1. Comparision of Mutant Selection Window Hypothesis with Traditional
Pharmacodynamics
One of the questions frequently asked is how the selection window hypothesis relates to traditional PK/PD ideas. The short answer is that the selection window is a type of in vitro pharmacodynamics that is directed at predicting the propensity of particular bacterial-compound combinations for enrichment of mutants.

2. Pharmacodynamic Correlates with Cure
Cmax/MIC
Cmax
Time above MIC
AUC above MIC
Serum drug concentration
MIC
Time
Traditional pharmacodynamics can be described in three ways, i.e. there are three measures that correlate with a favorable patient outome. One is the time above MIC, another is the AUC/MIC, and the third is Cmax/MIC. Each of these parameters measures the cumulative attack of susceptible bacteria. What you notice is that none restricts the time at which the concentration falls inside the selection window. Thus traditional pharmacodynamics differs conceptually from the selection window idea.
Time post-administration

3. Dosing Strategies and Mutant Enrichment
>MPC
Cmax/MIC and AUC/MIC
Serum or tissue drug concentration
MPC
Mutant
Selection
Window
Indeed, when you consider the traditional pharmacodynamic strategies within the context of the selection window, you see that unlike the MPC idea, they allow concentrations to be inside the window. In principle, you could have a very high value for a pharmacodynamic parameter and remain inside the window throughout therapy. This is because traditional pharmacodynamics is aimed at curing disease, not stopping resistance. In microbiological terms, traditional pharmacodynamics uses drug activity against susceptible cells (MIC) as its point of reference rather than activity against resistant mutants. Another important feature is the empirical nature of traditional pharmacodynamic measurements: values were obtained from clinical studies that revealed thresholds for successful therapy with respect to cure. For such studies patient numbers in the hundreds were deemed suitable. Using an empirical approach to define doses that will block the increase in resistance prevalence may require testing many, many more patients because on a per-patient basis resistance generally arises rarely.
MIC
Time post-administration

4. Comparison of MPC Approach and Traditional Pharmacodynamics
Designed to block
resistance
Indexed to mutant
growth
Conceptual threshold
Clinical correlates
unknown
Traditional PD
Designed to cure
patients
Indexed to susceptible
cell growth
Empirical threshold
Based on clinical data
The important differences between the mutant prevention concentration and standard pharmacodynamics are summarized.

5. Applications to Streptococcus neumoniae
Now let’s focus on Streptococcus pneumoniae. This organism is involved in roughly half the pneumonia cases, and it has exhibited resistance to the compounds that have traditionally been used to cure pneumonia. Recently the fluoroquinolones have become popular anti-pneumococcal agents.

6. Fluoroquinolone StructureOH F N HN H
ciprofloxacin O O F OH N N N O
H3C
CH3
levofloxacin O O O
The structures of four quinolones are shown. The compounds differ in many features. What may be most important with respect to resistance is the moiety at the C-8 position, as we discussed above with mycobacteria. O F OH H OH N N N N HN O O H
H3C F
H3C F
Moxifloxacin
Garenoxacin

7. levo cipro Pharmacodynamic Comparison with S. pneumoniae10
MPC
cipro
Plasma drug concentration (fold of MIC)
MIC 1
In the early 1990s, soon after ciprofloxacin entered the market, it was used for S. pneumoniae. It did not work very well, and it was withdrawn from this indication. In retrospect we can understand why it did not do well. When you plot the ciprofloxacin pharmacokinetic curve relative to MIC and MPC, you see that ciprofloxacin concentration barely exceeds the MIC (filled circles). Levofloxacin, however, (open circles), climbs much higher relative to the MIC, and so it is expected to give a better cure rate. Indeed, it is considered to be quite effective. However, you will notice that levofloxacin is inside the selection window throughout therapy. Thus we predicted (14) that widespread resistance to levofloxacin would arise.
The question of which compound would selectively enrich mutants more rapidly is complex because non-topoisomerase mutants tend to be recovered in great numbers when drug concentrations are near the MIC. When drug concentrations are higher in the window, the abundant non-topoisomerase mutants are not recovered. Moreover, the higher concentrations tend to more effectively eliminate susceptible cells, thereby elevating the chance that host defenses will remove resistant mutants. It is not likely to be helpful for ciprofloxacin concentration to drop well below the MIC where selective pressuure is weak because susceptible cell outgrowth will occur.
0.1 5 10 15 20 25
Time post-administration (hr)

