**1. Introduction**

422 A Bird's-Eye View of Veterinary Medicine

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Bovine mastitis is a disease that affects dairy herd production, characterized by considerable economical loss due to diminished milk secretion, potential productive cow damage, increase in production costs and milk contamination. Intramammary infection (IMMI) is the most common reason for the use of antimicrobials in dairy cows. Antimicrobials (ATMs) have been used to treat mastitis for more than fifty years, but consensus about the most efficient, safe, and economical treatment is still lacking.

*Staphylococcus aureus* is considered one of the main bacteria causing bovine mastitis, which is widely distributed in different countries. *S. aureus* is Gram-positive cocci, catalase-positive and facultative anaerobe. The *Staphylococcus* genus comprises more than thirty species which are able to colonize many environments and are part of the cutaneous or mucousal flora of various animals and humans. The intracellular survival of *S. aureus* is believed to contribute to the recurrence of some infections such as mastitis. Some publications reported the ability of this pathogen to colonize multiple cell types. However the precise fate of intracellular *S. aureus* is still poorly understood.

The general lack of therapeutic success against subclinical mastitis caused by *S. aureus* has prompted a reevaluation of treatment strategies. Despite the availability of several antibiotics with good in vitro activity, cure rates are poor, suggesting that inadequate concentrations of active antibiotic are coming into contact with the infecting bacteria for sufficient time and /or adequate concentrations to be effective.

Over the last few years, much concern has been raised regarding the optimization of antibiotic use, owing to the worrying increase of bacterial resistance. In this context, progress in the field of anti-infective pharmacology has led to the emergence of a new discipline, referred to as pharmacokinetics/pharmacodynamics (PK/PD) of antibiotics, the discipline that strives to understand the relationships between drug concentrations and effects, both desirable (bacterial killing) and undesirable (bacterial resistance). Over the past 15 years, three key PK/PD parameters have been elaborated, which determine how

Pharmacokinetic-Pharmacodynamic Considerations for Bovine Mastitis Treatment 425

Antimicrobial PK/PD relationship reflects a correlation between the drug concentrations in the blood, the concentration of the biologically active drug at the site of the infection, and the microbial or clinical outcome (Levison, 2004). The PK component describes the processing of the drug by the host (absorption, distribution, metabolism, and elimination). The PD component describes the effect of the drug on the bacterial pathogen. By identifying an association between PK and PD for specific host-drug-microbe combinations, the PK/PD approach provides a valuable guide for estimating the doses and dosage regimens that can

The extent to which a drug has access into milk when given systemically, or is absorbed and distributes throughout the udder when given intramammarily, depends on its properties: lipid solubility, degree of ionization, and extent of binding to serum and udder proteins. High lipid solubility, poor degree of ionization and less plasma protein binding contributes to a better transfer into milk. With IMM preparations, the type of vehicle is also important. Weak organic bases like macrolides and sulfonamidestend to accumulate in milk in the ionized form after parenteral administration, and attain concentrations higher than those in blood. On the other hand, concentrations of weak acids like penicillins and cephalosporins in milk are significantly lower than those in blood. In the concentration-dependent group of ATMs (e.g. aminoglycosides and fluoroquinolones) concentration of several times the MIC for the target organisms at the infection site increases the efficacy. In the time dependent group (e.g. penicillins, cephalosporins and classical macrolides) the efficacy depends on the time during which the concentration of the drug exceeds the MIC, but high concentrations

While there are a number of factors that contribute to the PK/PD indexes (e.g. in vitro MIC of the drug, its post-antibiotic effects –PAE-, and sub-MIC effects), there are also several factors that the traditional PK/PD approach does not describe. For example, the in vitro MIC, which is the basis for the PD component of these indices, does not provide information on time to kill, time to maximum kill, log change within a fixed time, or the maximum reduction in viable bacterial counts. There are also many others in vivo factors that influence ATM effectiveness (e.g. anti-inflammatory effects, presence of bacterial biofilms, the drug´s ability to interfere with bacterial colonization on epithelial surface, and influence of the drug on toxin production and release). Furthermore, plasma drug concentrations do not necessarily reflect a compound´s ability to diffuse into the site of infection and into the

An ideal drug for mastitis therapy should have a low MIC for mastitis pathogens. As treatment should be efficient and targeted towards specific infections, Gram-negative and Gram-positive infections in fact would require different ATMs. Antimastitic drugs should preferably have bactericidal action, as phagocites act normally immediately after milking, but as time elapses they incorporates fat globules becoming "engorged" thus diminishing its phagocytic capacity. As a consequence milk phagocytes are less effective than plasma ones.

The objective of this study was to make a bibliographic compilation on the PK of different ATM agents in milk, based in our experience and some other relevant publications, and to establish the relationship between the PK and the PD interaction with the target bacteria. We consider that non prudent use of ATMs in dairy farms can be fought through the

implementation of rational therapeutical procedures based on the following items:

optimize the bacteriological or clinical outcome.

do not increase efficacy.

bacterial cell.

