8th ISAP Symposium

Developments in Pharmacokinetics and Pharmacodynamics (PK/PD):

optimising efficacy and prevention of resistance

Nijmegen, The Netherlands, July 4th-6th, 2001

P Leesand

F Shojaee AliAbadiThe Royal Veterinary College, UK

Rational dosing: animals versus humans

Rational Dosing of Antimicrobial Drugs in Animals

• General considerations• Legal guidelines for PK-PD• Aspects of pharmacokinetics in animals• PK-PD integration of danofloxacin in ruminants

(tissue cage model)• PK-PD integration of danofloxacin in calf

pneumonia• Rational dosing: future perspectives

Pharmacology of Antimicrobial Therapy

Dose

Serum Conc.

Site of action

Conc.

Application of PK/PD integration to optimisation of antibacterial therapy

EfficacyEfficacyRecovery rateRecovery rate

ResistanceResistanceResiduesResiduesToxicityToxicity

CostCost

Antimicrobial Drug Treatment

Which drug ?

How much ?

How often ?

How long ?

0

1

2

3

4

0 4 8 12 16 20 24

Criteria for Setting Dose Schedules

Cmax > 2 x MIC90 for all bacterial species against which activity to be claimed

Plasma concentration > 1 x MIC90 for all bacterial species against which activity to be claimed for half inter-dose interval

Dosing schedules meeting these criteria will be effective in many patients but are unlikely to be optimal for any antimicrobial drug group

Aspects of Pharmacokinetics in Animals

• inter-species differences (half-life, clearance)• intra-species differences (breed)• age• depot formulations• residues in food producing species• distribution to udder (blood:milk barrier)• ruminants• chicken• fish• honey bee

Elimination Half-life of Antimicrobial Agents : Species Differences

HM = Hepatic metabolism, HE = hepatic excretion, RE= renal excretion Prescott, Baggot and Walker, 2000

DRUG ELIMINATION

MECHANISM

HALF-LIFE (h)

Man Dog Horse Cow

Trimethoprim

Sulphadiazine

Sulphadimethoxine

Metronidazole

Norfloxacin

HM + RE

HM + RE

HM + RE

HM + RE

HM + RE

10.6

9.9

40.0

8.5

5.0

4.6

5.6

13.2

4.5

3.6

3.2

3.6

11.3

3.9

6.4

1.3

2.5

12.5

2.8

2.4

Benzylpenicillin

Ampicillin

Gentamicin

RERE + HE

RE

1.0

1.3

2.8

0.5

0.8

1.3

0.9

1.2

2.2

0.7

1.0

1.8

Oral Dosing of Antimicrobial Drugs in Ruminants

PROPERTY CONSEQUENCE

Ruminal microflora metabolise many drugs

Low bioavailability

Dilutional effect in rumen (120 litres in adult cow, pH = 6.5)

Low bioavailability and/or slow absorption

Drug suppression of normal flora leading to overgrowth of pathogens

Life threatening disease and/or reduced fermentative digestion

Oral dosing usually restricted to pre-ruminant calves (aged <4-6 weeks)

Parenteral Dosing of Antimicrobial Drugs in Ruminants and Pigs

Use of depot formulations giving sustained release from intramuscular injection sites

a) aqueous suspensions e.g. Procaine benzylpenicillin, clavulanate potentiated amoxycillin

b) oily suspensions e.g. Procaine benzylpenicillin

c) high strength solutions in organic solvents, drug precipitating at injection site e.g. oxytetracycline

Use of Depot/Sustained Release Formulations

CONSEQUENCES FOR PHARMACOKINETICS, EFFICACY, TOXICITY AND DOSING:

• Commonly involve flip-flop pharmacokinetics

• Rising phase half-life represents elimination

• Declining phase half-life represents absorption

• Avoidance of peaks and troughs of concentration

• Maintenance of effective concentrations for 1-4 days

• Useful for time-dependent drugs (maintenance of T>MIC)

