Pharmacokinetics, urinary excretion and milk penetration of levofloxacin

in lactating goats

A. GOUDAH &

K. ABO-EL-SOOUD

Pharmacology Department, Faculty of veterinary Medicine, Cairo University, Giza, Egypt

(Paper received 4 March 2008; accepted for publication 18 June 2008)

K. Abo-EL-Sooud, Pharmacology Department, Faculty of veterinary Medicine, Cairo University, PO Box 12211, Giza, Egypt.

E-mail: kasooud@hotmail.com

Levofloxacin, a recently introduced third-generation fluoroqui-

nolone, is the L-isomer ofloxacin and possesses excellent activity

against Gram-positive, Gram-negative and anaerobic bacteria

(North et al., 1998). Compared with other fluoroquinolones

(FQs), it also has more pronounced bactericidal activity against

organisms such as Pseudomonas, Enterobacteriaceae and Klebsi-

ella spp. (Klesel et al., 1995). Several species of staphylococci,

streptococci including Streptococcus pneumoniae, bacteroides,

clostridium, haemophilus, moraxella, legionella, mycoplasma

and chlamydia are susceptible to levofloxacin (Langtry & Lamb,

1998). The bactericidal effect of levofloxacin is achieved through

reversible binding to DNA gyrase and subsequent inhibition of

bacterial DNA replication and transcription (Fu et al., 1992).

Levofloxacin distributes well to target body tissues and fluids in

the respiratory tract, skin, urine and prostrate, and its uptake by

cells makes it suitable for use against intracellular pathogens.

However, it penetrates poorly into the central nervous system

(Langtry & Lamb, 1998). FQs act by a concentration-dependent

killing mechanism, whereby the optimal effect is attained by the

administration of high doses over a short period of time (Drusano

et al., 1993). This concentration-dependent killing profile is

associated with a relatively prolonged postantibiotic effect

(Aliabadi & Lees, 2001). The drug undergoes a limited metab-

olism in rats and human (Langtry & Lamb, 1998) and is

primarily excreted by kidney mainly as active drug. Inactive

metabolites (N-oxide and demethyl metabolites) represent <5%

of the total dose (Hurst et al., 2002). The pharmacokinetics of

levofloxacin has been fully investigated in humans (Chulavatna-

tol et al., 1999), rabbits (Destache et al., 2001), cats (Albarellos

et al., 2005) and calves (Dumka & Srivastava, 2006, 2007).

However, there is no information available on the pharmacoki-

netics of levofloxacin in goats. In view of the marked species

variation in the kinetic data of antimicrobial drugs, the present

study was undertaken to determine the pharmacokinetics,

urinary excretion and milk penetration of levofloxacin following

single intravenous (i.v.) and intramuscular (i.m.) administration

in lactating goats.

Tavanic� [100 mL vial of solution of levofloxacin hemihy-

drate equivalent to 500 mg (5 mg ⁄ mL) levofloxacin] was

purchased from Aventis, Frankfurt, Germany and Mueller–

Hinton agar from Mast Group Ltd., Merseyside, UK.

Six adult lactating goats weighing 27–35 kg and aged from 3

to 5 years were determined to be clinically healthy before the

study based on physical examination. The goats were fed on

barley, alfalfa hay and wheat straw with free access to food and

water. The animals did not receive any drug treatment before the

study. The study was approved by the Bioethics Committee of the

Faculty of Veterinary Medicine, Cairo University. The study was

performed in two phases, following a crossover design (2 · 2)

