**4.1.2 Penethamate**

442 A Bird's-Eye View of Veterinary Medicine

We determined once again the T>MIC90 in the milk of each quarter (MIC90= 6.4:1.6 µg.ml-1) (See Table 7). It was observed that concentrations achieved in cows receiving combination therapy (IM + IMM) were higher than those in cows treated intramammarly alone, which

Fig. 4. Mean concentrations of AMX-CLA in milk of healthy and mastitic quarters after administration of 3 IMM syringes (200 mg-50 mg each) in combination with three 3.5 mg.kg-

Significant effects of quarter health status on the PK parameters were found. The Cmax resulted higher in milk from sick quarters (P= 0.0384). The level of production had significant effect over milk AMX Cmax and Tmax, these were higher in the mammary quarters of low production cows. The level of production also affected the PK profile of CLA, the Cmax, Tmax and AUC0-∞ were higher in animals of low production. The differences observed between CL/Fmam suggest that the ATMs removal rate was higher in the quarters of high production cows. Significant effect of the mammary quarter health status and production level on the T>MIC90 was found. It should be emphasized that the T>MIC determined after the combined treatment (IM + IMM) exceeded those calculated

**T>MIC90 (%)** 62.64 ± 8.87 78.25 ± 23.16 65.31 ± 20.33 79.48 ± 18.67 Table 7. T>MIC90 (%): percentage of the period between 0 and 12 h post-administration during which the concentration was ≥ the MIC90; Amx:Cla 4:1, the MIC90 was considered as

Owens et al. (1988) found higher cure rates in *S. aureus* mastitis with combined treatment as compared with IMM treatment only. Recently, the therapeutical effects of parenteral, IMM and combination treatments with AMX-CLA have been compared by *Perner et al.* 2002 too. They found the combination treatment to be superior than parenteral or IMM treatment only. In this paper, the bacteriological cure rate for all causing agents and mastitis types (acute, subclinical and chronic), was 75.3%. We found also low bacteriological cure rates (62.5%) after AMX-CLA IMM infusion alone, but after the combined treatment the cure rate

**Xmastitic ± SD Xhealthy ± SD XM high prod. ± SD XM low prod. ± SD** 

1 of 15% AMX-CLA IM administrations every 12 h

was a logical finding.

after IMM infusion alone.

the ratio 4:1 (MIC90= 6.4:1.6 µg.mL-1)

was complete (100%) (Lucas et al., 2009a, b).

Penethamate hydriodide (PNTM) is a diethylaminoethyl ester of penicillin which, unlike salts of penicillin, is unionised and so exists in a neutral state. It is only weakly water soluble forming a suspension in an aqueous environment. After its intramuscular administration, is rapidly absorbed from the site of injection and on entering the blood, partially dissociates by hydrolysis into penicillin G and diethylaminoethanol. At the blood pH (7.4), equilibrium is established where 90% of the active drug is present in its hydrolyzed form (penicillin G) with the remainder persisting as PNTM. As PNTM leaves the circulation due to its neutral and lipophilic properties and its high affinity to milk, this equilibrium is maintained by reassociation of penicillin G and diethylaminoethanol until excretion is complete. PNTM easily passes the milk-blood barrier due to the pH gradient between milk (pH 6.6-6.8) and plasma (pH 7.2-7.4) and its weakly basic properties (pKa = 8.4). This is further facilitated by its highly lipophylic characteristics which facilitates its passage across the lipo-proteic blood-milk barrier. PNTM starts to dissociate as it passes over the barrier and this process continues during diffusion of the drug through the udder, releasing increasing quantities of penicillin G (PENG). PENG is rapidly ionised in the udder (pKa = 2.8) so limiting its return to the circulation. It therefore becomes "trapped" in the udder in increasing concentrations.

The same pH gradient between blood and milk presides in the case of mild to moderate udder inflammation such as in sub-clinical mastitis, the pH gradient between blood and milk is the same than in healthy animals thus generating similar PK behaviors to those which take place in the healthy udder. In acute mastitis, although the pH of milk is nearer that of blood due to a breakdown of the blood-milk barrier, higher concentrations of PNTM are still found in mastitic milk than in blood due to its lipophilic properties. Not only does undissociated PNTM rapidly and easily penetrate the udder whether inflammed or not, but its liposoluble nature gives it advantage, compared with other beta-lactam antibiotics such as amoxicillin and aminoglycosides to diffuse through the parenchyma of the udder, pass into the milk and penetrate the lactogenic cells. This diffusion through the udder is supported by the mechanism of "ion trapping" mentioned above and so explains the different penetration of PNTM compared with PENG (Friton et al., 2003).

It must be remembered, however, that *S. aureus* survives in acidic media, including phagolysosomes. Controversial in vitro/in vivo data exist on its susceptibility to antibiotics in such environments. We performed some studies to evaluate the effect of the pH variation on the antibacterial activity of penicillin against strains of *S. aureus* isolated from mastitic quarters (Moncada Cárdenas et al., 2009). MIC of *S. aureus* field strains and *S. aureus* ATCC 25923 were tested at pH 7.4, 6.5 and 5.0, in order to simulate the conditions of acidity of subcellular structures which are commonly associated with *S. aureus* intracellular persistence. The PEN MIC90 at pH 7.4 was consistent with those reported by CLSI 2007 (0.5 μg/mL) but at pH 5.0 (phagolysosomes) the activity of PENG increased markedly and almost linearly (~10 fold decrease in MIC -0.06 μg/mL) (Figure 5).

#### **4.1.3 Cloxacillin**

Cloxacillin (CLX) is used in the treatment or prevention of staphylococcal bovine mastitis. Its ATM activity against *S.aureus* is higher than that of PENG. There are strains of *S. aureus* resistant to isoxazolilpenicillins (oxacillin or methicillin resistant *S. aureus* –MRSA). These strains are a menace to public health.

Pharmacokinetic-Pharmacodynamic Considerations for Bovine Mastitis Treatment 445

In a recent study, we obtained an MIC90 of 2 μg.mL-1 against *S.aureus* (isolated from cattle

Fig. 6. Mean CLX concentrations in milk of dairy cows after IMM infusion of 250 mg CLX

**PARAMETER MEAN SD** 

**B (µg.mL-1)** 1288.557 464.800

**ß (h-1)** 0.160 0.016

**T 1/2ß (hs)** 4.370 0.458

**MTR (hs)** 6.982 1.469

Table 8. Mean pharmacokinetic parameters obtained in milk after IMM infusion of 250 mg

An experiment was conducted in cows with staphylococcal subclinical mastitis at drying off for the purpose of studying the pharmacokinetics behaviour of CLX benzathine in the dry

In Table 9 the CLX PK parameters in udder dry secretion are presented. We determined the T>MIC90 in the mammary dry secretion of each quarter. Normally, T>MIC90 is expressed as the percentage of time of the inter-dose interval during which the concentration remains above the MIC. Since we used a single dose of benzathine CLX in a slow-release formulation, there was no inter-dose interval. Therefore, with the objective of evaluating the PK behaviour based on the MIC, we estimated T>MIC90 as hours post-treatment during

The milk:plasma passage rate was very low and the CLX concentrations in plasma could not be measured. For mastitis treatment by IMM route it is desirable the permanence of the

udder secretion after a single IMM infusion (Lucas et al., 2009b) (Fig. 7).

ATM in the mammary compartment or glandular tissue for a long time.

**AUC0-all (µg.h/ml)** 7372.005 2062.654

with subclinical mastitis) (Lucas et al., 2009a).

three times at 12 hour intervals in each mammary quarter.

CLX in each quarter three times at 12 hour intervals.

which the ATM concentration was above the MIC.

Fig. 5. Inhibition of *S. aureus* exposed to different concentrations (expressed as Log10) of PEN (0.25 MIC; 0.5MIC; 1MIC; 2MIC; 4MIC and 8 x MIC) in function of the time and the medium pH (5; 6.5 and 7.4).

Benzathine CLX is used to treat bovine mastitis by IMM route at drying off and may be associated to other compounds, eg antiinflammatory agents (prednisolone) or other ATMs (eg. AMX, AMP, streptomycin, cephalosporins, etc.). Intramammary products with combinations of two or even three ATMs were introduced due to suggested synergistic action and broad spectrum. The evidence of their efficacy against clinical mastitis is many times lacking and synergistic action has never been proven *in vivo* (Taponen *et al.* 2003; Ødegaard & Sviland, 2001). For mastitis treatment during lactation the formulations contain CLX as sodium salt and, in general, the drug is not combined with other compounds. After IMM administration, sodium CLX binds scarcely to the mammary tissue (25%), with a moderate passage milk:plasma.

We evaluated the pharmacokinetic behavior of sodium CLX in milk after its IMM administration in healthy lactating cows of low production. The experimental cows received syringes containing 250 mg sodium CLX IMM, three times at 12 hour intervals in each quarter. Milk samples were obtained at different post administration times (Fig. 6).