8. Two Situations Producing Concentrations Inside the Window
levo with
S. pneumoniae 10
MPC
cipro with
S. aureus
Plasma drug concentration
(fold of MIC)
MIC 1
Our prediction of levofloxacin resistance was based partially on the behavior of ciprofloxacin with Staphylococcus aureus. When this organism was treated with ciprofloxacin, resistance developed very rapidly as resistant strains spread through hospitals (1). Although the selection window hypothesis does not bear on dissemination of resistant strains, those strains had to come from somewhere. The window hypothesis predicts that ciprofloxacin-resistant S. aureus would arise readily because of the location of the pharmacokinetic curve relative to the window (filled circles). Notice that data for levofloxacin with S. pneumoniae are similar to data for ciprofloxacin and S. aureus. This correlation does not mean that levofloxacin resistance in S. pneumoniae will develop as rapidly as ciprofloxacin resistant S. aureus since dissemination factors are probably different. However, this correspondence is very unsettling.
0.1 5 10 15 20 25
Time post-administration (hr)

9. Number of isolates tested
Levofloxacin resistance among clinical isolates of S. pneumoniae
Geographic region
Dates of sample
Number of isolates tested
% resistant
USA
1523
0.3
1596
0.5
1531
0.7
2950
0.1
4296
0.6
9499
6362
0.8
USA/Canada
3854
1999
1201
0.9
Canada
7224
0.4
2000
2245
Japan
218
China
124
Germany
283
Hong Kong
180 10
Brooklyn
1997,1999
138
1.4
We can take a look at surveillance data to see whether worrying about levofloxacin-resistant S. pneumoniae is justified. Several large surveys are listed; in each you can see that the prevalence of resistance to levofloxacin is quite low. Thus there should be no problem using the drug to cure people. However, when you ask whether the prevalence of resistance has been increasing, you see that it has. For example, in the first set of data the prevalence doubled between 1994 and In the next set it went up by a factor of 8 between 1997 and Of course we need to be cautious with these numbers because they are so small. Indeed, the increases are unlikely to be statistically significant, and the authors of these studies do not emphasize increases in resistance. But these data do suggest that we should watch next year's data carefully. At the single-patient level studies from Don Low’s group show that levefloxacin resistance can develop in a single patient during levofloxacin therapy.

10. Recovery of Fluoroquinolone-resistant S. pneumoniae
Levo 1
Moxi
10-2
MIC99
MIC99
10-4
Fraction of cells recovered
10-6
10-8
Next we can ask whether levofloxacin is the best fluoroquinolone for treatment of S. pneumoniae with respect of resistance. Since fluoroquinolones with a methoxy moiety at the C-8 position show enhanced activity against resistant mutants with other bacteria., we carried out a comparison of moxifloxacin and levofloxacin for the ability to select resistant mutants by placing a large number of cells on agar plates containing a variety of fluoroquinolone concentrations. We found that the MPC for moxifloxacin was about 1/5 that of levofloxacin. In this case we estimated MPC as the concentration that allows no colony formation when at least 1010 cells are applied to an agar plate.
10-10 
0.1 1
[Fluoroquinolone] (μg/ml)

11. MPC and Fluoroquinolone Pharmacokinetics1 2 3 4 5 6 7 8 9 10 10
Moxifloxacin
Levofloxacin 9
MPC90 8 7
750 mg 6
500 mg
Plasma drug concentration (mg/ml) 5 4 3
MPC90 2
MIC90
In collaboration with Joe Blondeau we also measured MPC for about 150 clinical isolates of S. pneumoniae to compare fluoroquinolones. Since activity measurements are meaningless without consideration of pharmacokinetics, we show MPC relative to drug concentrations achieved with standard doses. What you see is that moxifloxacin is above its MPC for roughly half a day while levofloxacin, at the standard 500 mg dose, never reaches MPC. Thus we would predict that moxifloxacin would be more effective than levofloxacin at restricting the development of resistance.
MIC90 1 10 20 30 40 50 10 20 30 40 50
Time post-administration (hr)