with a 15-day washout period between the two phases. Three

animals were given a single i.v. injection into the left jugular

vein at a dose of 4 mg ⁄ kg bodyweight (b.w.) levofloxacin, and

the other three were injected i.m. into the semimembranous

muscle with the drug at the same dose. Five millilitre venous

whole blood samples were taken by jugular venepuncture into

10 mL heparinized Vacutainers (Becton Dickinson Vacutainer

Systems, Rutherford, NJ, USA). The sampling times were 0

(blank sample), 0.08, 0.166, 0.33, 0.5, 0.75, 1, 2, 4, 6, 8, 10,

12, 18, 24, 36, 48 and 72 h after treatment. All the blood

samples were centrifuged at 3000 g for 15 min to separate the

plasma. The plasma samples were frozen at )20 �C until

analysis. After a washout period of 2 weeks, the animals that

had been injected i.v. with the drug were injected i.m. and vice

versa. Blood was collected and processed as above. Urine and

milk samples were also collected simultaneously from the same

animals at various predetermined time intervals of 0.5, 1, 2, 4,

6, 8, 10, 12, 18, 24, 36, 48 and 72 h postadministration. The

urine samples were collected via a rubber balloon catheter

(Folatex No.12; Sewoon Medical Co., Ltd, Seoul, Korea) previ-

ously inserted in the bladder and their volumes were measured.

Milk samples were collected by hand stripping both halves of the

udder. Complete evacuation of the udder was carried out after

each sampling. The concentration of levofloxacin in plasma,

urine and milk samples was estimated by a standard microbi-

ological assay (Bennett et al., 1966) using Escherichia coli ATCC

10536 as test micro-organism. This method estimated the level

of drug having antibacterial activity, without differentiating

between the parent drug and its active metabolites. The reasons

why we selected the bioassay are: (i) bioassay measures the total

activity which could be more practical for pharmacodynamic

evaluations than HPLC (McKellar et al., 1999); (ii) the bioassay

method is precise, reproducible and does not require neither

J. vet. Pharmacol. Therap. 32, 101–104, doi: 10.1111/j.1365-2885.2008.01001.x. SHORT COMMUNICATION

� 2008 The Authors. Journal compilation � 2008 Blackwell Publishing Ltd 101

exhausted extraction nor toxic solvents (Ev Lda & Schapoval,

2002) and (iii) because there is no report on the clinically

relevant active metabolites in rats or human beings, the

application of the microbiological assay for measuring levoflox-

acin concentration is suitable (Albarellos et al., 2005). Each

sample was measured in triplicate and a standard curve was

prepared using normal goat plasma. Serial twofold dilutions from

25 to 0.05 lg ⁄ mL were used. Inhibition zones around the

sample wells were measured and compared with inhibition zones

produced by the standards. Urine samples were diluted with

phosphate buffer before assaying as they had higher concentra-

tions and the dilution factor was recorded. The limit of

quantification was 0.05 lg ⁄ mL in different media. The method

was linear between 0.05 and 12.5 lg ⁄ mL (r = 0.995). The

mean percentage of inter- and intra-assay rate of eliminations of

variation was 8.76% and 3.65% respectively. The mean

percentage recoveries of levofloxacin from plasma milk and

urine were 95.78 ± 3.11%, 92.02 ± 5.26% and 98 ± 3.44%

respectively. The extent of protein binding was determined

in vitro according to the method described previously by Craig

and Suh (1991). This method was based on the diffusion of free

antibiotic into the agar medium. To estimate the protein binding

of levofloxacin, the drug was dissolved in phosphate buffer (pH

6.2) and antibiotic free goat’s plasma and milk at different

concentrations. The differences in the diameter of the inhibition

zone between the solutions of the drugs in the buffer and plasma

and milk samples were calculated. A computerized curve-

stripping program (R Strip; Micromath Scientific Software, Salt

Lake City, UT, USA) was used to analyse the concentration–time

curves for each individual animal after the administration of

levofloxacin by different routes. For the i.v. data, the appropriate

pharmacokinetic model was determined by the application of

Akaike’s Information Criterion (AIC) (Yamaoka et al., 1978).