The PK analysis was performed after the last administration. Milk elimination half-life was rather prolonged (T½β = 4.37 ± 0.458 h), with levels in milk at 60 h post-last administration ≥0.35 µg.mL-1. In low production animals the elimination of the ATM is even slower, with longer milk half life (Table 8). Various benzathine CLX containing IMM suspensions are registered for use in cattle as antibiotics for IMM use at drying off. These formulations are recommended for routine use in cows at drying off to treat existing IMM infections and to provide prolonged protection against new infections during the next lactation.

The excretion through the mammary gland depends on the characteristics of the pharmaceutical excipient, production level (high vs. low), molecule characteristic and udder health status (Mestorino, 1993a). The elimination rate is affected also by the binding level to dry udder secretion, which is very high (80%) ( Ødegaard & Sviland, 2001).

Fig. 5. Inhibition of *S. aureus* exposed to different concentrations (expressed as Log10) of PEN (0.25 MIC; 0.5MIC; 1MIC; 2MIC; 4MIC and 8 x MIC) in function of the time and the medium

Benzathine CLX is used to treat bovine mastitis by IMM route at drying off and may be associated to other compounds, eg antiinflammatory agents (prednisolone) or other ATMs (eg. AMX, AMP, streptomycin, cephalosporins, etc.). Intramammary products with combinations of two or even three ATMs were introduced due to suggested synergistic action and broad spectrum. The evidence of their efficacy against clinical mastitis is many times lacking and synergistic action has never been proven *in vivo* (Taponen *et al.* 2003; Ødegaard & Sviland, 2001). For mastitis treatment during lactation the formulations contain CLX as sodium salt and, in general, the drug is not combined with other compounds. After IMM administration, sodium CLX binds scarcely to the mammary tissue (25%), with a

We evaluated the pharmacokinetic behavior of sodium CLX in milk after its IMM administration in healthy lactating cows of low production. The experimental cows received syringes containing 250 mg sodium CLX IMM, three times at 12 hour intervals in each

The PK analysis was performed after the last administration. Milk elimination half-life was rather prolonged (T½β = 4.37 ± 0.458 h), with levels in milk at 60 h post-last administration ≥0.35 µg.mL-1. In low production animals the elimination of the ATM is even slower, with longer milk half life (Table 8). Various benzathine CLX containing IMM suspensions are registered for use in cattle as antibiotics for IMM use at drying off. These formulations are recommended for routine use in cows at drying off to treat existing IMM infections and to

The excretion through the mammary gland depends on the characteristics of the pharmaceutical excipient, production level (high vs. low), molecule characteristic and udder health status (Mestorino, 1993a). The elimination rate is affected also by the binding level to

quarter. Milk samples were obtained at different post administration times (Fig. 6).

provide prolonged protection against new infections during the next lactation.

dry udder secretion, which is very high (80%) ( Ødegaard & Sviland, 2001).

pH (5; 6.5 and 7.4).

moderate passage milk:plasma.

In a recent study, we obtained an MIC90 of 2 μg.mL-1 against *S.aureus* (isolated from cattle with subclinical mastitis) (Lucas et al., 2009a).

Fig. 6. Mean CLX concentrations in milk of dairy cows after IMM infusion of 250 mg CLX three times at 12 hour intervals in each mammary quarter.


Table 8. Mean pharmacokinetic parameters obtained in milk after IMM infusion of 250 mg CLX in each quarter three times at 12 hour intervals.

An experiment was conducted in cows with staphylococcal subclinical mastitis at drying off for the purpose of studying the pharmacokinetics behaviour of CLX benzathine in the dry udder secretion after a single IMM infusion (Lucas et al., 2009b) (Fig. 7).

In Table 9 the CLX PK parameters in udder dry secretion are presented. We determined the T>MIC90 in the mammary dry secretion of each quarter. Normally, T>MIC90 is expressed as the percentage of time of the inter-dose interval during which the concentration remains above the MIC. Since we used a single dose of benzathine CLX in a slow-release formulation, there was no inter-dose interval. Therefore, with the objective of evaluating the PK behaviour based on the MIC, we estimated T>MIC90 as hours post-treatment during which the ATM concentration was above the MIC.

The milk:plasma passage rate was very low and the CLX concentrations in plasma could not be measured. For mastitis treatment by IMM route it is desirable the permanence of the ATM in the mammary compartment or glandular tissue for a long time.

Pharmacokinetic-Pharmacodynamic Considerations for Bovine Mastitis Treatment 447

additional antibiotic to extend the spectrum of the product. There are cephalosporins that, when administered systemically penetrates in significantly low levels to the mammary gland of the producing cow. Is the case of ceftiofur. Ceftiofur is an interesting tool because it can be used in cows that are producing milk to treat infections that are located out of the

The toxicity of chloramphenicol (CAP) has been the cause of its use being banned or strictly regulated worldwide. This has accentuated the need for an effective broad-spectrum antibiotic to be used in food producing animals. Florfenicol (FLF) is a synthetic fluorinated CAP analogue that is exclusively used in food animals for treatment of infectious diseases. PK studies showed that high concentrations, both in serum and milk, are obtained following IMM administration in cows, which led to propose its use for the treatment of bovine mastitis. The FLF MIC90 among *S. aureus* obtained by San Martin was 2 μg.mL-1 (2002). We

TAP is a structural analogue of CAP with marked toxicological differences (Mestorino et al., 1993b). TAP has a greater in vitro activity against some bacteria that are resistant to CAP. Although TAP has similarities in its antibacterial spectrum with that of CAP, there are marked pharmacological differences between the two drugs. TAP is more stable in solution, is not appreciably protein bound in the body (16%) and does not undergo significant biotransformation. The drug diffuses into intracellular spaces, the central nervous system and the aqueous humour (Mestorino et al., 1993a; b). We performed a complete study about the TAP PK behavior in lactating healthy Holstein cows after its administration by intravenous, intramuscular, subcutaneous routes (Fig. 8) and after IMM infusion in a unique quarter and in the complete gland (in the four quarters) (Mestorino et al., 1993a; 1993b; 1995). Serum PK after IV administration was described following a bicompartmental model. The antibiotic showed a rapid distribution, with a half life of 8.84 ± 4.34 min. Half life of elimination was 1.95 ± 0.55 h, indicating that TAP is rapidly eliminated in the bovine. Volume of distribution was high, 1404.18 ± 428.19 ml.kg-1. Milk concentrations were high, when the drug was administered systemically, with a mean time of gland penetration (T½P) rather fast of 36.58 ± 9.72 min and a half life of elimination from the gland of 3.62 ± 0.97 h, indicating that accumulation of TAP takes place in the mammary gland. A milk permanency

A monocompartmental model was used to describe the PKs of TAP after its IM and SC administration. After IM administration, TAP was rapidly absorbed, with a half life of 6.53 ± 6.25 min and a half life of elimination of 2.9 ± 0.50 h. Maximum concentration was 29.38 ± 6.90 µg.mL-1 and was found at 0.32 ± 0.09 h. Area under the curve was 68.45 ± 16.27 µg.h/mL and bioavailability 86.33 ± 19.08%. Milk therapeutic levels were maintained between 0.5 and 8 h with a Cmax of 17.05 ± 2.73 µg.mL-1 reached at 3.42 ± 0.19 h. Mammary gland half life of penetration was 51.11 ± 6.10 min and half life of elimination 5.31 ± 4.27 h. Ratio CmaxS:CmaxM was 1.81 and the ratio AUCM:AUCS 1.19. Theoretical milk permanency

After SC administration a maximum serum concentration of 19.83 ± 3.57 µg.mL-1 was obtained at 0.72 ± 0.12 h with a half life of absorption of 10.09 ± 5.37 min. Half life of

obtained a thianphenicol (TAP) MIC90 higher than FLF (>16 µg.mL-1) (Lucas, 2009b).

mammary gland (metritis, foot infections), without discarding milk.

time of 4.13 ± 1.13 days was determined (Table 10).

time was 4.79 ± 1.40 days.

**4.2 Phenicols** 

Therefore, these findings are acceptable considering that the formulation used is indicated for the IMM treatment of mastitis at drying off. The level of binding of CLX to dry udder secretion is high (86 ± 6.5 %), acting as a reservoir and increasing the persistence of the ATM inside the udder. The experimental animals maintained concentrations above the limit of quantitation by microbiological methods (0.03µg.mL-1) during the first 24-31 days. Oliver et al. (1990) found that after IMM administration of CLX at drying off, the secretion samples had detectable levels until 28-35 days post-treatment in some animals and until 42-49 in others. This finding shows a marked variability in CLX concentrations at drying off. The determinants of this variability are varied and complex: udder size, seasonal effects, body condition of cows, physiological changes in body condition at the start of the dry period and other factors affecting gland and body.