• Problem of injection site residue in muscle

Residues of Antimicrobial Drugs in Food Producing Species

• Muscle, fat, liver, kidney, eggs, milk

• In EU maximum residue limits (MRLs) set by CVMP

• MRLs set on basis of residue concentration anda) No Observable Effect Level (NOEL) in animal toxicity studiesb) No microbiological effect level (NMEL)

• Acceptable daily intake (ADI) determined using safety factor (SF) of 100 to 1000 (NOEL) or 10 (NMEL)

Drug Pharmacokinetics in Mastitis Therapy in Cattle

Good 1) Lipid soluble neutral molecules

2) Lipid soluble weak organic bases

Poor 1) Ionised molecules (strong acids and bases)

2) Lipid soluble weak organic acids

3) Lipid insoluble weak organic bases

PASSAGE OF BLOOD : MILK BARRIER

Blood pH = 7.35 – 7.40Milk pH = 6.50 – 6.80  1 + 10 (pHm – pKa)

Rm/p = --------------------------- Weak acid1 + 10 (pHp - pKa)

Milk : Plasma Concentration Ratios in Cattle

DRUG

 

LIPID

SOLUBILITY

 

PKa

 

 

Milk ultrafiltrate : Plasma Ultrafiltrate

Theoretical

Experimental

ACIDS*

Benzylpenicillin

Sulphadimethoxine

Sulphamethazine

Low

Moderate

Moderate

2.7

6.0

7.4

 

0.25

0.20

0.58

0.13-0.26

0.23

0.59

BASES**

Trimethoprim

Spiramycin

Streptomycin

Polymyxin B

High

High

Low

Very low

7.3

8.2

7.8

10.0

 

2.32

3.57

3.13

3.97

 

2.90

4.60

0.50

0.30* Improved penetration in mastitis** Improved/reduced penetration in mastitis

Prescott, Baggot and Walker, 2000

Drug Pharmacokinetics in Mastitis Therapy in Cattle

                                     

Intramammary Dosing of Antimicrobial Drugs in Ruminants

Use of intramammary infusion tubes for treatment of mastitis in cattle

a) lactation therapy

b) dry cow therapy

PHARMACOKINETIC (and Efficacy ?) Variability of Antimicrobial Drugs in

Poultry

• MEDICATION

- Continuous dosing in water

- Pulse dosing in water

- In feed medication (pelletted food)

• VARIABILITY

- Inherent inter-animal variation in A, D, M, E

- Inter-animal variation in intake

AbsorptionBlood

Protein bound

Metabolism

Excretion

Metabolites

Distribution

Tissue bound

Poultry

Unique Anatomical Considerations

Poultry Unique Anatomical Considerations

1) Haematology / immunology

2) Respiratory / Pulmonary system

3) Musculoskeletal system

4) Reproductive system

5) Integument

6) Gastrointestinal

7)7) Excretory / urinary systemsExcretory / urinary systems

Excretory / urinary systems

1)1) Bilateral tri-lobed elongated kidneys, “resting” in Bilateral tri-lobed elongated kidneys, “resting” in synsacrum / retroperitoneal fossae synsacrum / retroperitoneal fossae

2)2) No bladder (ureters traverse to cloaca) No bladder (ureters traverse to cloaca)

3)3) Histologically “chaotic”, without specific medullary / Histologically “chaotic”, without specific medullary / cortical regions cortical regions

4)4) Urine produced is rich in uric acid. Uric acid / urates Urine produced is rich in uric acid. Uric acid / urates are a “white cap” on darker GI excretaare a “white cap” on darker GI excreta

5)5) Renal portal system supplies peritubular capillary Renal portal system supplies peritubular capillary network network

Enrofloxacin

Absorption:Absorption:

OralOral

Rapidly absorbed in monogastric species, Rapidly absorbed in monogastric species, preruminant calves and chicken. preruminant calves and chicken.

Absorption in adult ruminants is variable Absorption in adult ruminants is variable and has ranged from 10 to 50%. and has ranged from 10 to 50%.

Enrofloxacin

Distribution:Distribution:

Rapidly and widely distributed into all Rapidly and widely distributed into all measured body tissues and fluids in many measured body tissues and fluids in many species, including cats, cattle, chickens, dogs, species, including cats, cattle, chickens, dogs, horses, and rabbits. horses, and rabbits.