The plasma concentration–time relationship was best estimated

as a two-compartment open model:

Cp ¼ Ae�at þ Be�bt

Where Cp is the concentration of drug in the plasma at time t;

A is the intercept of the distribution phase with the concentra-

tion axis expressed as lg ⁄ mL; B is the intercept of the elimination

phase with the concentration axis expressed as lg ⁄ mL; a is the

distribution rate constant expressed in units of reciprocal time

( ⁄ h); b is the elimination rate constant expressed in units of

reciprocal time ( ⁄ h) and e is the natural logarithm base. The i.m.

data were analysed by adopting a one-compartment open model.

This program also calculated noncompartmental parameters

using the statistical moment theory (Gibaldi & Perrier, 1982).

The maximum plasma concentration (Cmax) and time of

maximum plasma concentration (Tmax) were taken directly from

the curve. The area under plasma concentration–time curve

(AUC) and the area under the first moment curve (AUMC) were

calculated by using the method of trapezoids, and extrapolation

to infinity was performed.

The milk time–concentration data were analysed with R Strip

programme using the drug concentration at each sampling time

interval. The extent of drug penetration from the blood into

the milk was expressed as the ratios of AUCmilk ⁄ AUCplasma and

Cmax-milk ⁄ Cmax-plasma (Ziv et al., 1995).

Pharmacodynamic efficacy of levofloxacin was determined by

calculating the Cmax ⁄ MIC90 and AUC24 ⁄ MIC90 ratios following

i.m. administration using the respective mean MIC value for

susceptible organisms. There are no studies reporting MIC90

values for levofloxacin for bacteria isolated from goats. So, to

calculate the PK ⁄ PD efficacy predictors, the MIC90 value

(MIC £ 0.17 lg ⁄ mL) was used based on other veterinary FQs

against sensitive strains isolated from field of veterinary impor-

tance (Watts et al., 1997).

The mean plasma pharmacokinetic variables for levofloxacin

were statistically compared by nonparametric analysis, using the

Mann–Whitney test and INSTANT version 3.00 (GraphPad

Software). The mean values were considered significantly

different at P < 0.05, 0.01 and 0.001.

Clinical examination of all animals before and after each trial

did not reveal any abnormalities. No local or systemic adverse

reaction to levofloxacin occurred after i.v. or i.m. administration.

The mean plasma, milk and urine concentration–time profiles of

levofloxacin following single i.v. and i.m. administrations of

4 mg ⁄ kg b.w. are presented graphically in Fig. 1. The

mean ± SD values of plasma and milk pharmacokinetic param-

eters estimated from the curve fitting after i.v. or i.m. admin-

istration are shown in Table 1.

As there is no report of significant active metabolites in rats or

human beings, the application of the microbiological assay for

measuring levofloxacin concentration is suitable.

Plasma levofloxacin disposition curves after i.v. injection were

best fit to an open bicompartmental model in all the animals,

which is in accordance with the results reported for calves

(Dumka & Srivastava, 2007). The K12 ⁄ K21 ratio was 1.13

indicating a faster drug transportation rate from the central to

the peripheral compartment than redistribution from the

peripheral to the central compartment. The Vdss for levofloxacin

0 10 15 20 25 30 35 400.01

0.1

1

10

100

Plas

ma

and

urin

e co

ncen

trat

ions

(µg

/mL

)

Time (h)

0 10 20 2515 30 35 400.01

0.1

1

10

Milk

con

cent

ratio

ns (

µg/m

L)

Time(h)5

5

Fig. 1. Mean ± SD of plasma (�i.v.,•i.m.), urine (D i.v.,.i.m.) and milk

(oi.v.,•i.m.) concentrations of levofloxacin in lactating goats after i.v. and

i.m. administration of 4 mg ⁄ kg body weight (n = 6).

102 A. Goudah & K. Abo-El-Sooud

� 2008 The Authors. Journal compilation � 2008 Blackwell Publishing Ltd

was 0.73 L ⁄ kg in lactating goats indicating a relatively wide

distribution after i.v. administration and it was consistent with

those that reported for moxifloxacin by Fernandez-Varon et al.