Fig. 7. Average CLX milk concentrations in dry udder secretion (mastitic vs. healthy) after the administration of one IMM syringe in each quarter at drying off (600 mg)


Table 9. CLX PKs in dry udder secretion in mastitic quarters vs. healthy quarters after its administration at dose of 600 mg in each quarter at drying off. T>CIM90: post-administration hours during which CLX concentrations remained above the MIC90 (2 μg.mL-1)

#### **4.1.4 Cephalosporins**

Several IMM products for the IMM treatment of dairy cattle contain cephalosporins, most commonly as a single product (e.g., cefquinome; cephapirin), but also in combination (cephalexin and kanamycin). The cephalosporins are semisynthetic antibiotics derived from cephalosporin C (produced by *Cephalosporium acremonium*). There are currently 4 generations of cephalosporins, which vary from the narrow-spectrum first generation through the expanded-spectrum fourth generation (Hornish and Kotarski, 2002), with all generations being used in veterinary medicinal products. When using a first-generation cephalosporin in an IMM preparation, there is a strong rationale for combining it with an additional antibiotic to extend the spectrum of the product. There are cephalosporins that, when administered systemically penetrates in significantly low levels to the mammary gland of the producing cow. Is the case of ceftiofur. Ceftiofur is an interesting tool because it can be used in cows that are producing milk to treat infections that are located out of the mammary gland (metritis, foot infections), without discarding milk.

#### **4.2 Phenicols**

446 A Bird's-Eye View of Veterinary Medicine

Therefore, these findings are acceptable considering that the formulation used is indicated for the IMM treatment of mastitis at drying off. The level of binding of CLX to dry udder secretion is high (86 ± 6.5 %), acting as a reservoir and increasing the persistence of the ATM inside the udder. The experimental animals maintained concentrations above the limit of quantitation by microbiological methods (0.03µg.mL-1) during the first 24-31 days. Oliver et al. (1990) found that after IMM administration of CLX at drying off, the secretion samples had detectable levels until 28-35 days post-treatment in some animals and until 42-49 in others. This finding shows a marked variability in CLX concentrations at drying off. The determinants of this variability are varied and complex: udder size, seasonal effects, body condition of cows, physiological changes in body condition at the start of the dry period and other factors affecting gland and body.

Fig. 7. Average CLX milk concentrations in dry udder secretion (mastitic vs. healthy) after

**Parameter Unit Mean mastitic ± SD Mean healthy ± SD T½λ** h 48.13 ± 51.27 39.53 ± 27.29 **ABC0-24h** µg.h.mL-1 2130.24 ± 797.31 2144.09 ± 850.06 **ABC0-<sup>∞</sup>** µg.h.mL-1 5003.07 ± 3292.88 5358.52 ± 3398.00 **TMR** h 62.98 ± 47.49 57.42 ± 35.07 **T>CIM90** 257.14 ± 188.45 231.54 ± 153.82 Table 9. CLX PKs in dry udder secretion in mastitic quarters vs. healthy quarters after its administration at dose of 600 mg in each quarter at drying off. T>CIM90: post-administration

Several IMM products for the IMM treatment of dairy cattle contain cephalosporins, most commonly as a single product (e.g., cefquinome; cephapirin), but also in combination (cephalexin and kanamycin). The cephalosporins are semisynthetic antibiotics derived from cephalosporin C (produced by *Cephalosporium acremonium*). There are currently 4 generations of cephalosporins, which vary from the narrow-spectrum first generation through the expanded-spectrum fourth generation (Hornish and Kotarski, 2002), with all generations being used in veterinary medicinal products. When using a first-generation cephalosporin in an IMM preparation, there is a strong rationale for combining it with an

the administration of one IMM syringe in each quarter at drying off (600 mg)

hours during which CLX concentrations remained above the MIC90 (2 μg.mL-1)

**4.1.4 Cephalosporins** 

The toxicity of chloramphenicol (CAP) has been the cause of its use being banned or strictly regulated worldwide. This has accentuated the need for an effective broad-spectrum antibiotic to be used in food producing animals. Florfenicol (FLF) is a synthetic fluorinated CAP analogue that is exclusively used in food animals for treatment of infectious diseases. PK studies showed that high concentrations, both in serum and milk, are obtained following IMM administration in cows, which led to propose its use for the treatment of bovine mastitis. The FLF MIC90 among *S. aureus* obtained by San Martin was 2 μg.mL-1 (2002). We obtained a thianphenicol (TAP) MIC90 higher than FLF (>16 µg.mL-1) (Lucas, 2009b).

TAP is a structural analogue of CAP with marked toxicological differences (Mestorino et al., 1993b). TAP has a greater in vitro activity against some bacteria that are resistant to CAP. Although TAP has similarities in its antibacterial spectrum with that of CAP, there are marked pharmacological differences between the two drugs. TAP is more stable in solution, is not appreciably protein bound in the body (16%) and does not undergo significant biotransformation. The drug diffuses into intracellular spaces, the central nervous system and the aqueous humour (Mestorino et al., 1993a; b). We performed a complete study about the TAP PK behavior in lactating healthy Holstein cows after its administration by intravenous, intramuscular, subcutaneous routes (Fig. 8) and after IMM infusion in a unique quarter and in the complete gland (in the four quarters) (Mestorino et al., 1993a; 1993b; 1995). Serum PK after IV administration was described following a bicompartmental model. The antibiotic showed a rapid distribution, with a half life of 8.84 ± 4.34 min. Half life of elimination was 1.95 ± 0.55 h, indicating that TAP is rapidly eliminated in the bovine. Volume of distribution was high, 1404.18 ± 428.19 ml.kg-1. Milk concentrations were high, when the drug was administered systemically, with a mean time of gland penetration (T½P) rather fast of 36.58 ± 9.72 min and a half life of elimination from the gland of 3.62 ± 0.97 h, indicating that accumulation of TAP takes place in the mammary gland. A milk permanency time of 4.13 ± 1.13 days was determined (Table 10).

A monocompartmental model was used to describe the PKs of TAP after its IM and SC administration. After IM administration, TAP was rapidly absorbed, with a half life of 6.53 ± 6.25 min and a half life of elimination of 2.9 ± 0.50 h. Maximum concentration was 29.38 ± 6.90 µg.mL-1 and was found at 0.32 ± 0.09 h. Area under the curve was 68.45 ± 16.27 µg.h/mL and bioavailability 86.33 ± 19.08%. Milk therapeutic levels were maintained between 0.5 and 8 h with a Cmax of 17.05 ± 2.73 µg.mL-1 reached at 3.42 ± 0.19 h. Mammary gland half life of penetration was 51.11 ± 6.10 min and half life of elimination 5.31 ± 4.27 h. Ratio CmaxS:CmaxM was 1.81 and the ratio AUCM:AUCS 1.19. Theoretical milk permanency time was 4.79 ± 1.40 days.

After SC administration a maximum serum concentration of 19.83 ± 3.57 µg.mL-1 was obtained at 0.72 ± 0.12 h with a half life of absorption of 10.09 ± 5.37 min. Half life of

Pharmacokinetic-Pharmacodynamic Considerations for Bovine Mastitis Treatment 449

*diphtheriae*, *Diplococcus pneumoniae*, *Staphylococcus albus*, *Streptococcus pyogenes*, *Streptococcus viridans*, *Bacteroides, Fusobacterium, Bordetella, Brucella, Haemophilus, Neisseria, Pasteurella, Shigella* and some vibrio strains. Some Bacilli, *Erysipelothrix*, *Staphylococcus aureus* and *Streptococcus faecalis* are sensitive to moderate concentrations of TAP (FAO: JECFA 47, 1997). When TAP was administered by IMM route in an individual quarter high concentrations in the nonmedicated quarters and in serum were determined. Although the experimental animals were milked every 12 h, TAP levels above the minimum inhibitory concentration for the majority of pathogens were maintained during 36 h in the treated quarter and between 8 and 12 h in the nontreated quarters and during less than 4 h in the case of blood (Mestorino et al., 1995). TAP passes easily and completely from the treated quarter to the homolateral quarter by simple diffusion, to the heterolateral quarters, on the other hand, it arrives through circulation. Time elapsed between the administration and the first measured concentration (Lag Time) was shorter in the case of the homolateral quarter (rear-left –RL-: 0.25 h), intermedial in the case of serum (0.54 h) and rear-right –RR- (0.58 h) and longer in the case of the front-right quarter –FR- (1.92 h). In the homolateral quarter, the main Cmax and the highest bioavailability were recorded. Maximum TAP concentrations of 75.30, 15.06, 19.03 and 15.79 µg.mL-1 in the RL, FR, RR and serum respectively, and the ratios of AUCtreat:AUCnontreat of 10.61, 1.81, 2.78 and 1.35 in the RL, FR, RR and serum respectively, suggests that the largest passage takes place from the administered quarter to the homolateral one, and that the arrival of TAP to the contralateral quarters is via the blood due to that the longitudinal medial intermammary septum is a highly impermeable (Fig. 9).