Enrofloxacin Concentration in chicken tissues (Enrofloxacin Concentration in chicken tissues (g/ml or g) g/ml or g) 1 h post treatment (O.S. 10mg/kg)1 h post treatment (O.S. 10mg/kg)

Brain 1.1

Lung 2.4

Heart 2.8

Spleen 2.5

Kidney 1

Liver 4.6

Muscle 2

Skin 1.1

Serum 1.4

Enrofloxacin

Enrofloxacin is metabolised to

ciprofloxacin

in chickens.

EnrofloxacinPreparation of dosage form:

1) The stock solutions should be prepared fresh daily.

2) Protect stock solution or medicated water from:1) Freezing2) Direct sunlight.

3) Use water with low hardness.

4) Galvanized metal watering systems or containers should not be used to carry or store this product (possible chelation with metal ions)

5) Chlorinators should not be operated while administering this medication.

Time (h)

Co

nce

ntr

atio

n (

g/m

l)Enrofloxacin serum concentrations after continuous

dosage at a dose rate of 50 ppm

0

0.2

0.4

0.6

0.8

1.0

1.2

0 24 48 72 96

PK/PD integration for enrofloxacin in chickens following continuous PK/PD integration for enrofloxacin in chickens following continuous OS administration at a dose rate of 50 ppm (10 mg/kg)OS administration at a dose rate of 50 ppm (10 mg/kg)

PK/PD

Integration

Mycoplasma

spp

Staphylococcus

spp

E.coli Salmonella

spp

MIC 50 0.25 0.12 0.06 0.03

Cmax/MIC 4.40 9.17 18.33 36.67

AUC24h/MIC 82.80 172.50 345.00 690.00

MIC 90 (100?) 1.00 1.00 0.50 0.50

Cmax/MIC 1.10 1.10 2.20 2.20

AUC24h/MIC 20.70 20.70 41.40 41.40

I Need Some Fresh Air

PHARMACOKINETIC (and Efficacy ?) Variability of Antimicrobial Drugs in Fish

• MEDICATION e.g. FURUNCULOSISBaths:

Short-term baths Long-term baths

Topical treatmentsInjectionMEDICATED FOOD

• VARIABILITY- Inherent inter-animal variation in A, D, M, E- Inter-animal variation in intake- Temperature dependency of pharmacokinetics- Temperature dependency of residues

Salmon Farming

Elimination Half-life of Antimicrobial Agents in Fish : Species and

Temperature DifferencesDRUG SPECIES TEMPERATURE

(C)

HALF-LIFE

(h)

Sulphadimidine Carp

Carp

Rainbow trout

Rainbow trout

10

20

10

20

50

26

21

15

Trimethoprim Carp

Carp

10

24

41

20

Oxytetracycline Rainbow trout

African catfish

12

25

90

80

Antibacterial Therapy in Fish

Antibacterials Dose(mg /kg/day - in feed)

Species Withdrawal time(Degree days)

Amoxycillin 40-80 for 10 days Salmon

Atlantic Salmon

50 or 80

40

Oxolinic acid 10 for 10 days Salmon, trout 500

Oxytetracycline 75 for 4-10 days Salmon, trout 400 - 500

Florfenicol 10 for 10 days Atlantic Salmon 150

Sarafloxacin 10 for 5 days Atlantic Salmon 150

Major indications: Furunculosis

Bacterial Disease in Bees

AMERICAN FOULBROOD• Spore forming bacterium: Paenibacillus larvae• Brood disease• High contagious• Treatment: burning hives and contaminated equipment

EUROPEAN FOULBROOD• Non-spore forming bacterium: Melissococcus pluton• “Stress” disease, most prevalent in spring/early summer• Treatment: oxytetracycline, ampicillin, mirosamicin

European Foulbrood

• Objective is to deliver drug to young larvae

• Drug must have good bioavailability after oral intake in adult bee

• Secreted from the jelly glands to jelly which is acidic (pH~4)

• Lipophilic and/or basic drugs are likely to achieve high concentrations in jelly by ion-trapping