Milk should not interfere with ATM activity.

antibiotic concentrations reached in body fluids over time (as predicted from the PK profile of the drug) compare with potentially effective antibiotic concentrations (as deduced from the minimal inhibitory concentration (MIC) or minimal bactericidal concentration (MBC) of antibiotics in vitro). The first parameter is the time during which concentrations of the ATM are above the MIC (t > MIC), it links bactericidal effects to time and is critically dependent on the half-life of the drug, dosage and frequency of administration over a given period of time. The second parameter is the peak plasma concentration divided by the MIC (Cmax/MIC), it relates bactericidal effects to concentration, and is primarily dependent on the dose. The third parameter is the area under the concentration-time curve divided by the MIC (AUC/MIC), and it combines both types of effects, since it corresponds to the total amount of drug to which bacteria are exposed over the time period, and is directly related to the total dose given during that period and inversely proportional to the drug clearance (Van Bambeke et al., 2006).

These parameters are critical in predicting antibiotic activity and, therefore, in establishing dosages on a rational basis. The application of these parameters, however, have so far been limited to extracellular infections in well-vascularized tissues, because they are all based on serum antibiotic levels.

Antimicrobials exhibit three major patterns of ATM activity. The first pattern in characterized by concentration dependent killing and moderate to prolonged persistent effects. Higher concentrations would kill organisms more rapidly and more extensively than lower levels. The prolonged persistent effects would allow for the administration of large doses with long inter-dose periods. Microorganism regrowth is not immediate at the time in which the drug concentrations fall below the MIC. This called post antibiotic effect is variable between different drug types but always present for ATMs exhibiting this kind of killing. This pattern is observed with aminoglycosides, fluoroquinolones, daptomycin, ketolides, and amphotericin B. The goal of a dosing regimen for these drugs would be to maximize concentrations. The peak level and the AUC should be the pharmacokinetic parameters that would determine in vivo efficacy (Andes et al., 2001; Craig, 2001).

The second pattern is characterized by time-dependent killing and minimal to moderate persistent effects. High drug levels would not kill organisms better than lower concentrations. Furthermore, organism regrowth would start very soon after serum levels fall below the MIC. This pattern is observed with β-lactams, macrolides, clindamycin, and oxazolidinones. The goal of a dose regime for these drugs would be to optimize the duration of exposure. The duration of time that serum levels exceed some minimal value such as the MIC should be the major parameter determining the in vivo efficacy of these drugs (Andes et al., 2001; Craig, 2001).

The third pattern is also characterized by time-dependent killing, but the duration of the persistent effects is much prolonged. This can prevent any regrowth during the dosing interval. This pattern is observed with azithromycin, tetracyclines, quinupristin-dalfopristin, glycopeptides, and fluconazole. The goal of a dose regime is to optimize the amount of drug administered to ensure that killing occurs for part of the time and there is no regrowth during the dosing interval. The AUC should be the primary pharmacokinetic parameter that would determine in vivo efficacy (Andes et al., 2001; Craig, 2001).

antibiotic concentrations reached in body fluids over time (as predicted from the PK profile of the drug) compare with potentially effective antibiotic concentrations (as deduced from the minimal inhibitory concentration (MIC) or minimal bactericidal concentration (MBC) of antibiotics in vitro). The first parameter is the time during which concentrations of the ATM are above the MIC (t > MIC), it links bactericidal effects to time and is critically dependent on the half-life of the drug, dosage and frequency of administration over a given period of time. The second parameter is the peak plasma concentration divided by the MIC (Cmax/MIC), it relates bactericidal effects to concentration, and is primarily dependent on the dose. The third parameter is the area under the concentration-time curve divided by the MIC (AUC/MIC), and it combines both types of effects, since it corresponds to the total amount of drug to which bacteria are exposed over the time period, and is directly related to the total dose given during that period and inversely proportional to the drug clearance

These parameters are critical in predicting antibiotic activity and, therefore, in establishing dosages on a rational basis. The application of these parameters, however, have so far been limited to extracellular infections in well-vascularized tissues, because they are all based on

Antimicrobials exhibit three major patterns of ATM activity. The first pattern in characterized by concentration dependent killing and moderate to prolonged persistent effects. Higher concentrations would kill organisms more rapidly and more extensively than lower levels. The prolonged persistent effects would allow for the administration of large doses with long inter-dose periods. Microorganism regrowth is not immediate at the time in which the drug concentrations fall below the MIC. This called post antibiotic effect is variable between different drug types but always present for ATMs exhibiting this kind of killing. This pattern is observed with aminoglycosides, fluoroquinolones, daptomycin, ketolides, and amphotericin B. The goal of a dosing regimen for these drugs would be to maximize concentrations. The peak level and the AUC should be the pharmacokinetic

parameters that would determine in vivo efficacy (Andes et al., 2001; Craig, 2001).