Fig. 9. Averaged milk and serum TAP concentrations for six lactating cows following IMM

The prototype of the macrolide class of antibiotics was introduced in 1952 under the name of erythromycin. Macrolides are frequently used in Argentina for bovine mastitis treatment, since high concentrations in milk are obtained following parenteral administration. Erythromicin, oleandomycin, tylosin and the modern tilmycosin, azithromycin and tulathromycin have been extensively used in bovines. The last three are drugs with

infusion in the front-left mammary quarter at dose of 25 mg.kg-1.

substantial improvements respect the older ones.

**4.3 Macrolides** 

elimination was 2.60 ± 0.60 h. AUC of 56.17 ± 7.33 µg.h/mL was calculated. Bioavailability was of 82.33 ± 13.36 %. Milk concentrations after SC administration were maintained above the MIC between 1 and 8 h. The Cmax was of 13.19 ± 3.47 µg.mL-1 at 3.42 ± 0.19 h. The half life of penetration was 41.97 ± 4.55 min and the half life of elimination 7.57 ± 4.47 h. ratio CmaxS:CmaxM and AUCM:AUCS were 1.57 and 1.03 respectively.

Fig. 8. Averaged serum and milk TAP concentrations for six lactating cows following intravenous, intramuscular and subcutaneous TAP administration at dose of 25 mg.kg-1


Table 10. TAP PKs in serum and milk after its administration at dose of 25 mg.kg-1 by IV, IM and SC routes in lactating Holstein cows. Withdrawal is the withdrawal time needed to obtain milk with TAP levels of less than 20 ng.mL-1.

As a conclusion, TAP is a highly bioavailable antibiotic, independently of the administration route, it arrives with ease to the mammary gland, reaching high levels there. That is why it can be considered an excellent option to use in mastitis therapeutics, especially against Gram negative pathogens. It has a bacteriostatic action against a broad range of microorganisms, although it may be bactericidal for some species under some conditions, and in concentrations 3 to 5 times higher than the bacteriostatic ones. Among the bacteria inhibited *in vitro* by relatively low concentrations of TAP are *Clostridium*, *Corynebacterium*  *diphtheriae*, *Diplococcus pneumoniae*, *Staphylococcus albus*, *Streptococcus pyogenes*, *Streptococcus viridans*, *Bacteroides, Fusobacterium, Bordetella, Brucella, Haemophilus, Neisseria, Pasteurella, Shigella* and some vibrio strains. Some Bacilli, *Erysipelothrix*, *Staphylococcus aureus* and *Streptococcus faecalis* are sensitive to moderate concentrations of TAP (FAO: JECFA 47, 1997).

When TAP was administered by IMM route in an individual quarter high concentrations in the nonmedicated quarters and in serum were determined. Although the experimental animals were milked every 12 h, TAP levels above the minimum inhibitory concentration for the majority of pathogens were maintained during 36 h in the treated quarter and between 8 and 12 h in the nontreated quarters and during less than 4 h in the case of blood (Mestorino et al., 1995). TAP passes easily and completely from the treated quarter to the homolateral quarter by simple diffusion, to the heterolateral quarters, on the other hand, it arrives through circulation. Time elapsed between the administration and the first measured concentration (Lag Time) was shorter in the case of the homolateral quarter (rear-left –RL-: 0.25 h), intermedial in the case of serum (0.54 h) and rear-right –RR- (0.58 h) and longer in the case of the front-right quarter –FR- (1.92 h). In the homolateral quarter, the main Cmax and the highest bioavailability were recorded. Maximum TAP concentrations of 75.30, 15.06, 19.03 and 15.79 µg.mL-1 in the RL, FR, RR and serum respectively, and the ratios of AUCtreat:AUCnontreat of 10.61, 1.81, 2.78 and 1.35 in the RL, FR, RR and serum respectively, suggests that the largest passage takes place from the administered quarter to the homolateral one, and that the arrival of TAP to the contralateral quarters is via the blood due to that the longitudinal medial intermammary septum is a highly impermeable (Fig. 9).

Fig. 9. Averaged milk and serum TAP concentrations for six lactating cows following IMM infusion in the front-left mammary quarter at dose of 25 mg.kg-1.

#### **4.3 Macrolides**

448 A Bird's-Eye View of Veterinary Medicine

elimination was 2.60 ± 0.60 h. AUC of 56.17 ± 7.33 µg.h/mL was calculated. Bioavailability was of 82.33 ± 13.36 %. Milk concentrations after SC administration were maintained above the MIC between 1 and 8 h. The Cmax was of 13.19 ± 3.47 µg.mL-1 at 3.42 ± 0.19 h. The half life of penetration was 41.97 ± 4.55 min and the half life of elimination 7.57 ± 4.47 h. ratio

Fig. 8. Averaged serum and milk TAP concentrations for six lactating cows following intravenous, intramuscular and subcutaneous TAP administration at dose of 25 mg.kg-1

**Parameter Serum IV Serum IM Serum SC Milk IV Milk IM Milk SC**  β (h-1) – Ke (h-1)M 0.40±0.16 0.24±0.05 0.28±0.06 0.21±0.067 0.18±0.07 0.15±0.10 T½β (h)- T½E(h)M 1.95±0.55 2.88±0.50 2.60±0.60 3.62±0.97 5.31±4.27 7.57±4.47 Kab(h-1)- KP(h-1)M 14.26±8.72 5.06±2.00 1.23±0.37 0.83±0.10 1.00±0.10 T½ab -T½PM (min) 6.53 ±6.25 10.09±5.37 36.58±9.72 51.11±6.10 41.97±4.55 AUC (µg.mL/h) 50.59 ±7.56 68.45±16.27 56.17±7.33 85.77±21.58 75.31±9.85 52.85±17.50 Cmax (µg.mL-1) 29.38±6.90 19.83±3.57 23.09±3.12 17.05±2.73 13.19±3.47 Tmax (h) 0.32±0.09 0.72±0.12 2.50±0.29 3.42 ±0.19 3.42±0.19 Lag time (h) 0.05±0.02 0.15±0.02 1.08±0.51 0.78 ±0.23 1.42±0.34

MIC90

Cmax-S/Cmax-M 1.81±0.64 1.57±0.39 AUCM/AUCS 1.72±0.47 1.19±0.41 1.03±0.26 Withdrawal (d) 4.13±1.13 4.79±1.40 3.23±0.86 Table 10. TAP PKs in serum and milk after its administration at dose of 25 mg.kg-1 by IV, IM and SC routes in lactating Holstein cows. Withdrawal is the withdrawal time needed to

As a conclusion, TAP is a highly bioavailable antibiotic, independently of the administration route, it arrives with ease to the mammary gland, reaching high levels there. That is why it can be considered an excellent option to use in mastitis therapeutics, especially against Gram negative pathogens. It has a bacteriostatic action against a broad range of microorganisms, although it may be bactericidal for some species under some conditions, and in concentrations 3 to 5 times higher than the bacteriostatic ones. Among the bacteria inhibited *in vitro* by relatively low concentrations of TAP are *Clostridium*, *Corynebacterium* 

CmaxS:CmaxM and AUCM:AUCS were 1.57 and 1.03 respectively.

F (%) 86.33±19.08 82.33±13.36

obtain milk with TAP levels of less than 20 ng.mL-1.

The prototype of the macrolide class of antibiotics was introduced in 1952 under the name of erythromycin. Macrolides are frequently used in Argentina for bovine mastitis treatment, since high concentrations in milk are obtained following parenteral administration. Erythromicin, oleandomycin, tylosin and the modern tilmycosin, azithromycin and tulathromycin have been extensively used in bovines. The last three are drugs with substantial improvements respect the older ones.

