 9, September 2000
Foals should be given ERY before
they are fed hay. Administration
of ERY to foals from which food
was withheld overnight apparently
provides plasma concentrations of
erythromycin A that exceed the
minimum inhibitory concentration
of Rhodococcus equi for
approximately 5 hours. The dosage
of 25 mg/kg every 8 hours, PO,
appears appropriate.
29
Why a possible low oral
bioavailability
• Poor stability in the stomach
– pH effect
• Poor absorption
– Physiological origin
– Binding to cellulosis
• Hepatic first-pass effect
– Can be predicted from the blood clearance
• Drug interaction
31
In vitro binding (%) of TMP and sulphachlorpyridazine to
hay, grass silage and concentrate
Medium
(3h at 37C)
% Binding
Trimethoprim
% Binding
Sulphachlorpyridazine
Concentrations
4 mg/ml
100 mg/ml
4 mg/ml
100 mg/ml
Hay
82
63
90
67
Grass silage
73
47
71
33
Concentrate
64
36
86
64
Van Duijkeren, 1996
The pH effect
(stomach)
33
Poor stability of the AM in the
stomach: the case of erythromycin
• Inactivated by gastric acid thus:
– Enteric-coated formulations
– Esters (prodrugs) with improved acid stability but
requiring hydrolysis by esterases
• Estolate
• Stearate
• ethyl succinate
However a horse and a man can be different and
extrapolation misleading
34
Gastric pH
7
8
6
7
6
4
5
pH
pH
5
3
4
3
2
2
1
1
0
0
Time
Fasted
Low pH (average of 1.6)
Continuous secretion
Time
Hay ad libitum
Buffering capacity of hay and saliva (at each peak)
35
Erythromycin: bioinequivalence of the
different forms
• Three possible forms for an oral administration
– Erythromycin base
– Erythromycin salt (lactobionate, phosphate…)
– Erythromycin esters absorbed by the GIT (estolate,
etylsuccinate)
– Erythromycin ester hydrolysed in the GIT (stearate)
Phosphate
Estolate
Stearate
Ethylsuccinate
(salt)
(ester)
(ester)
ester
AUC (µg*h/mL)
295
176
302
308
Cmax (µg/mL)
2.3
0.4
2
0.3
T1/2, (min)
149
145
Poor
absorption
110
221
Slow hydrolysis
36
Effect of age on bioavailability
37
Age effect:
Bioavailability of IG Cefadroxil in foal
Age
(months)
0.5
1
2
3
5
F%
99.6
67.6
35.1
19.5
14.4
Tmax (h)
2.1
1.6
1.6
.96
.90
Duffee JVPT 1997 20 427
38
Effect of age on bioavailability of
oral penicillins in the horse
Drug
F (%) In foal
F (%) in adult
Penicillin V
(phenoxymethyl
penicillin)
16.00
2.00
Amoxycillin
36-42
5 - 10
Why a possible low oral
bioavailability
• Poor stability in the stomach
– pH effect
• Poor absorption
– Physiological origin
– Binding to cellulosis
• Hepatic first-pass effect
– Can be predicted from the blood clearance
• Drug interaction
40
Poor absorption due to drug-drug
interaction
Association of AMD
Clarithromycin ± Rifampin
•
•
•
•
After RIF comedication, relative bioavailability of CLR decreased by more than 90%.
the drastic lowering of the average CLR plasma concentrations by more than 90% have
resulted from induction of hepatic and intestinal CYP3A4 and intestinal ABCB1 and
probably
ABCC2. efflux transport seems to be the major reason for lower bioavailability
there are many doubts from a pharmacokinetic point of view that combination therapy of CLR with
RIF might really be superior to other eradication protocols as suggested by the results of a
retrospective clinical study in foals (Gigue`re et al., 2004). The absence of major drug interactions as
shown in our recent pharmacokinetic study with tulathromycin and RIF should be confirmed before
a combination treatment is launched in clinical practice (Venner et al., 2010).
42
Poor bioavailability due to a
hepatic first-pass effect
43
The 3 segments of the digestive tract in
terms of first-pass effect
Buccal cavity
No
first-pass
effect
Small intestine/large bowel
Full First pass-effect
Rectal
Limited
first-pass
effect
44
Hepatic first pass effect
Eythromycin
Dose
Liver
30%
Fmax = 1 - Eh
Eh~70%
Fraction eliminated by first pass effect
• Fmax = 1 – Eh=1 - [Clh / Qh]=1-[17/24]=0.30
45
Plasma erythromycin after an IG administration of a
salt (phosphate) or an ester (estolate) of
erythromycin (food withheld)
F% from Phosphate:16±3.5%
F% from estolate: 14.7±11%
Both are very low: why?
Plasma clearance of
erythromycin is very large
(17.5ml/kg/min) suggesting a
likely large hepatic first-pass
effect in horse
47
Intramuscular administration
IV administration of sodium benzylpenicillin
Penicillin G potassium vs. Penicillin G procaine
Flip-flop kinetics
Procaine benzylpenicillin (
procaine penicillin) is an ester
of benzylpenicillin and the local
anaesthetic agent procaine.
