**2. General pharmacokinetic considerations**

An important question regarding the treatment of mastitis is whether the ATM should accumulate in milk or in the udder tissue (Erskine et al., 2003). The target site may depend on the causative agent: streptococci are known to remain in the milk compartment, but *S. aureus* penetrates udder tissue and causes deep infection (Table 1). The most common route of administration of ATMs in mastitis is the IMM route. Efficacy of IMM treatment varies according to the pathogen, with the best therapeutic response being shown for mastitis caused by streptococci, coagulase-negative staphylococci, and Corynebacterium spp.


Table 1. Where to target ATM therapy in clinical mastitis due to different pathogens (Erskine et al., 2003)

#### **2.1 Parenteral antimicrobial treatments**

Systemic administration for treatment of mastitis was first used in the 70's (Ziv, 1980a). In acute cases, the IMM administration often fails due to poor and uneven distribution of the drug, either by the growth of breast parenchyma or blockage caused by the products of inflammation. In these circumstances, parenteral therapy is preferred (Mestorino, 1993a).

From the clinical point of view, the success of parenteral therapy depends mainly on the ATM passage from blood to milk (Ziv, 1980a). The ATM concentrations in highly vascularized tissues are equivalent to those determined in blood plasma. By contrast, in places where the irrigation is poor or those which are separated from the central compartment by biological membranes, drug levels are not equivalent (Baggot, 1986). The time during which concentrations in the mammary gland are effective depends largely on

1. Knowledge on the microbiological profile of mastitis. Clinical and laboratory diagnosis (isolation, typification and antibiogram) is one of the basic pillars for the rational use of

2. Knowledge on the PK and PD of antibiotics in milk producing animals, both healthy and mastitic. Understanding the relation PK/PD in these animals will result in an increase of ATM efficiency and a decrease in the selection of resistant strains, two

3. More efficient and safer prescription of antimastitic drugs. The development of new products should involve more than just the discovery of a new substance; it should deal with the full utilization of the effects of the agents in the organism. The way a drug is transported to the target location should be considered; once there, it should be

4. Establishment of the correct withdrawal periods for the antibiotics tested, following

An important question regarding the treatment of mastitis is whether the ATM should accumulate in milk or in the udder tissue (Erskine et al., 2003). The target site may depend on the causative agent: streptococci are known to remain in the milk compartment, but *S. aureus* penetrates udder tissue and causes deep infection (Table 1). The most common route of administration of ATMs in mastitis is the IMM route. Efficacy of IMM treatment varies according to the pathogen, with the best therapeutic response being shown for mastitis

**Milk/ducts Udder tissue Cow** 

caused by streptococci, coagulase-negative staphylococci, and Corynebacterium spp.

*Streptococcus agalactiae* +++ --- --- Other streptococci +++ + --- *Staphylococcus aureus* + +++ --- Coagulase-negative staphylococci +++ --- --- Arcanobacterium pyogenes (summer mastitis) --- ++ +++ Coliforms + -- +++ Table 1. Where to target ATM therapy in clinical mastitis due to different pathogens

Systemic administration for treatment of mastitis was first used in the 70's (Ziv, 1980a). In acute cases, the IMM administration often fails due to poor and uneven distribution of the drug, either by the growth of breast parenchyma or blockage caused by the products of inflammation. In these circumstances, parenteral therapy is preferred (Mestorino, 1993a).

From the clinical point of view, the success of parenteral therapy depends mainly on the ATM passage from blood to milk (Ziv, 1980a). The ATM concentrations in highly vascularized tissues are equivalent to those determined in blood plasma. By contrast, in places where the irrigation is poor or those which are separated from the central compartment by biological membranes, drug levels are not equivalent (Baggot, 1986). The time during which concentrations in the mammary gland are effective depends largely on

important aspects leading to the success of the therapy.

internationally agreed protocols.

**2. General pharmacokinetic considerations** 

available in the appropriate concentration and time to be effective.

ATM agents.

