**3. Antimicrobial pharmacodynamic concepts**

Successful ATM chemotherapy depends on a correct diagnosis, selection of the appropriate ATM agent, and its administration with an adequate dosing scheme.

When considering the choice of ATM agent and dosage regimen, we need to consider the PKs of the chosen drug in the target animal species and the PD indices that drive its clinical effectiveness. For example, penicillins, like all β-lactam ATMs (penicillins, cephalosporins, carbapenems and monobactams), exhibit time-dependent killing. This means that maximum clinical effectiveness is achieved by ensuring that the free serum concentration of the selected β-lactam exceeds the MIC of the pathogen for the appropriate percentage of the dosing interval. If the pathogen is a Gram-positive organism, the targeted duration is usually ≥40% of the dosing interval. Instead, concentrations of most β-lactams should exceed the MIC of the pathogen by ≥80% of the dosing interval when the infectious agent is a Gram-negative organism. In other words, for drugs exhibiting time-dependent killing, increasing the concentration of the drug in excess of the MIC of the pathogen does not increase the killing rate. Rather, the extent of killing is dictated by the duration of time that bacteria are exposed to the drug.

On the other hand, other bactericidal ATM agents, as fluoroquinolones and aminoglycosides, exhibit concentration-dependent killing. In this situation, the rate of killing increases as the drug concentration increases above the MIC of the bacterial pathogen. Thus, ATM agents may be classified as those that exhibit time-dependent killing with null or brief post-antibiotic effect (e.g. β-lactams), time-dependent killing with prolonged post-antibiotic effect (e.g. glycopeptides), concentration dependent killing (e.g. fluorquinolones and aminoglycosides), and those that are generally considered to be bacteriostatic (e.g. tetracyclines, macrolides, lincosamides and phenicols).

Pharmacokinetic-Pharmacodynamic Considerations for Bovine Mastitis Treatment 433

**Azithromycin** " Prolonged AUC24/MIC **Lincosamides** " Brief AUC24/MIC

> Static and cidal (S.pneumoniae, S.pyogenes)

synthesis Cidal Time-

synthesis (30S) Static Time-

Static alone Cidal with sulfonamides

Static (Staph. and Enterococcus) Cidal (most Strep.)

Table 3. Relationships among drug, drug effects, and the PD surrogate most closely aligned to its clinical response (Martinez et al, 2006). \*Brief: less than one hour. Prolonged: up to six

Cidal(slower than β-lactams)

Static Time-

Time-

dependent

Time-

Cidal Concentration

Cidal Concentration

Static Time-

Static Time-

Time-

Time-

**effect Duration PAE PD** 

dependent Brief\* T>MIC

dependent Prolonged AUC24/MIC

Gram- bacteria: null or brief, Gram+: may be prolonged

dependent Prolonged AUC24/MIC



dependent Prolonged AUC24/MIC

dependent Prolonged AUC24/MIC

dependent Brief T>MIC

dependent Brief T>MIC

dependent Brief T>MIC

**Parameter** 

T>MIC

Cmax/MIC

Cmax/MIC

**Drug Mechanism Activity Bacterial** 

Binds to 50S ribosomes (inhibits proteins synthesis)

Binds to 50S ribosomes and some 30S ribosomal unit activity

Inhibits cell wall

Inhibits cell wall synthesis

Inhibit DNA gyrase, prents transcription and replication

Binds to 30S ribosome (inhibits protein synthesis) and disrupts biofilms

Inhibits protein synthesis by inhibition peptydyltransferase (50S)

Inhibits folic acid synthesis by inhibiting dihydrofolate reductase

PABA analogue interferes with folic acid synthesis

Inhibits initiation of protein synthesis (50S)

**Tetracyclines** Inhibits protein

**Macrolides Erythromycin,** 

**Azalides** 

**Ketolides (telithromycin)** 

**Β-lactams(e.g. penicillins, cephalosporins, carbapenems, monobactams)** 

**Glycopeptides (e.g.vancomycin)** 

**fluoroquinolone (e.g. enrofloxacin, ciprofloxacin, danofloxacin)** 

**Aminoglycosides (e.g.gentamycin, streptomycin)** 

**Phenicols (e.g.chloramphen icol, florfenicol, thiamphenicol)** 

**Trimethoprim** 

**Sulfonamides** 

**Oxazolidinones (linezolid)** 

hours.

Mouton et al. (2005) published an attempt to standardize the interpretation n of these various PK/PD parameters (Fig. 1). Some of the basic definitions are as follow:


Fig. 1. Illustration of the main PK/PD parameters that correlate with efficacy against extracellular infections.

The units associated with the AUC/MIC ratio are hours, by dividing this value by the dosing interval (e.g. 24 hour), we obtain the average plasma concentration over the steadystate 24-hour dosing interval relative to MIC, which may be far more informative than the traditional method for expressing this value.