Following deep intramuscular
injection, it is slowly absorbed
into the circulation and
hydrolysed to benzylpenicillin
This combination is aimed at
reducing the pain and discomfort
associated with a large
intramuscular injection of
penicillin.
Influence of the injection site on bioavailability of Penicillin
(administration of procaine benzylpenicilin)
Semi-membrane / semi-tendineux
M. serratus
M. biceps
M. pectoralis
M. gluteus
M. Subcutaneous
Concentrations (UL/mL)
4
3
2
1
(Time)
0
0
2
4
Firth et al. 1986, Am. J. Vet. Res.
6
8
10
12
24h
Terminal half-life and bioavailability of
procaine benzylpenicillin in the horse
Injection site
Subcutaneous
Intramuscular :
M.gluteus
M.pectoralis
M.biceps
M.serratus
Intravenous
Terminal half-life
(h)
Bioavailability (%)
21.8
78.4
12.8
14.9
14.9
8
3.72
78.4
94.2
97.6
113.2
100
The terminal half-life is much more longer after an extravascular administration:
The so-called flip-flop phenomenon
Intra- vs intermuscular administration
• The best site for IM administration is the 5th
cervical vertebra, ventral to the funicular part
of the ligamentum nuchae but dorsal to the
brachiocephalic muscle
True IM
Boyd et al,1987, Vet. Rec.
Intra- vs intermuscular administration
• Injection in the 4th space but the ventral
injection has traversed to the 6th vertebral
space
Boyd et al,1987, Vet. Rec.
3Preanalytical method 06 54
Procaine penicillin adverse effects
• PP is associated with incidence of severe
adverse reactions with distress…...but much
less frequently with water-soluble salts of
Penicillin.
– Anaphylactic reaction: rare in horses
• Penicillin have affinity to proteins and may form hapten
• Hypersensitivity is the most common cause of negative
reaction to penicillin
– Procaine toxicity: frequent in horses
• Due to action of the free procaine on the CNS
56
Procaine penicillin adverse effects
• Procaine is hydrolysed by plasma esterase to
non toxic metabolite (Para-aminobenzoic acid
and Diaminoethanol)
• Toxicity is observed if the rate of Procaine
absorption exceeds the hydrolyzing capacity
– Inadvertent IV route after an IM administration
– Poor esterase activity (next slide)
– Some formulations have high free procaine
concentration (vehicule) and this is increase by
high room temperature (stability issue)
57
PP adverse effects:
esterase activity
Poor esterase activity
in horses havingADR
58
The question of medication/doping
control for penicillin procaine
• Normally, no routine screening for doping
control for the AMD
• But procaine is controlled (as a local
anesthetic)
– What about penicillin procaine?
– Can be very long in urine (several months)
The EHSLC web site
Click on the image
Local tolerance of AMD
• Poorly tolerated
– aminoglycosides
– TMP/sulfate
– macrolides
– tétracyclines
• Well tolerated
– Penicillines (peni-procaine better than
penicillin G)
Inhalation
63
Many devices: are they equivalent?
Cortic 00A.64
Cortic 00A.65
Cefquinome inhalation:
high local concentration
• Very high local drug concentrations of
cefquinome was achieved in horses using a jet
nebulizer, but cefquinome was not detectable
after 4 h in the majority of horses
– This is likely true for any drug that was not
specifically developed for inhalation (e.g.
dexamethasone) because pulmonary absorption is
very fast due to a very high blood flow.
66
Inhalation treatment:
an user safety issue?
• During exhalation, some degree of air
pollution of the drug was evident and user
safety was accounted for by ventilating the
room sufficiently during administration
67
Drug elimination and PK selectivity
Selectivity of antimicrobial drugs in
veterinary medicine
Selectivity
PD
PK
Narrow spectrum
Selective distribution of
the AB to its biophase
Almost all oral and parenterally administered antimicrobials have been
linked with antimicrobial associated diarrhoea (AAD) in both man and
horses, although some antimicrobials clearly pose a higher risk:
• Macrolides ( erythromycine, tylosine, …)
• Tetracyclines (doxycyclin, OTC…)
• Bêtalactams (Penicillin G, ampicillin, ceftiofur..)
AMD effect on the enteric anaerobes
• The potential of an antimicrobial to induce
AAD is largely dependent on its effect on the
enteric anaerobes, which in turn reflects its
spectrum of antibacterial activity, and the
concentration of active drug within the
intestine
– lincosamides, macrolides and b-lactams have
efficacy against anaerobes
Factors determining AMD
concentration in the gut
• the route of administration
– IV vs. oral for oxytetracyclines
• the % of drug absorbed from the intestine
– Low bioavailability of many AMD
– Food effect
• The % excreted in bile or mucus
– Macrolides (bile), doxycycline (enterocytes)
– Large differences between quinolones (enro vs. cipro)
• The extent to which the drug is inactivated by the
intestinal contents
• Anecdotally, there appear to be geographical
differences in the susceptibility of the local
equine population to develop AAD after
administration of a particular antimicrobial
• This mayreflect regional differences in the
composition of the enteric flora
Both hospitalisation and the use of AMD were associated with prevalence of AMR
among E coli isolated from the feces of horse (Dunowska at al JAVMA 2006 228 1909
Pharmacodynamic of antibiotic in
horses
A fundamental relationship
PK
PK
(0 to 1)
!