Pharmacokinetic-Pharmacodynamic Considerations for Bovine Mastitis Treatment 451

 Fig. 10. **(A)** Mean concentrations of SPM in mastitic quarters and healthy quarters after two doses of 3.75 mg.kg-1 IM with a 24-h interval. **(B)** Mean concentrations of SPM in plasma in high-producing and low-producing cows; and quarters of high-producing cows and low-

**Parameter Milk High-Pr Milk low-Pr Plasma High-Pr Plasma Low-Pr**  Cmáx1 *µ*g.mL-1 4.26 ± 0.66 3.57 ± 0.61 0.12 ± 0.01 0.12 ± 0.01 Tmáx1 h 8.67 ± 2.31 9.33 ± 1.97 0.50 ± 0.00 0.50 ± 0.00 Cmáx2 *µ*g.mL-1 3.92 ± 0.71 3.01 ± 0.71 0.08 ± 0.02 0.11 ± 0.04 Tmáx2 h 36.00 ± 0.00 36.00 ± 0. 00 32.00 ± 6.93 32.00 ± 6.93 T½*λ*<sup>1</sup> h 12.26 ± 2.11 13.64 ± 4.20 27.83 ± 18.,38 25.19 ± 11.03 T½*λ*<sup>2</sup> h 21.17 ± 8.65 36.60 ± 7.61 7.70 ± 2.06 14.07 ± 5.56 ABC0-24h *µ*g.h/mL 70. 54 ± 10.62 56.44 ± 8.41 1.77 ± 0.30 2.14 ± 0.42 ABC0-<sup>∞</sup> *µ*g.h/mL 169.76 ± 30.66 171.80 ± 25.61 3.10 ± 1. 14 4.04 ± 0.83 TMR h 43.54 ± 10.93 55.96 ± 9.83 22.66 ± 0.99 28.78 ± 5.83 FM 57.73 ± 13.61 43.91 ± 10.30 - -

producing cows after two doses of 3.75 mg.kg-1 IM with a 24-h interval

Table 11. Milk and plasma PK parameters obtained after two doses of SPM IM administration (3.75 mg.kg-1 every 24h) in high and low producing lactating cows.

2009b; 2009c).

Due to the described PK behaviour, AZT proved to be an interesting tool with potential for infections in soft tissues, although this should be backed by efficacy trials. Classically, macrolides have been considered drugs of choice to treat IMM infections not only because of their antibacterial efficacy but because of their favourable PK profile. On the basis of these antecedents we decided to investigate the distribution of AZT in plasma and milk after IV, IM and IMM administration in healthy and mastitic lactating Holstein cows and cows at drying-off (Turic et al., 2003a; 2003b; 2006; Errecalde et al., 2003;Lucas et al.,

There are no AZT susceptibility studies on S. aureus isolated from bovine mastitis cases. However, there are data for other macrolides. Usually when running antibiograms, an erythromycin disk is used to evaluate susceptibility to the macrolide family. Variable percentages of macrolide resistant S. aureus ranging from 1.9% to 26.3% have been reported in several studies (Ziv., 1980a; Owens et al., 1997; Andrade et al., 2000). At present there are

## **4.3.1 Spiramycin**

Spiramycin (SPM) is a macrolide antibiotic that is active against most of the microorganisms isolated from the milk of mastitic cows. SPM is a very soluble weak base (pKa 8.2) which crosses lipid membranes with ease.

Our team investigated the disposition of SPM in plasma and milk after intramuscular administration. Lactating Holstein cows with subclinical mastitis caused by *S. aureus* were given two injections of SPM adipate at a dose of 16,000 IU/kg (3.75mg.kg-1) with 24 hours of intervals. The experimental animals were allocated by production level (high vs. low production) and the quarters were grouped by health state (Fig. 10 A and B).

In susceptibility studies, usually erythromycin is used as a representative of the macrolide group of compounds. However, when evaluating SPM susceptibility, it is not recommended to use erythromycin disks, because it has been shown that proteins involved in macrolideribosome interactions are different. The binding site of erythromycin is the L22 protein while for SPM the protein is the L27 (Lucas et al., 2007; 2009b). The MIC90 calculated for *S. aureus* field isolates was 4µg.mL-1(17 IU.mL-1) (Lucas et al., 2009b). Other authors have reported MICs of 3.25 µg.mL-1 (15 IU.mL-1) (Renard et al., 1996) and 8 µg.mL-1 (Friis et al., 1988). After IM SPM administration, milk concentrations were higher than those in plasma. The maximum concentration in milk (CmaxM) was 3.91µg.ml-1 at 9 hours post-first administration and 3.46 µg.ml-1 12 hours post-second administration. Whereas in plasma the measured concentrations were far below those in milk (0.12µg.ml-1 at 0.5 h and 0.097µg.ml-1 at 8 h post first and second administration respectively). An average milk-to-plasma ratio of 46.35 ± 11.09 was calculated by comparison of the areas under the concentration vs time curves. Our results were coincident with other authors (Renard et al., 1996). The level of production factor exert a significant effect on Cmax, T½λ, AUC0-24, MRT and Fmilk (Table 11). SPM in milk of high production animals reached higher concentration with AUC0-24h also higher in this group. However, T½λ was longer in the milk of low production cows. The average milk-to-plasma ratio was higher in high producing cows. Since the milk production level has a significant effect on efficacy predictors such as T>MIC90 (41.60) and AUC0- 24/MIC90 (15.87) milk SPM concentrations obtained were insufficient to achieve optimal PK/PD relationships.

### **4.3.2 Azithromycin**

Azithromycin (AZT) is a semisynthetic compound in which the lactone ring has been expanded to a 15-member structure and is considered the prototype of the new macrolide structures identified as azalides (Lucas et al., 2007; 2009c). AZT formulations are not available for use in production animals but could possess advantages for the treatment of certain bovine infections such as those produced in the mammary gland. We performed a complete study about its pharmacokinetic behaviour in blood and milk following administration by different routes, both in healthy cows and cows with mastitis. The results observed after its administration by any of the analyzed routes was typical of the macrolides, with low plasma concentrations and very high concentrations in milk and soft tissues; a great volume of distribution and a prolonged terminal half-life both in blood and milk. The fact that AZT tends to accumulate in inflammatory cells, has a kinetic incidence in the results, especially after IMM administration.

Spiramycin (SPM) is a macrolide antibiotic that is active against most of the microorganisms isolated from the milk of mastitic cows. SPM is a very soluble weak base (pKa 8.2) which

Our team investigated the disposition of SPM in plasma and milk after intramuscular administration. Lactating Holstein cows with subclinical mastitis caused by *S. aureus* were given two injections of SPM adipate at a dose of 16,000 IU/kg (3.75mg.kg-1) with 24 hours of intervals. The experimental animals were allocated by production level (high vs. low

In susceptibility studies, usually erythromycin is used as a representative of the macrolide group of compounds. However, when evaluating SPM susceptibility, it is not recommended to use erythromycin disks, because it has been shown that proteins involved in macrolideribosome interactions are different. The binding site of erythromycin is the L22 protein while for SPM the protein is the L27 (Lucas et al., 2007; 2009b). The MIC90 calculated for *S. aureus* field isolates was 4µg.mL-1(17 IU.mL-1) (Lucas et al., 2009b). Other authors have reported MICs of 3.25 µg.mL-1 (15 IU.mL-1) (Renard et al., 1996) and 8 µg.mL-1 (Friis et al., 1988). After IM SPM administration, milk concentrations were higher than those in plasma. The maximum concentration in milk (CmaxM) was 3.91µg.ml-1 at 9 hours post-first administration and 3.46 µg.ml-1 12 hours post-second administration. Whereas in plasma the measured concentrations were far below those in milk (0.12µg.ml-1 at 0.5 h and 0.097µg.ml-1 at 8 h post first and second administration respectively). An average milk-to-plasma ratio of 46.35 ± 11.09 was calculated by comparison of the areas under the concentration vs time curves. Our results were coincident with other authors (Renard et al., 1996). The level of production factor exert a significant effect on Cmax, T½λ, AUC0-24, MRT and Fmilk (Table 11). SPM in milk of high production animals reached higher concentration with AUC0-24h also higher in this group. However, T½λ was longer in the milk of low production cows. The average milk-to-plasma ratio was higher in high producing cows. Since the milk production level has a significant effect on efficacy predictors such as T>MIC90 (41.60) and AUC0- 24/MIC90 (15.87) milk SPM concentrations obtained were insufficient to achieve optimal

Azithromycin (AZT) is a semisynthetic compound in which the lactone ring has been expanded to a 15-member structure and is considered the prototype of the new macrolide structures identified as azalides (Lucas et al., 2007; 2009c). AZT formulations are not available for use in production animals but could possess advantages for the treatment of certain bovine infections such as those produced in the mammary gland. We performed a complete study about its pharmacokinetic behaviour in blood and milk following administration by different routes, both in healthy cows and cows with mastitis. The results observed after its administration by any of the analyzed routes was typical of the macrolides, with low plasma concentrations and very high concentrations in milk and soft tissues; a great volume of distribution and a prolonged terminal half-life both in blood and milk. The fact that AZT tends to accumulate in inflammatory cells, has a kinetic incidence in

production) and the quarters were grouped by health state (Fig. 10 A and B).

**4.3.1 Spiramycin** 

PK/PD relationships.

**4.3.2 Azithromycin** 

the results, especially after IMM administration.

crosses lipid membranes with ease.

Fig. 10. **(A)** Mean concentrations of SPM in mastitic quarters and healthy quarters after two doses of 3.75 mg.kg-1 IM with a 24-h interval. **(B)** Mean concentrations of SPM in plasma in high-producing and low-producing cows; and quarters of high-producing cows and lowproducing cows after two doses of 3.75 mg.kg-1 IM with a 24-h interval


Table 11. Milk and plasma PK parameters obtained after two doses of SPM IM administration (3.75 mg.kg-1 every 24h) in high and low producing lactating cows.