The PD parameter providing the most appropriate surrogate for drug effectiveness is dependent on several factors. This includes: mechanism of action of the different drugs, whether its effects are time or concentration dependent, and the PAE duration (Table 3).

Mouton et al. (2005) published an attempt to standardize the interpretation n of these

 AUC (area under the concentration versus time curve). Should be expressed in terms of unbound drug. If multiple dosing regimens are applied, AUC should be measured over a 24 hour dosing interval at steady state. It should be noted that for compounds exhibiting linear PK, the AUC over a single dosing interval at steady state (AUC0-τ) is equal to AUC extrapolated to infinity (AUC0-∞) following single administration. AUC/MIC (AUC divided the minimum inhibitory concentration). Although sometimes given the dimension of time (generally 18 to 24 hours), this ratio can be more

 T>MIC (period of time during which the drug concentrations exceed the MIC). The cumulative percentage of a 24-hour period that the free drug concentration exceeds the

In vitro PAE (post-antibiotic effect). The period of suppression of bacterial growth after

 In vivo PAE. The difference in time for the number of bacteria in a tissue of treated versus control animals to increase 1 log10 over values when drug concentration in serum or at the infection site fall below the MIC (unit = time). The in vivo PAE includes any effect associated with sub-MIC concentrations. Sub MIC effect. Any effect of an

 Post-antibiotic sub-MIC effect. The effect of sub-MIC drug concentrations on bacterial growth following serial exposure to drug concentrations exceeding the MIC (unit =

short exposure of an organism to an ATM compound (unit = time).

ATM on a microorganism at concentrations below the MIC (unit = time)

Fig. 1. Illustration of the main PK/PD parameters that correlate with efficacy against

The units associated with the AUC/MIC ratio are hours, by dividing this value by the dosing interval (e.g. 24 hour), we obtain the average plasma concentration over the steadystate 24-hour dosing interval relative to MIC, which may be far more informative than the

The PD parameter providing the most appropriate surrogate for drug effectiveness is dependent on several factors. This includes: mechanism of action of the different drugs, whether its effects are time or concentration dependent, and the PAE duration (Table 3).

various PK/PD parameters (Fig. 1). Some of the basic definitions are as follow:

conveniently expressed as a dimensionless value.

MIC at steady-state pharmacokinetic conditions.

time)

extracellular infections.

traditional method for expressing this value.


Table 3. Relationships among drug, drug effects, and the PD surrogate most closely aligned to its clinical response (Martinez et al, 2006). \*Brief: less than one hour. Prolonged: up to six hours.

Pharmacokinetic-Pharmacodynamic Considerations for Bovine Mastitis Treatment 435

mutational event that could lead to the genesis of a less susceptible population. In infectious disease processes where there is a high bacterial burden (inoculum effect), the risk of a mutational event is increased due simply to the laws of probability (Craig and Dalhoff, 1998). In these cases, to ensure maximum killing, the targeted Cmax/MIC ratios are approximately 10 to 12 (Drusano et al., 1993). Such ratios ensure increased killing of susceptible organisms and an increased killing or inhibition of organisms with higher MICs. The goal in these situations is to reduce bacterial numbers to a level where the host can effectively handle those pathogens not killed by the ATM agent. While a high Cmax/MIC ratio (e.g. 10) is correlated with a high rate of bacterial kill for compounds exhibiting concentration-dependent killing, there are conditions under which AUC/MIC may be as or more predictive of a sustained ATM activity. AUC/MIC could be considered a major PK/PD parameter when the infection is caused by relatively slow growing bacteria, when there is little or no PAE that will contribute to inhibition of bacterial re-growth or when the

High drug concentrations relative to the MIC may contribute to an increase in the duration of the in vitro and in vivo PAE. For those bacteria/drug combinations that exhibit a PAE, in vivo PAEs have been shown to be longer than in vitro PAEs for most organisms. Thus, optimizing the Cmax/MIC ratio will delay the re-growth of the pathogen, sometimes by several hours. This type of dosing schemes results in fewer organisms remaining that can

For many compounds, the duration of the in vivo and in vitro PAE is substantially greater for Gram-positive than for Gram-negative pathogens. Because the duration of the in vitro and in vivo PAE of β-lactams tends to be negligible for Gram-negative species, it is recommended that concentrations of drug remain above the MIC of the pathogen for >80% of the dosing interval to combat this type of organisms. While a T>MIC of about 40% is

This difference in the duration of the in vitro and in vivo PAE may also be one of the reasons why the in vivo AUC/MIC for FQ tends to be less for Gram-positive than for Gramnegative pathogens. For Gram-negative organisms, the estimated AUC/MIC ratios needed to ensure effective treatment and prevent the selection of resistant strains is estimated to be approximately 100 to 125 (Forrest et al., 1993). In contrast, the AUC/MIC ratio for Grampositive bacteria is considerably lower, approximately 30 to 50 for a number of drugpathogen combinations (Wright et al., 2000). Studies involving the third and fourth generation FQ suggest that for Gram-positive organisms AUC/MIC values are substantially