• Drug should be stable in jelly

eggs are laidby the queen

3rd daythe eggs hatch

into worm-like larvae

7th dayFully grown larvae

Cells sealed offby workers

Only Royal Jelly Royal Jelly (Queen)

Royal Jelly + Pollen and Honey (Worker)

Honey Crop Stomach

absorptionJelly glands

Pollen / Syrup

Royal Jelly

Ventricle

Larva

Worker

Nakajima C, Sakogawa T, Okayama A, et al. Disposition of mirosamicin in honeybee hives. J Vet Pharmacol Ther (England), Aug 1998, 21(4) p269-73

Ventricle

Body nutrients

Disposition profile of Ampicillin (ABPC) in larvae after single dose of 30 mg/hives in syrup or paste.

Nakajima C, Okayama A, Sakogawa T, et al. Disposition of ampicillin in honeybees and hives. J Vet Med Sci (Japan), Sep 1997, 59(9) p765-7

--- Syrup

Paste

Disposition profile of mirosamicin (MRM) in honey bees after continuous administration for a week at a dosage of 200

mg/hive/week in pollen-substitute paste (hive 1-6).

Nakajima C, Sakogawa T, Okayama A, et al. Disposition of mirosamicin in honeybee hives. J Vet Pharmacol Ther (England), Aug 1998, 21(4) p269-73

Days after termination of dosing

Nakajima C, Sakogawa T, Okayama A, et al. Disposition of mirosamicin in honeybee hives. J Vet Pharmacol Ther (England), Aug 1998, 21(4) p269-73

Days after termination of dosing

Disposition profile of mirosamicin (MRM) in honey bees after continuous administration for a week at a dosage of 200

mg/hive/week in pollen-substitute paste (hive 1-6).

Tissue Cage Model of Acute Sterile Inflammation

• Implantation of perforated tissue cages at subcutaneous sites (4 per animal)

• Internal volume = 35 ml (calf, camel, horse)= 15 ml (pig)= 10 ml (sheep, goat)

• After >30 days, stimulation of granulation tissue by intracaveal injection 0.5 ml 1% carrageenan solution

• Withdrawal at pre-determined times of inflammed fluid (exudate)

• Withdrawal of non-inflammed fluid from control tissue cages (transudate)

PK/PD Integration of Fluoroquinolones

Tissue Cage Model of Sterile Inflammation

IV or IM administration (ruminant species) Measurement of serum, exudate and transudate drug

concentration-time relationships Calculation of PK parameters Measurement of ex vivo antibacterial activity in serum,

exudate and transudate against e.g. M. haemolytica (PD)

Integration of PK/PD data (sigmoidal E-max equation)

Mean ex vivo antibacterial activity of danofloxacin against M.haemolytica WOO251 in goat serum following

IM administration at a dose rate of 1.25mg/kg

1.E+00

1.E+03

1.E+06

1.E+09

0 6 12 18 24

Incubation time (h)

Lo

g c

fu/m

l

Con.

1 h

3 h

6 h

9 h

12 h

24 h

48 h

Ex vivo time killing curve and PK/PD integration in serum of goat 5, 9 h

sampleCss = 0.065 g/ml

1.00E+00

1.00E+01

1.00E+02

1.00E+03

1.00E+04

1.00E+05

1.00E+06

1.00E+07

1.00E+08

0 6 12 18 24

Incubation time (h)

Lo

g c

fu/m

l

Ex vivo AUC24h = 0.065 x 24 = 1.56 g.h/mlEx vivo AUC24h/MIC = 1.56 / 0.030 = 52

Sample Conc.

Bacterial Count

In vivo PK/PD integration in serum of goat 5 after single IM injection at a dose rate of 1.25

mg/kg Cmax / MIC = 12

T>MIC =13.5 h

0.001

0.010

0.100

1.000

0 6 12 18 24

Time (h)

Dan

ofl

oxa

cin

co

nce

ntr

atio

n (g

/ml)

Serum Conc.