Due to the described PK behaviour, AZT proved to be an interesting tool with potential for infections in soft tissues, although this should be backed by efficacy trials. Classically, macrolides have been considered drugs of choice to treat IMM infections not only because of their antibacterial efficacy but because of their favourable PK profile. On the basis of these antecedents we decided to investigate the distribution of AZT in plasma and milk after IV, IM and IMM administration in healthy and mastitic lactating Holstein cows and cows at drying-off (Turic et al., 2003a; 2003b; 2006; Errecalde et al., 2003;Lucas et al., 2009b; 2009c).

There are no AZT susceptibility studies on S. aureus isolated from bovine mastitis cases. However, there are data for other macrolides. Usually when running antibiograms, an erythromycin disk is used to evaluate susceptibility to the macrolide family. Variable percentages of macrolide resistant S. aureus ranging from 1.9% to 26.3% have been reported in several studies (Ziv., 1980a; Owens et al., 1997; Andrade et al., 2000). At present there are

Pharmacokinetic-Pharmacodynamic Considerations for Bovine Mastitis Treatment 453

**Parameter Serum healthy Serum mastitic Milk healthy Milk mastitic** 

**λz (h-1)** 0.019±0.006 0.013±0.001 0.007±0.003 0.007±0.001 **t½λz (h)** 38.6±9.11 53.07±3.36 113.3±39.5 111.2±21.1

**AUC0-∞ (h.µg/ml)** 89.0±13.4 84.8±2.65 599.4±141.3 511.2±113.3 **MRT(h)** 52.1±8.94 69.5±7.61 163,6±58,6 159,6±34,7 **Tmax (h)** 9.0±3.29 12.0±9.86 **Cmax (µg/ml)** 3.82±0.49 3.13±0.66 **AUCM/AUCS** 389.4±69.4 232.3±52.7 Table 12. Serum and milk PK parameters obtained after one IV administration of AZT at a

**Parameter Serum healthy Serum mastitic Milk healthy Milk mastitic λz (h 1)** 0.018±0.004 0.015±0.003 0.008±0.003 0.005±0.001 **t½λz (h)** 39.11±7.64 47.31±9.75 105.19±40.18 135.96±39.49 **AUC0-∞ (h.µg/ml)** 36.26±8.59 26.09±1.71 561.89±60.55 713.36±249.99 **MRT(h)** 53.43±6.80 63.22±10.92 158.38±54.21 201.03±56.06 **Tmax (h)** 3.00±1,549 2.17±0.41 17.33±10.56 13.67±5.13 **Cmax (µg.ml-1)** 0.92±0.11 0.66±0.03 4.35±2.17 3.48±0.42

**AUCM/AUCS** 1041.57±516.7 1422.57±350. **Cmax M /Cmax S** 4.85±2.67 5.26±0.73 Table 13. Serum and milk PK parameters obtained after one IM administration of AZT at a

When comparing PK parameters by grouping quarters according to health status, it was observed that AZT was eliminated more slowly from and AUC0-∞ was substantially higher in mastitic quarters. Although this was an unexpected finding (the pKa partition hypothesis suggests the opposite), it is coincident with previously reported data (Turic et al., 2003a). Milk pH in the experimental animals ranged between 6.5 and 7.5 with the majority of values around 7.0. Average pH from all mastitic quarters was 7.13 ± 0.23 and from healthy quarters was 6.90 ± 0.21 (see Table 14). This is a normal finding for animals carrying subclinical mastitis. AZT is a weak base with a pKa value of 8.74, as a consequence, by application of the Henderson–Hasselbach equation, there would be approximately double AZT molecule dissociation in mastitic milk and more than three times in milk of healthy animals in comparison with plasma (see Table 14). Alkaline drugs (like AZT) are trapped in acidic compartments. This theoretical considerations could not, however, be confirmed by the experimental findings reported here. Azithromycin (IM) gave rise to very low plasma AUCs, which could be explained by its very high liposolubility and penetration into tissues. Although higher AUC was expected in milk of healthy animals (more acidic), which is a

**Varea (ml.kg-1)** 6325.6±1467.9 6486.3±318.6

dose of 10 mg.kg-1 in healthy and mastitic lactating Holstein cows

**F (%)** 41.38±13.98 18.860±2.51

dose of 10 mg.kg-1 in healthy and mastitic lactating Holstein cows

highly specific methods for susceptibility testing of veterinary pathogens (CLSI, 2008). The AZT MIC50 calculated for the 51 S. aureus isolations was 0.5 µg.mL-1 and the MIC90 was 1 µg.mL-1 (Lucas et al, 2009c). Although it is not advisable to compare MICs of different ATM agents, it is worth stating that erythromycin MIC90 for bovine isolated *S. aureus* was reported as 0.5 µg.mL-1 in several publications (Gianneechini et al., 2002; Russi et al., 2008).

The most prominent pharmacokinetic characteristic of AZT is the presence of high tissue concentrations which are maintained a long time after serum concentrations decline to very low levels (Fig 11 A y B). This characteristic was demonstrated after its IV and IM administration (Tables 12 and 13) (Turic et al., 2003a; 2003b), and was coincident with some authors in goats (Cárceles et al., 2005) and humans (Foulds et al., 1991). AZT T½λ was long, an expected finding, according to the characteristics of this ATM.

Milk AZT levels resulted much higher than those found in serum (Fig. 11 A y B), after each IV and IM administration which is a logical finding according to the lipophilicity and wide distribution of the drug.

Fig. 11. Mean concentration of AZT in serum and milk of healthy and mastitic lactating cows after IV (A) and IM (B) administration (10 mg.kg-1)

AZT exhibited major penetration into milk and it was cleared rather slowly. PK parameters indicated a high retention of the drug in peripheral compartments. The T½λ in milk after each route of administration was always at least four times longer that in plasma. AZT T½<sup>λ</sup> clearly suggested that milk concentrations decrease more slowly than plasma ones.

Later we performed other assay with AZT, but in this case it was administered intramuscularly in two doses of 10 mg.kg-1 body weight with a 48 h interval (Lucas et al., 2009c). The experimental animals were allocated by production level (high and low production levels) and the quarters were grouped by health state (Fig. 12 A and B). The T½<sup>λ</sup> in milk after first administration was at least four times longer than that in plasma. AZT T½<sup>λ</sup> suggested that milk concentrations exhibited a tendency to decrease more slowly than plasma ones. The same pattern was observed after a single 10 mg.kg-1 IM dose of AZT to lactating Holstein cows (Turic et al., 2003a).

highly specific methods for susceptibility testing of veterinary pathogens (CLSI, 2008). The AZT MIC50 calculated for the 51 S. aureus isolations was 0.5 µg.mL-1 and the MIC90 was 1 µg.mL-1 (Lucas et al, 2009c). Although it is not advisable to compare MICs of different ATM agents, it is worth stating that erythromycin MIC90 for bovine isolated *S. aureus* was reported

The most prominent pharmacokinetic characteristic of AZT is the presence of high tissue concentrations which are maintained a long time after serum concentrations decline to very low levels (Fig 11 A y B). This characteristic was demonstrated after its IV and IM administration (Tables 12 and 13) (Turic et al., 2003a; 2003b), and was coincident with some authors in goats (Cárceles et al., 2005) and humans (Foulds et al., 1991). AZT T½λ was long,

Milk AZT levels resulted much higher than those found in serum (Fig. 11 A y B), after each IV and IM administration which is a logical finding according to the lipophilicity and wide

Fig. 11. Mean concentration of AZT in serum and milk of healthy and mastitic lactating cows

AZT exhibited major penetration into milk and it was cleared rather slowly. PK parameters indicated a high retention of the drug in peripheral compartments. The T½λ in milk after each route of administration was always at least four times longer that in plasma. AZT T½<sup>λ</sup>

Later we performed other assay with AZT, but in this case it was administered intramuscularly in two doses of 10 mg.kg-1 body weight with a 48 h interval (Lucas et al., 2009c). The experimental animals were allocated by production level (high and low production levels) and the quarters were grouped by health state (Fig. 12 A and B). The T½<sup>λ</sup> in milk after first administration was at least four times longer than that in plasma. AZT T½<sup>λ</sup> suggested that milk concentrations exhibited a tendency to decrease more slowly than plasma ones. The same pattern was observed after a single 10 mg.kg-1 IM dose of AZT to

clearly suggested that milk concentrations decrease more slowly than plasma ones.

as 0.5 µg.mL-1 in several publications (Gianneechini et al., 2002; Russi et al., 2008).

an expected finding, according to the characteristics of this ATM.

after IV (A) and IM (B) administration (10 mg.kg-1)

lactating Holstein cows (Turic et al., 2003a).

distribution of the drug.