The second pattern is characterized by time-dependent killing and minimal to moderate persistent effects. High drug levels would not kill organisms better than lower concentrations. Furthermore, organism regrowth would start very soon after serum levels fall below the MIC. This pattern is observed with β-lactams, macrolides, clindamycin, and oxazolidinones. The goal of a dose regime for these drugs would be to optimize the duration of exposure. The duration of time that serum levels exceed some minimal value such as the MIC should be the major parameter determining the in vivo efficacy of these drugs (Andes

The third pattern is also characterized by time-dependent killing, but the duration of the persistent effects is much prolonged. This can prevent any regrowth during the dosing interval. This pattern is observed with azithromycin, tetracyclines, quinupristin-dalfopristin, glycopeptides, and fluconazole. The goal of a dose regime is to optimize the amount of drug administered to ensure that killing occurs for part of the time and there is no regrowth during the dosing interval. The AUC should be the primary pharmacokinetic parameter that

would determine in vivo efficacy (Andes et al., 2001; Craig, 2001).

(Van Bambeke et al., 2006).

serum antibiotic levels.

et al., 2001; Craig, 2001).

Antimicrobial PK/PD relationship reflects a correlation between the drug concentrations in the blood, the concentration of the biologically active drug at the site of the infection, and the microbial or clinical outcome (Levison, 2004). The PK component describes the processing of the drug by the host (absorption, distribution, metabolism, and elimination). The PD component describes the effect of the drug on the bacterial pathogen. By identifying an association between PK and PD for specific host-drug-microbe combinations, the PK/PD approach provides a valuable guide for estimating the doses and dosage regimens that can optimize the bacteriological or clinical outcome.

The extent to which a drug has access into milk when given systemically, or is absorbed and distributes throughout the udder when given intramammarily, depends on its properties: lipid solubility, degree of ionization, and extent of binding to serum and udder proteins. High lipid solubility, poor degree of ionization and less plasma protein binding contributes to a better transfer into milk. With IMM preparations, the type of vehicle is also important.

Weak organic bases like macrolides and sulfonamidestend to accumulate in milk in the ionized form after parenteral administration, and attain concentrations higher than those in blood. On the other hand, concentrations of weak acids like penicillins and cephalosporins in milk are significantly lower than those in blood. In the concentration-dependent group of ATMs (e.g. aminoglycosides and fluoroquinolones) concentration of several times the MIC for the target organisms at the infection site increases the efficacy. In the time dependent group (e.g. penicillins, cephalosporins and classical macrolides) the efficacy depends on the time during which the concentration of the drug exceeds the MIC, but high concentrations do not increase efficacy.

While there are a number of factors that contribute to the PK/PD indexes (e.g. in vitro MIC of the drug, its post-antibiotic effects –PAE-, and sub-MIC effects), there are also several factors that the traditional PK/PD approach does not describe. For example, the in vitro MIC, which is the basis for the PD component of these indices, does not provide information on time to kill, time to maximum kill, log change within a fixed time, or the maximum reduction in viable bacterial counts. There are also many others in vivo factors that influence ATM effectiveness (e.g. anti-inflammatory effects, presence of bacterial biofilms, the drug´s ability to interfere with bacterial colonization on epithelial surface, and influence of the drug on toxin production and release). Furthermore, plasma drug concentrations do not necessarily reflect a compound´s ability to diffuse into the site of infection and into the bacterial cell.

An ideal drug for mastitis therapy should have a low MIC for mastitis pathogens. As treatment should be efficient and targeted towards specific infections, Gram-negative and Gram-positive infections in fact would require different ATMs. Antimastitic drugs should preferably have bactericidal action, as phagocites act normally immediately after milking, but as time elapses they incorporates fat globules becoming "engorged" thus diminishing its phagocytic capacity. As a consequence milk phagocytes are less effective than plasma ones. Milk should not interfere with ATM activity.

The objective of this study was to make a bibliographic compilation on the PK of different ATM agents in milk, based in our experience and some other relevant publications, and to establish the relationship between the PK and the PD interaction with the target bacteria. We consider that non prudent use of ATMs in dairy farms can be fought through the implementation of rational therapeutical procedures based on the following items:

Pharmacokinetic-Pharmacodynamic Considerations for Bovine Mastitis Treatment 427

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

*AUC*

*AUC*

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

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

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

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

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

1 10

1 10

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

 

*milk a plasma a pH pK pH pK* 

> *a milk a plasma*

*pK pH pK pH* 

(2)

(3)

Ratio milk:plasma = 1 10

Ratio milk:plasma= 1 10

0( ) 0( ) *milk*

 

*serum*

(1)

the ratio AUC0-∞ milk / AUC0-∞ plasma, as shown in the following equation:

*milk*

gradient and the lipid-to-water partition coefficient of the ATM (Ziv, 1980b).

proteins or tissues are not able to cross membranes (Ziv, 1980b).

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

*F*

for reaches the milk compartment (Mestorino, 1993a).

passive diffusion or specialized transport.

penicillin G (pKa = 2.8) is the following:

plasma (Erskine, 2002b).