AUC24h = 2.32 g.h/mlAUC24h/MIC = 2.32 / 0.030 = 77

In vivo and ex vivo PK/PD integration in serum of goat 5 after single IM injection at a

dose rate of 1.25 mg/kg D

ano

flo

xaci

n c

on

cen

trat

ion

(g

/ml)

Cmax / MIC = 12

T>MIC =13.5 h

0.001

0.010

0.100

1.000

0 6 12 18 24

Time (h)

Serum Conc.

Bacterial Count

AUC24h = 2.32 g.h/mlAUC24h/MIC = 2.32 / 0.030 = 77

-7

-6

-5

-4

-3

-2

-1

0

1

0 50 100 150 200 250 300

AUC24h / MIC

Observed

Predicted

Bacteriostatic AUC24h / MIC= 18

Bactericidal AUC24h / MIC = 39

Elimination AUC24h / MIC = 90L

og

cfu

/ml

dif

fere

nce

Sigmoidal Emax relationship for bacterial count versus ex vivo AUC24h / MIC in goat 1 serum

AUC24h / MIC Values and Hill Constants for Danofloxacin Determined Ex Vivo in

the Goat

AUC24h / MIC SERUM

EXUDATE TRANSUDATE

Bacteriostatic 22.61.7 18.3±1.6 23.4±4.0

Bactericidal 29.62.5 28.2±2.4 36.7±1.6

Elimination 52.48.1 55.1±4.4 55.5±2.0

Slope* 15.5±2.9 11.±3.9 13.4±3.9*Hill Constant

Values are mean standard error of the mean (n=6)

Ex Vivo AUC24h/MIC Values for Danofloxacin in Serum from Ruminant Species

AUC24h / MIC SPECIESCalf Sheep Goat Camel

Bacteriostatic 15.92.0

17.81.7 22.61.7 17.23.6

Bactericidal 18.11.9

20.21.7 29.62.5 21.23.7

Elimination 33.53.5 28.71.8 52.48.1 68.715.6Values are mean standard error of the mean (n=6)

Preliminary Study of Danofloxacin AUC/MIC in Calf Model of Pneumonia• M.haemolytica model of calf pneumonia (MIC = 0.03 g/ml)

• 18 calves allocated randomly to 3 groups of 6

- no treatment

- AUC / MIC = 15* (0.29 mg/kg danofloxacin)

- AUC / MIC = 120 ** (2.30 mg/kg danofloxacin)

* Prediction of poor response ) from in vivo pharmacokinetic

** Prediction of bacterial ) and ex vivo

and clinical cure ) pharmacodynamic data

Median in vivo antibacterial activity of a fluoroquinolone against M.haemolytica in bronchial secretions

-6

-4

-2

0

2

4

6

0 6 12 18 24

Controls

AUC / MIC = 15

AUC / MIC = 120

Ch

ang

e in

bac

teri

al c

ou

nt

(cfu

/ml)

Proposal for Future Studies

Clinical Disease e.g. Calf Pneumonia

Measurement of population pharmacokinetics

Determination of distribution of MICs

Distribution of MIC

• Normal

• Lognormal

• Bimodal

• Other

0

0.5

1

1.5

2

2.5

3

0 6 12 18 24

Time (h)

Co

nce

ntr

atio

n (

g/m

l)

Possible toxicity

Optimal efficacyPossible resistance

Simulated plasma concentration-time curves and their mean in a small population

PK/PD Integration of Antimicrobial Drugs in Clinical

TrialsDrug treatment of disease in given

populationPopulation PK measurementsDetermination of disease outcomeIntegration of PK/PD data using sigmoidal

E-max equation combined with appropriate mathematical modelling and statistical evaluation

Acknowledgments

• Pfizer Animal Health (Collaboration in and support of calf studies)– Tim Rowan – Simon Sunderland– Patxi Sarasola

• Moredun Research Institute (Collaboration in study of calf model of pneumonia)– Quintin McKellar – Willie Donachie

Animal•Anatomy•Physiology•Biochemistry

Disease •Microbiology•Clinical

Drug PK•Dissolution•Absorption•Distribution•Metabolism•Excretion•Residue

Drug PD•Spectrum•Type of activity•Potency•Resistance•Host toxicity

Rational Dosing of Antimicrobial Drugs