Blood concentrations and MIC data alone cannot predict drug effectiveness. For example, using human and bovine estimated breakpoints for cephapirin and oxytetracycline, Constable and Morin (2002) showed that the MIC values predicted that the causative pathogens would be susceptible to both agents. However, these compounds were not effective in the treatment of acute bovine mastitis. In the same line, compounds effective in the treatment of acute bovine mastitis may be ineffective in the treatment of chronic bovine

evolve into a resistant subpopulation and can be managed by the host defenses.

lower when Cmax/MIC values are ≥10 (Nightingale et al., 2000).

MIC for the pathogen is relatively low.

**3.1 Post-Antibiotic Effects (PAE)** 

sufficient for staphylococcal species.

mastitis (Owens et al., 1997).

Whether a drug exhibits concentration-dependent or time-dependent killing is largely a function of the shape of its concentration-effect curve, the steeper the curve, the less will be the impact of increasing drug concentrations on the ATM response. Conversely, the more shallows the curve, the greater the relationships between the rates of bacterial kill versus the ATM drug concentration. This relationship can be described using a sigmoidal Emax model, also known as the Hill model, which can be described as follows (Toutain, 2002):

$$E(t) = E\_0 \frac{E \max \times C^h(t)}{EC\_{50}{}^h + C^h(t)} \tag{4}$$

Where:

*E(t)* is the effect observed for a given concentration at time t (*C* (*t*)) ; *Emax* is the maximal effect attributable to the drug; *EC*50 is the plasma concentration producing 50% of *Emax* ; *h* is the Hill coefficient, which adjust the degree of sigmoidicity in the curve; and *E*0 describes the rate of spontaneous cure. When *h*= 1, the Hill model reduces to the *Emax* model, which corresponds to a hyperbolic function. While there are certain characteristics common to all ATMs within a given drug class, there can be important differences in the PK/PD ratios needed to achieve a desired effect. Within

the fluoroquinolones (FQ), it has been demonstrated that the rate of kill and the duration of the in vitro PAE (Finberg et al., 2004; Firsov et al., 1998b) can be markedly different across compounds and microbial species. In some cases, the PK/PD relationship necessary to achieve a 2-log kill can also vary as a function of the microbial strain (Andes and Craig, 2002). Similarly, the AUC/MIC ratio of 100-125 frequently quoted as a target for FQ ATM activity may be an appropriate predictor of success for many Gram-negative infections, but lower AUC/MIC ratios (e.g. 35 to 40) may be appropriate for infections due to Gram positive organisms (Wright et al., 2000). With regard to the β-lactams, while there tends to be a substantial in vivo PAE for *S. aureus*, a substantially shorter PAE is associated with Gram negative organisms (Craig, 1993).

Within any given bacterial population, the possibilities of bacterial subpopulations that are less susceptible to the ATM agent exist. As demonstrated by Drusano (2004), unless these less susceptible pathogens are killed, succeeding microbial generations will re-populate the infection site with pathogens whose MIC values are higher than those found within the initial infection.

Accordingly, ensuring adequate exposure following an initial dose of a FQ is as important as insuring that high drug concentrations occur after repeated administration. Drug concentrations need to be adequate to either destroy the existing bacterial population at the site of the infection or to reduce its size to the point where the host defense mechanism can successfully control and eliminate the remaining pathogens.

For drugs exhibiting concentration-dependent killing, Cmax/MIC ratios may be particularly important when the pathogen has a high MIC value or is rapidly proliferating (Craig and Dalhoff, 1998). Rapidly proliferating bacteria have a greater likelihood of undergoing a mutational event that could lead to the genesis of a less susceptible population. In infectious disease processes where there is a high bacterial burden (inoculum effect), the risk of a mutational event is increased due simply to the laws of probability (Craig and Dalhoff, 1998). In these cases, to ensure maximum killing, the targeted Cmax/MIC ratios are approximately 10 to 12 (Drusano et al., 1993). Such ratios ensure increased killing of susceptible organisms and an increased killing or inhibition of organisms with higher MICs. The goal in these situations is to reduce bacterial numbers to a level where the host can effectively handle those pathogens not killed by the ATM agent. While a high Cmax/MIC ratio (e.g. 10) is correlated with a high rate of bacterial kill for compounds exhibiting concentration-dependent killing, there are conditions under which AUC/MIC may be as or more predictive of a sustained ATM activity. AUC/MIC could be considered a major PK/PD parameter when the infection is caused by relatively slow growing bacteria, when there is little or no PAE that will contribute to inhibition of bacterial re-growth or when the MIC for the pathogen is relatively low.