Table 12. Serum and milk PK parameters obtained after one IV administration of AZT at a dose of 10 mg.kg-1 in healthy and mastitic lactating Holstein cows


Table 13. Serum and milk PK parameters obtained after one IM administration of AZT at a dose of 10 mg.kg-1 in healthy and mastitic lactating Holstein cows

When comparing PK parameters by grouping quarters according to health status, it was observed that AZT was eliminated more slowly from and AUC0-∞ was substantially higher in mastitic quarters. Although this was an unexpected finding (the pKa partition hypothesis suggests the opposite), it is coincident with previously reported data (Turic et al., 2003a). Milk pH in the experimental animals ranged between 6.5 and 7.5 with the majority of values around 7.0. Average pH from all mastitic quarters was 7.13 ± 0.23 and from healthy quarters was 6.90 ± 0.21 (see Table 14). This is a normal finding for animals carrying subclinical mastitis. AZT is a weak base with a pKa value of 8.74, as a consequence, by application of the Henderson–Hasselbach equation, there would be approximately double AZT molecule dissociation in mastitic milk and more than three times in milk of healthy animals in comparison with plasma (see Table 14). Alkaline drugs (like AZT) are trapped in acidic compartments. This theoretical considerations could not, however, be confirmed by the experimental findings reported here. Azithromycin (IM) gave rise to very low plasma AUCs, which could be explained by its very high liposolubility and penetration into tissues. Although higher AUC was expected in milk of healthy animals (more acidic), which is a

Pharmacokinetic-Pharmacodynamic Considerations for Bovine Mastitis Treatment 455

groups according to median production allowed us to identify observations that may be extrapolated to situations of major differences in productive levels (Fig. 12 A). The AUC0-<sup>∞</sup> was higher in quarters of low-producing cows than in quarters of high-producing cows. It is possible that a lower milk production causes a slower antibiotic elimination, with a lower Clmam F value and more prolonged T½λ. On the basis of these results, we could suggest that low-producing cows have a high tendency to exceed LMR in milk while high-producing cows can eliminate (and dilute) the drug fast enough so as to diminish "contact time" and

AZT is a time dependent bacterial killing antibiotic with prolonged persistence (PAE, PASME). Thus, for this group, both t>MIC and AUC24h/MIC ratio play an important role in planning the dosage regimens. According to microbiological results, the MIC for *S. aureus* of bovine udder origin was 0.5µg.mL-1. The AUC24/MIC was greater than 100 in both healthy as mastitis cows (Table 15). The times above MIC were longer than 95 h for all cases. Six healthy and six mastitic lactating cows received one IMM AZT syringe containing 125 mg in each mammary quarter for three consecutive milkings; and six healthy and six mastitic Holstein cows at drying-off received one IMM AZT syringe containing 500 mg in

For the study in lactating cows, serum profiles evolved with three peaks and a final elimination phase after the last infusion. Serum penetration from milk was fast and the three peaks were in the same order of magnitude both in healthy and mastitic animals (Fig. 13 A). AUC serum concentration was however higher in healthy animals than mastitic animals. This resulted an unexpected finding, since AZT is a basic drug. No significant differences

**Parameters Mastitic ± SD Healthy ± SD H-prod ± SD L-prod ± SD AUC0-24h/MIC90** 156.53 ± 39.68 152.46 ± 33.19 152.53 ± 38.93 155.11 ± 31.51 **T>MIC90** 96.09 ± 0.74 96.48 ± 1.20 95.83 ± 0.00 96.88 ± 1.80

Table 15. PK PD parameters of AZT in mastitic and healthy quarters; and in quarters of high-producing cows and quarters of low-producing cows after two 10 mg. Kg-1 IM doses

Milk profiles, on the other hand, showed interesting differences. Peak concentrations resulted much higher in mastitic animals (Fig. 13 A). T ½ <sup>λ</sup> and MRT resulted in the same order of magnitude in both groups and the differences lacked statistical significance. Areas under the curves, however, showed major differences. In the case of the healthy animals AUC0-∞ was in the order of 503.03 and 1615.65 µg.h.mL-1 in mastitic animals, the difference being statistically significant (Table 16). This difference in AUC was coincident with the

Once again we found higher AZT concentrations in milk of mastitic animals than in milk of healthy animals after IMM administration, similarly as the situation occurred after IM

differences found in serum, although these were not statistically significant.

each mammary quarter (Turic et al., 2003a; Errecalde et al., 2003).

were found either in T ½ λ or MRT between healthy and mastitic animals.

\*MIC90 of AZT against the 51 S. aureus isolated

**4.3.2.1 AZT IMM administration** 

with a 48-h interval

clinical efficacy possibilities.

common finding with the classic macrolide antibacterials, we found exactly the opposite. In our experiment, the highest concentrations were determined in the milk of mastitic animals, with an AUCmilk AUCplasma ratio extremely high. Our explanation for this unexpected finding is the amount of somatic cells (SCC) present in mastitic milk in comparison with the normal milk. In the former case, the number of SCC was several times above those in normal milk. Mastitic milk normally exhibits very high cell counts as consequence of the inflammatory reaction. As it is known, AZT is able to reach high concentrations at infected sites, as a result of increased delivery from phagocytes (Lucas et al, 2009c). On this basis, we consider that the inflammatory reaction (and the high amount of cells) in the infected quarters is the main reason for the differences found between mastitic and healthy quarters.


Table 14. Average plasma pH, milk pH (mastitic vs. healthy), dissociation as a function of AZT pKa (8.74), theoretical ratio milk plasma and experimental AUC ratio milk plasma

Fig. 12 **(A)** Mean plasma and milk concentrations of AZT in high-producing and lowproducing cows after two 10 mg.kg-1 IM doses with a 48-h interval. **(B)** Mean concentrations of AZT in mastitic quarters and healthy quarters after two 10 mg.kg-1 IM doses with a 48-h interval

A significant AZT fraction could be trapped in the milk-cell compartment without participating of the plasma:milk equilibrium, largely dependent on the pKa – pH relationship. The AUC0-∞(P ≤ 0.05) and the MRT were higher in whole milk from mastitic quarters, which may indicate that the drug is present in higher amounts and persist during longer time in mastitic quarters than in healthy ones. At the same time, the Fmilk of AZT was higher in the mastitic quarters indicating a different PK profile of AZT depending on the quarter status. The previous data, reported after a single 10 mg.kg-1 IM dose of AZT to lactating Holstein cows, support our observations (Turic et al., 2003a). The Clmam F showed that AZT elimination was faster in healthy quarters than in mastitic quarters. Separating the

common finding with the classic macrolide antibacterials, we found exactly the opposite. In our experiment, the highest concentrations were determined in the milk of mastitic animals, with an AUCmilk AUCplasma ratio extremely high. Our explanation for this unexpected finding is the amount of somatic cells (SCC) present in mastitic milk in comparison with the normal milk. In the former case, the number of SCC was several times above those in normal milk. Mastitic milk normally exhibits very high cell counts as consequence of the inflammatory reaction. As it is known, AZT is able to reach high concentrations at infected sites, as a result of increased delivery from phagocytes (Lucas et al, 2009c). On this basis, we consider that the inflammatory reaction (and the high amount of cells) in the infected quarters is the main reason for the differences found between mastitic and healthy quarters.

**pH** 7.40 7.13 6.90 **Dissociated/non dissociated molecules** 21.88/1 40.74/1 69.18/1 **Theoretical ratio of dissociated molecules** 1 2.86 3.16 **Experimental Milk/plasma AUC** 743.60 482.72

Table 14. Average plasma pH, milk pH (mastitic vs. healthy), dissociation as a function of AZT pKa (8.74), theoretical ratio milk plasma and experimental AUC ratio milk plasma

(A) (B)

Fig. 12 **(A)** Mean plasma and milk concentrations of AZT in high-producing and lowproducing cows after two 10 mg.kg-1 IM doses with a 48-h interval. **(B)** Mean concentrations of AZT in mastitic quarters and healthy quarters after two 10 mg.kg-1 IM doses with a 48-h

A significant AZT fraction could be trapped in the milk-cell compartment without participating of the plasma:milk equilibrium, largely dependent on the pKa – pH relationship. The AUC0-∞(P ≤ 0.05) and the MRT were higher in whole milk from mastitic quarters, which may indicate that the drug is present in higher amounts and persist during longer time in mastitic quarters than in healthy ones. At the same time, the Fmilk of AZT was higher in the mastitic quarters indicating a different PK profile of AZT depending on the quarter status. The previous data, reported after a single 10 mg.kg-1 IM dose of AZT to lactating Holstein cows, support our observations (Turic et al., 2003a). The Clmam F showed that AZT elimination was faster in healthy quarters than in mastitic quarters. Separating the

interval

**Plasma Mastitic milk Healthy milk** 

groups according to median production allowed us to identify observations that may be extrapolated to situations of major differences in productive levels (Fig. 12 A). The AUC0-<sup>∞</sup> was higher in quarters of low-producing cows than in quarters of high-producing cows. It is possible that a lower milk production causes a slower antibiotic elimination, with a lower Clmam F value and more prolonged T½λ. On the basis of these results, we could suggest that low-producing cows have a high tendency to exceed LMR in milk while high-producing cows can eliminate (and dilute) the drug fast enough so as to diminish "contact time" and clinical efficacy possibilities.