PD
X MIC
PK
(0 to 1)
A dose can be determined rationally
using a PK/PD approach
76
CLSI breakpoints for the horse 2014
(µg/mL)
Conditions
Antibiotics
Pathogens
S
I
R
Comments
Enterobacteriaceae
≤2
4
≥8
≤2
4
≥8
Breakpoints derived from microbiological, pharmacokinetic
(using accepted clinical doses), and pharmacodynamic data.
For horses, the dose of gentamicin modeled was 6.6 mg/kg
every 24 hours, IM.
≤2
4
≥8
Gentamicin
Pseudomonas
aeruginosa
Actinobacillus spp.
Horses Respiratory
Disease
Ampicillin
Horses (Respiratory, Soft
Tissue)
Penicillin
Streptococcus equi
subsp.
≤0.25
zooepidemicus and
subsp. equi
≤0.25
Staphylococcus spp.
≤0.5
1
≥2
Streptococcus spp.
≤0.5
1
≥2
Horses (respiratory,
genital tract)
Cefazolin
Streptococci – βhemolytic group
Escherichia coli
Horses Respiratory
Disease
Ceftiofur
Streptococcus equi
subsp. zooepidemicus
≤2
≤0.25
For horses, the dose of ampicillin sodium modeled was 22
mg/kg IM every 12 hours
4
≥8
Breakpoints derived from microbiological, pharmacokinetic
data (using accepted clinical, but extra-label doses), and
pharmacodynamic data. The dose of procaine penicillin G
modeled was 22 000 U/kg, IM, every 24 hours.
Cefazolin breakpoints were determined from an examination
of MIC distribution of isolates and PK-PD analysis of cefazolin.
The dosage regimen used for PK-PD analysis of cefazolin was
25 mg/kg administered every six hours intravenously in
horses and dogs.
In vitro veritas
MICs estimated with different inoculmum densities, relative to
that MIC at 2x105
Ciprofloxacin
Gentamicin
Linezolid
Oxacillin
Daptomycin
Vancomycin
In vitro veritas?
Evaluation of tulathromycin in the treatment of
pulmonary abscesses (Rhodococcus equi) in foals
Azithromycin+Rifampin
Tulathromycin
The combination of a macrolide and rifampin is synergistic both in vitro and in vivo, and the use of the 2
classes of drugs in combination reduces the likelihood of R. equi esistance to either drug
Venner et al Vet J 2006
Tulathromycin: MIC (ng/mL) in MHB vs. calf serum
25%,50%,75% and 100%
25%
50%
75%
100 %
The serum effect
For azithromycin (closely related to tulathromycin) the
presence of 40% serum during the MIC test decreased
MICs by 26-fold for serum-resistant Escherichia coli and
15-fold for Staphylococcus aureus.
Rhodococcus equi:
Clarithromycin is the macrolide of
choice for foals
• Clarithromycin is the macrolide of choice for foals with severe disease,
given the most favorable minimum inhibitory concentration against R equi
isolates obtained from pneumonic foals (90% of isolates are inhibited at
0.12, 0.25, and 1.0 mcg/mL for clarithromycin, erythromycin, and
azithromycin, respectively).
• In foals with R equi pneumonia, the combination of clarithromycin (7.5
mg/kg, PO, bid) and rifampin is superior to erythromycin-rifampin and
azithromycin-rifampin.
• Foals treated with clarithromycin-rifampin have improved survival rates
and fewer febrile days than foals treated with erythromycin-rifampin and
azithromycin-rifampin. Reported adverse effects of clarithromycinrifampin include diarrhea in treated foals. The duration of antimicrobial
therapy typically is 3–8 wk.
In vitro veritas
the case of combination
• The combination of a macrolide (erythromycin,
azithromycin, or clarithromycin) with rifampin is
the recommended treatment for infection caused
by R. equi, based on in vitro activity data,
pharmacokinetic studies, and retrospective
studies.
• The level of evidence for this recommendation is
moderate, with no randomized controlled studies
available to substantiate it.
Association Clarithromycin + Rifampin
a major PK interaction
•
•
•
•
After RIF comedication, relative bioavailability of CLR decreased by more than 90%.
the drastic lowering of the average CLR plasma concentrations by more than 90% have
resulted from induction of hepatic and intestinal CYP3A4 and intestinal ABCB1 and
probably
ABCC2. efflux transport seems to be the major reason for lower bioavailability
there are many doubts from a pharmacokinetic point of view that combination therapy of CLR with
RIF might really be superior to other eradication protocols as suggested by the results of a
retrospective clinical study in foals (Gigue`re et al., 2004). The absence of major drug interactions as
shown in our recent pharmacokinetic study with tulathromycin and RIF should be confirmed before
a combination treatment is launched in clinical practice (Venner et al., 2010).
87
La cinquième édition (2013) du livre de
référence en antibiothérapie vétérinaire avec
un chapitre chez le cheval
88