(2006) in lactating goats (0.79 L ⁄ kg). Levofloxacin’s clearance

in lactating goats was (0.18 L ⁄ h ⁄ kg) similar to the value

reported in calves (0.19 L ⁄ h ⁄ kg, Dumka & Srivastava, 2007).

The extent of renal elimination varies across the FQs. Levoflox-

acin is eliminated primarily by the kidney, with the renal

clearance exceeding creatinine clearance by approximately 60%

(Martinez et al., 2006) suggesting the involvement of both

glomerular filtration and tubular section (Okazaki et al., 1991).

The finding was confirmed by a 24–35% decrease in renal

clearance of levofloxacin following doses of probenecid (Amini-

manizani et al., 2001). Elimination half-lives were (2.95 and

3.64 h) close to those reported for levofloxacin in calves after i.v.

and i.m. dosing respectively (Dumka & Srivastava, 2006, 2007).

The drug was eliminated from plasma after i.m. treatment at a

significantly slower rate than after i.v. treatment, suggesting the

presence of a ‘flip–flop’ effect at least after i.m. administration

(Toutain & Bousquet-Melou, 2004). In that model, the last phase

of the curve is determined by the absorption rate constant and

not by the apparent elimination constant, because the rate of

absorption is a limiting factor for the elimination process.

The peak plasma level of levofloxacin attained in the present

study was approximately 19-fold higher than the MIC of

levofloxacin and the drug was detected above the minimum

therapeutic plasma level up to 24 h of administration. Similar to

our findings, a peak plasma concentration of 3.07 lg ⁄ mL was

attained after single i.m. injection of levofloxacin in calves

(Dumka & Srivastava, 2006). The systemic bioavailability of

levofloxacin in lactating goats after i.m. administration was 85%

indicating good absorption of the drug from that injection site.

This value was lower than that reported for other FQs in

nonlactating goats such as moxifloxacin (97%; Fernandez-Varon

et al., 2006), difloxacin (90%; Marin et al., 2007) and danoflox-

acin (110%; Aliabadi & Lees, 2001). This variation may be

attributed to the more rapid elimination of fluoroquinolone in

lactating animals (Petracca et al., 1993).

The mean binding of levofloxacin to the plasma proteins of

goats (22%) was in accordance with the corresponding values of

24% in human (Langtry & Lamb, 1998). Nevertheless, it was

relatively lower to that reported (17%) in calves (Dumka &

Srivastava, 2006). The extent of protein binding in milk was

higher (37%) than in plasma. Similar finding has been reported

for norfloxacin in lactating cows (Gips & Soback, 1999).

Levofloxacin, as with several other FQs, is amphoteric because

of the presence of a carboxylic acid and one or more basic amine

functional groups. Passive diffusion across biological membranes

is a function of fluoroquinolone lipophilicity relative to the pKa

values of the two ionizable moieties. The good penetration of

levofloxacin from the blood into the goat’s milk at pH 6.5–6.7

was predicted. From this data, levofloxacin could have been

successful against susceptible mastitic pathogens in goats after

parenteral administration. Levofloxacin urine concentrations

were (10–18 times) much higher than those of plasma and milk

and they could be detected in urine till 36 h postinjections by

both the routes. The concentration of levofloxacin-equivalent

inhibitory units in the urine was very high, by approximately 30

times the MIC90 even 24 h after administration. High urinary

concentration of danofloxacin (58.58 lg ⁄ mL) has also been

reported after i.v. doses of 1.25 mg ⁄ kg in goats (Atef et al.,

2001). Approximately 55% of the microbiological activity of the

administered drug was recovered in the urine of goats within

24 h. These findings suggest that levofloxacin may be an

appropriate drug for treating urinary tract infections in goats.