(Erskine et al., 2003)

**2.1 Parenteral antimicrobial treatments** 

the drug characteristics, the dose, the bioavailability of the molecule, the ability to penetrate the mammary gland and the microorganism susceptibility (Ziv, 1980b; Mestorino, 1993a).

The ability to penetrate the mammary gland or milk bioavailability (Fmilk) is determined by the ratio AUC0-∞ milk / AUC0-∞ plasma, as shown in the following equation:

$$F\_{milk} = \frac{ALIC\_{0-\infty \text{(milk)}}}{ALIC\_{0-\infty \text{(serum)}}} \tag{1}$$

This equation determines the relationship between the amount of ATM that is absorbed to the central compartment and the amount of ATM that passes through the mammary gland for reaches the milk compartment (Mestorino, 1993a).

Those antibiotics that have a high volume of distribution penetrate better into the mammary gland. However, differences in the degree of penetration blood: milk occur even among compounds that are chemically and structurally related. These differences can be explained by the principle of passive diffusion (Ziv, 1980b). ATMs cross biological membranes by passive diffusion or specialized transport.

Since the surface of the lipid portion of the membrane is extremely high, passive diffusion through membranes can be considered synonymous of diffusion through membrane lipids (Errecalde, 2004). The transfer in this case is directly proportional to the concentration gradient and the lipid-to-water partition coefficient of the ATM (Ziv, 1980b).

Weak organic acids and bases are found in milk and plasma as ionized or nonionized forms. The nonionized fraction is generally more soluble than the ionized one and diffuses better through the biological membrane (Ziv, 1980b; Errecalde, 2004; Mestorino, 1993a). The proportion of the drug in the nonionized form depends on the pKa of the molecule and the pH of the medium in which it is dissolved. When the molecules pass through the membrane by simple diffusion, are distributed according to their degree of ionization, the charge of their ionized form and the extent of protein binding. This is because the molecules bound to proteins or tissues are not able to cross membranes (Ziv, 1980b).

The theoretical relationship between the drug concentrations on both sides of a biological membrane can be calculated according to the Jacobs equation, that for organic acids such as penicillin G (pKa = 2.8) is the following:

$$\text{Ratio milk:}\\\text{plasma} = \frac{1 + 10pH\_{milk-pK\_a}}{1 + 10pH\_{plasma-pK\_a}} \tag{2}$$

And in the case of organic bases, such as spiramycin (pKa = 8.2) is:

$$\text{Ratio milk:}\\\text{plasma} = \frac{1 + 10pK\_a - pH\_{\text{millk}}}{1 + 10pK\_a - pH\_{\text{plasma}}} \tag{3}$$

The serum pH is 7.4 and the milk has a pH between 6.6 – 6.8. The organic bases administered by the parenteral route tends to accumulate in milk and remain there in its ionized form (ion trapping), thus achieving milk concentrations which exceed those in plasma. Instead, the concentrations of weak acids in milk are lower than those found in plasma (Erskine, 2002b).

Pharmacokinetic-Pharmacodynamic Considerations for Bovine Mastitis Treatment 429

In the case of a weak base, when administered parenterally, e.g intramuscularly, the rapid penetration from blood to milk is characterized by early appearance of measurable concentrations in this fluid, a ratio Cmax (plasma) /Cmax (milk) less than or equal to 1, a t-lag between the Cmax (plasma) and Cmax (milk) short and a ratio AUC (plasma) /AUC (milk) less than or

During the mastitis process, the chemical composition of milk presents changes consequence of the inflammatory process. There are ions, proteins and inflammatory cells passage from blood into the gland lumen, because of a great increase in the vascular permeability. The physical properties of milk are also affected and there is an increase of pH, conductivity and viscosity, while the density and redox potential decrease (Korhonen & Kaartinen, 1995).