AZT is a time dependent bacterial killing antibiotic with prolonged persistence (PAE, PASME). Thus, for this group, both t>MIC and AUC24h/MIC ratio play an important role in planning the dosage regimens. According to microbiological results, the MIC for *S. aureus* of bovine udder origin was 0.5µg.mL-1. The AUC24/MIC was greater than 100 in both healthy as mastitis cows (Table 15). The times above MIC were longer than 95 h for all cases.

Six healthy and six mastitic lactating cows received one IMM AZT syringe containing 125 mg in each mammary quarter for three consecutive milkings; and six healthy and six mastitic Holstein cows at drying-off received one IMM AZT syringe containing 500 mg in each mammary quarter (Turic et al., 2003a; Errecalde et al., 2003).

For the study in lactating cows, serum profiles evolved with three peaks and a final elimination phase after the last infusion. Serum penetration from milk was fast and the three peaks were in the same order of magnitude both in healthy and mastitic animals (Fig. 13 A). AUC serum concentration was however higher in healthy animals than mastitic animals. This resulted an unexpected finding, since AZT is a basic drug. No significant differences were found either in T ½ λ or MRT between healthy and mastitic animals.


\*MIC90 of AZT against the 51 S. aureus isolated

Table 15. PK PD parameters of AZT in mastitic and healthy quarters; and in quarters of high-producing cows and quarters of low-producing cows after two 10 mg. Kg-1 IM doses with a 48-h interval

#### **4.3.2.1 AZT IMM administration**

Milk profiles, on the other hand, showed interesting differences. Peak concentrations resulted much higher in mastitic animals (Fig. 13 A). T ½ <sup>λ</sup> and MRT resulted in the same order of magnitude in both groups and the differences lacked statistical significance. Areas under the curves, however, showed major differences. In the case of the healthy animals AUC0-∞ was in the order of 503.03 and 1615.65 µg.h.mL-1 in mastitic animals, the difference being statistically significant (Table 16). This difference in AUC was coincident with the differences found in serum, although these were not statistically significant.

Once again we found higher AZT concentrations in milk of mastitic animals than in milk of healthy animals after IMM administration, similarly as the situation occurred after IM

Pharmacokinetic-Pharmacodynamic Considerations for Bovine Mastitis Treatment 457

6.5 and 5.0, in order to simulate the conditions of acidity of plasma, tissue and subcellular structures which are commonly associated with *S. aureus* intracellular persistence. The results at pH 7.4, were consistent with those reported by CLSI 2008 (MIC: 1μg.mL-1).

> **Serum mastitic**

**λz ( h 1)** 0.078±0.04 0.050±0.02 0.01±0.00 0.02±0.00 **t½λz (h)** 11.76±7.31 16.20±7.20 59.67±12.48 46.85±20.87 **AUC0-∞ (h.µg/ml)** 3.51±2.25 2.74±1.11 503.03±49.47 1615.65±501.9 **MRT(h)** 17.37±10.56 24.69±11.20 76.37±18.32 52.41±28.39

**AUCM/AUCS** 143.31 589.65 Table 16. Serum and milk PK parameters obtained after one IMM AZT syringe containing 125 mg in each mammary quarter for three consecutive milkings in healthy and mastitic

> **Serum mastitic**

**λz ( h1)** 0.02±0.01 0.08±0.03 0.01±0.00 0.01±0.00 **t½λz (h)** 34.73±10.96 10.18±4.77 107.60±27.72 99.09±28.46 **AUC0-tlas (h. µg/ml)** 3.53±1.28 1.35±0.81 760.00±196.3 747.16±223.94 **AUC0-∞ (h.µg/ml)** 6.73±2.31 2.12±1.14 1136.11±262. 1056.71±171.4 **MRT(h)** 15.09±3.15 6.87±1.82 131.18±35.31 122.51±46.32

Table 17. Serum and milk pharmacokinetic parameters obtained after one IMM AZT syringe containing 500 mg in each mammary quarter of healthy and mastitic Holstein cows at

Tylosin (TYL), other antibiotic of the macrolide group, is commonly used in food animal practice. Because it is an organic base (pKa = 7.1), moderately bound by serum proteins (40%), with a high degree of lipid solubility (Lucas et al., 2007), TYL would be expected to be widely distributed in body fluids and tissues. The MIC of TYL for *S. aureus* was <1 µg.ml-1 for most isolates studied. We determined the elimination milk profile of TYL after IM

**Milk healthy** 

**Milk healthy**  **Milk mastitic** 

**Milk mastitic** 

However, MIC was approximately 16 times higher at pH 5.0 than at pH 7.4 (Fig. 14).

**Parameter Serum** 

lactating Holstein cows

**Parameter Serum** 

**AUCM/AUCS**

administration at multiple dose schemes.

drying off

**4.3.3 Tylosin** 

**healthy** 

**Tmax (h)** 22.75±0.27 22.67±0.26 **Cmax (µg/ml)** 0.23±0.05 0.13±0.02 **F (%)** 2.03±1.50 1.06±0.43

**healthy** 

**Tmax (h)** 1.25±0.61 2.08±2.18 **Cmax (µg/ml)** 0.22±0.08 0.21±0.19 **F (%)** 4.36±1.52 1.14±0.67

administration. As AZT is so penetrating in tissues and cells, and mastitic milk is so rich in somatic and inflammatory cells, the drug becomes included into the more acidic cellular compartment, thus hindering its participation in the milk-serum diffusion process (despite its pKa), and this retains high amounts of AZT in milk by cell trapping. All this evidence supports the fact that AZT is a penetrating azalide with concentrations several times higher in tissues and milk than in plasma. If we observe the ratios AUC(milk)/AUC(serum) and we compare them with those obtained when the drug was administered intramuscularly we could conclude that the high availabilities in milk compared to those of serum are independent of the route of administration.

But, in the study with cows at drying-off, we found some differences: AZT milk concentrations in mastitic and healthy animals were similar (Fig. 13 B and Table 17). One explanation for this is that physiology of the mammary gland during the dry period differs markedly from that during lactation. Very few cells (less than 2% are epithelial cells) and total leukocyte concentration increase rapidly in early involution and the milk fat and casein may decrease the leukocytes phagocytic function.

Fig. 13. Mean serum and milk AZT concentrations after one IMM AZT syringe containing 125 mg in each mammary quarter for three consecutive milkings in healthy and mastitic lactating cows (A). Mean serum and milk AZT concentrations after IMM syringe containing 500 mg in each mammary quarter of healthy and mastitic cows at drying off (B).

Finally, the excellent milk availability observed allows us to consider AZT as a potential antimastitic drug, although we have to fit the dosage through PK-PD modeling and corroborate with efficacy studies in the future. As was mentioned in previous paragraphs, the site where the pathogen is located represents one of the real challenges of ATM chemotherapy of the mammary gland. The pathogen can be in milk, or tissues. In the last case it can be in the interstitium or in cells. And in this case it can be in the cytoplasm or in fagolysosomes. As can be easily understood the deeper the location of the microorganism the more difficult will be to reach it by the ATM. Furthermore, if the ATM reaches the site of the microorganism, it has to exert its antibacterial effect and to do this it needs some special conditions, being the pH a critical one. And the pH becomes more acidic the deeper in tissues and cells it is measured. As illustrative figures we can mention the pH of plasma of 7.4, of interstitium 7.0, of cytoplasm 6.5 and of fagolysosome of 5.0. We evaluated the effect of the pH variation on the antibacterial activity of AZT against strains of *S. aureus* isolated of mastitic quarters. *S. aureus* strains isolated and *S. aureus* ATCC 25923 were tested at pH 7.4, 6.5 and 5.0, in order to simulate the conditions of acidity of plasma, tissue and subcellular structures which are commonly associated with *S. aureus* intracellular persistence. The results at pH 7.4, were consistent with those reported by CLSI 2008 (MIC: 1μg.mL-1). However, MIC was approximately 16 times higher at pH 5.0 than at pH 7.4 (Fig. 14).


Table 16. Serum and milk PK parameters obtained after one IMM AZT syringe containing 125 mg in each mammary quarter for three consecutive milkings in healthy and mastitic lactating Holstein cows


Table 17. Serum and milk pharmacokinetic parameters obtained after one IMM AZT syringe containing 500 mg in each mammary quarter of healthy and mastitic Holstein cows at drying off