It has been established that for concentration-dependant FQs, the

AUC0–24 ⁄ MIC90 is the most important efficacy predictor with the

rate of clinical cure being >80%, when this ratio is higher than

100–125 (Lode et al., 1998). A second predictor of efficacy for

concentration dependent antibiotic is the ratio Cmax ⁄ MIC90,

considering that values above 8–10 would lead to better clinical

results (Dudley, 1991). It is now accepted that high Cmax ⁄ MIC90

values are necessary to avoid bacterial resistance emergence

(Walker, 2000). MIC90 data of levofloxacin against caprine

Table 1. Pharmacokinetic parameters (mean ± SD) of levofloxacin in

lactating goats (n = 6) following i.v. and i.m. administration at a dosage

of 4 mg ⁄ kg bodyweight

Parameters Unit i.v. i.m.

Plasma

a (kab) h)1 2.1 ± 0.19 1.37 ± 0.18***

t1 ⁄ 2a (t1 ⁄ 2ab) h 0.31 ± 0.11 0.54 ± 0.10**

b(kel) h)1 0.24 ± 0.10 0.22 ± 0.02

t1 ⁄ 2b (t1 ⁄ 2el) h 2.95 ± 0.27 3.64 ± 0.42**

K21 h)1 0.87 ± 0.06 –

K12 h)1 0.98 ± 0.08 –

Vdss L ⁄ kg 0.73 ± 0.22 –

Cltot L ⁄ h ⁄ kg 0.18 ± 0.04 –

AUC0-¥ lgÆh ⁄ mL 23.94 ± 2.61 21.31 ± 1.24*

AUC0–24 lgÆh ⁄ mL 22.32 ± 3.11 21.14 ± 2.40

MRT h 3.74 ± 1.21 5.24 ± 1.12*

MAT h – 1.89 ± 0.71

Cmax lg ⁄ mL – 3.16 ± 0.46

Tmax h – 1.78 ± 0.32

F % – 84.91 ± 7.52

Cmax ⁄ MIC90 ratio – 18.6

AUC0–24 ⁄ MIC90 ratio – 124.5

Milk

Cmax lg ⁄ mL 3.65 ± 0.39 3.26 ± 0.34

Tmax h 0.45 ± 0.13 0.75 ± 0.21

t1 ⁄ 2b (t1 ⁄ 2el) h 3.67 ± 0.84 3.84 ± 0.76

AUC lgÆh ⁄ mL 18.25 ± 3.62 20.36 ± 2.91

Cmax milk ⁄ Cmax plasma Ratio NA 1.14 ± 0.0.12

AUCmilk ⁄ AUCplasma Ratio 0.81 ± 0.13 1.01 ± 0.18*

b, Elimination rate constant; t1 ⁄ 2a, distribution half-life; t½ab, absorption

half-life; t1 ⁄ 2b, elimination half-life; t½el, elimination half-life; K12 and

K21, first-order rate constants for drug distribution between the central

and peripheral compartments; Vdss, volume of distribution; Cltot, total

body clearance; AUC, area under the curve from zero to infinity by the

trapezoidal integral; MRT, mean residence time; MAT, mean absorption

time; Cmax, maximum plasma or milk concentration; Tmax, time to peak

concentration; F (%), bioavailability; MIC90, minimum inhibitory con-

centration of drug in plasma.

*P < 0.05; **P < 0.01; ***P < 0.001.

Pharmacokinetics, urinary excretion and milk penetration 103

� 2008 The Authors. Journal compilation � 2008 Blackwell Publishing Ltd

bacterial isolates have not been reported up to now. So if we take

into account, MICs of other veterinary FQs against sensitive

strains of different micro-organisms isolated from field of

veterinary importance (Watts et al., 1997) and using the

surrogate ratios ratios AUC0–24 ⁄ MIC90 (124.5) and Cmax ⁄ MIC90

(18.6), levofloxacin could be effective by the i.m. route at

4 mg ⁄ kg against bacterial isolates with MIC £ 0.17 lg ⁄ mL.

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