In mastitic milk it is expected that the passage of organic bases is diminished as the pH increases in detriment to the ion trapping in milk. However, the lipid solubility of this kind of molecules suggests that the relationship milk:plasma is always greater than 1 (Ziv, 1980b). In the case of organic acids, the penetration is favored by the increase of milk pH. However, these compounds will reach milk: plasma ratios above 1 only when the milk pH exceeds 7.4. This indicates that lipophilic weak bases parenterally administered may have a certain

The IMM infusion is the more used administration route in the ATM treatment of mastitis. However, many IMM products have been released to the market without the necessary scientific support about its PK behavior and studies about its clinical and bacteriological efficacy. The benefits of IMM administration are the high concentrations reached in milk and less loss due to drug absorption and transfer processes through biological membranes. While the disadvantages of this route may be the uneven distribution of various compounds within the udder, the risk of mammary contamination by bacteria inoculation through the teat canal and the possible irritation of tissue breast by the formulation (Gruet et al., 2001). Even in vitro studies have shown that ATMs administered by IMM route can negatively affect the phagocytic process in the mammary environment (Nickerson et al., 1985; 1986).

After administration of an IMM infusion, the contact between the ATM agent and the pathogen within the mammary gland is subject to a series of successive events (Ziv, 1980c;

1. Pharmaceutical Phase: begins after drug administration including the following steps:

2. Pharmacokinetic Phase: Assumes the presence of the drug in milk (drug availability)

3. Pharmacodynamic Phase: The effect of the drug against bacteria in the infection site.

advantage in distribution into milk in comparison with acids (Ziv, 1980b).

**2.2 Intramammary antimicrobial treatments (IMM)** 

equal to 1 (Ziv, 1980b).

Mestorino, 1993a):

Drug dissolution

Disintegration of the formulation

and includes the following events:

Liberation of the drug in milk

 Absorption (milk: plasma) Distribution (local and systemic)

 Metabolism (systemic) Excretion (local and systemic)

That is to say that the majority of the drugs can be ionized or nonionized according to its pKa and the pH of the surrounding environment. Nonionized compounds have a higher lipid-water partition coefficient than the ionized ones and thus it is easier for them to diffuse through lipidic membranes. The amphoteric molecules, such as danofloxacin (pKa = 6.2 to 9.4) does not depend on the relationship pK/pH and therefore its distribution is essentially determined by the degree of lipid solubility of the molecule and consequently by its lipidwater partition coefficient.

Table 2 shows different ATMs with its theoretical and experimental milk: plasma ratios. Observing this table we can conclude that the diffusion of organic acids into milk is highly predictable, but the diffusion of organic bases can be predicted only when these are largely nonionized in plasma and have a moderate degree of lipid solubility.


Table 2. Partition of ATMs in plasma and milk in lactating animals. From Ziv G. (1980b)

In the case of a weak base, when administered parenterally, e.g intramuscularly, the rapid penetration from blood to milk is characterized by early appearance of measurable concentrations in this fluid, a ratio Cmax (plasma) /Cmax (milk) less than or equal to 1, a t-lag between the Cmax (plasma) and Cmax (milk) short and a ratio AUC (plasma) /AUC (milk) less than or equal to 1 (Ziv, 1980b).

During the mastitis process, the chemical composition of milk presents changes consequence of the inflammatory process. There are ions, proteins and inflammatory cells passage from blood into the gland lumen, because of a great increase in the vascular permeability. The physical properties of milk are also affected and there is an increase of pH, conductivity and viscosity, while the density and redox potential decrease (Korhonen & Kaartinen, 1995).

In mastitic milk it is expected that the passage of organic bases is diminished as the pH increases in detriment to the ion trapping in milk. However, the lipid solubility of this kind of molecules suggests that the relationship milk:plasma is always greater than 1 (Ziv, 1980b).

In the case of organic acids, the penetration is favored by the increase of milk pH. However, these compounds will reach milk: plasma ratios above 1 only when the milk pH exceeds 7.4. This indicates that lipophilic weak bases parenterally administered may have a certain advantage in distribution into milk in comparison with acids (Ziv, 1980b).