12. Comparison of Fluoroquinolones by MPC-based Pharmacodynamicst
MPC
Serum Concentration
MIC
Time
Fluoroquinolone MPC90 (mg/ml) Time above MPC90 (hr)
Moxifloxacin
Gemifloxacin
Gatifloxacin
Levofloxacin
Determined with about 150 clinical isolates of Streptococcus pneumoniae. Source of data: J. Blondeau
The data in the previous slide can be expressed as time above MPC. When this is done, comparing the compounds is straightforward. The logic is that mutant growth is restricted by concentrations above MPC. The longer the threshold is exceeded, the better.
It may be that lethal activity for mutants will lower the threshold, but we don’t know that yet: the selection window hypothesis does not include consideration of lethal activity in order to cover compounds that only exhibit bacteriostatic activity.

13. Recovery of Fluoroquinolone-resistant S. pneumoniae
Levo 1
Moxi
10-2
MIC99
MIC99
Fraction of cells recovered
10-4
10-6
10-8
Shown is a repeat of an earlier slide to allow us to make another point. We isolated colonies from various points along these curves, which were generated for moxifloxacin and levofloxacin with S. pneumoniae. We then determined the nucleotide sequences of the QRDR (quinolone-resistance-determining regions) for the gyrA, gyrB, parC, and parE genes. The results, shown in the next slide, illustrate differences between the two target genes.
10-10 
0.1 1
[Fluoroquinolone] (ug/ml)

14. Topoisomerase Mutants Selected by Levofloxacin and Moxifloxacin
Fraction of Identity of mutants*
cells recovered Selected by moxi Selected by levo
as mutants gyrA gyrB parC parE gyrA gyrB parC parE
3.0 X none none none none
4.4 X none none none none
none none none none
1.8 X none none S79Y none
2.0 X none none none none none none S79Y none
none none S79Y none
none none D83H none
1.3 X none none none none
5.6 X none none none none none none none none
1.2 X none none none none none none S79Y none S81Y none none none none none S79Y none
2.9 X S81Y none none none
S81Y none none none
1.6 X S81Y none none none
* Changes in QRDR of the indicated genes
103
What you notice is that moxifloxacin selects gyrase mutants and levofloxacin selects topoisomerase IV mutants, but only at high concentrations. At low concentrations the mutants lack the classic mutations. While we have not defined the mutations responsible for the low-level resistance, our current guess is that they are involved in drug efflux. An interesting feature of this table is that the frequency at which gyrase mutants are recovered with moxifloxacin is about 1000 times lower than topoisomerase IV mutants with levofloxacin. This difference may be due to the genetics of resistance for the two targets. Gyrase-mediated resistance is recessive while topoisomerase IV-mediated resistance is codominant. Recessive mutants remain susceptible to the lethal action of the drug until all sensitive protein has been replaced by new, resistant protein. Thus new gyrase mutants will be rarer than topoisomerase IV mutants. Another factor could be the 10-fold greater activity of moxifloxacin at killing resistant mutants. In either case fluoroquinolones that preferentially target gyrase are less likely to enrich resistant mutants.

15. Effect of a parCr mutation on recovery
of resistant mutants 1
MIC99
parCr
MIC99 wt
10-2
Fraction of cfu recovered
10-4
10-6
10-8
Once a parC mutation is fixed, a profound change occurs in the behavior of the strain with respect to selection of subsequent resistance mutations. The slide shows the effect of moxifloxacin concentration on selection of mutants with wild-type cells (open squaqres) and with a parC mutant (solid squares). The latter displays a much higher MPC and a broad, high plateau. Since the MIC is not raised very much in the parC mutant, such a strain might be susceptible to moxifloxacin treatment. However, we would expect moxifloxacin-resistant gyrA mutants to arise readily. Thus a first-step mutation is likely to accelerate the development of resistance. This phenomenon is likely to be important because first-step parC mutants are already widespread but not considered to be levofloxacin-resistant by standard approaches (7).
10-10
MPC
MPC
0.1 1 10
[Moxifloxacin] (mg/ml)
S. pneumoniae

16. Fluoroquinolone Challenge Adds Topoisomerase Mutations to S. pneumoniae
Strain Agent Changes in QRDR
  ParC ParE GyrA GyrB
70 D83G, K137N none E85K none
70 multiple D83G I460V E85K none
Gemi K137N, S79F E474K S81Y
70 Moxi-1 D83G, K137N none E85K, S81F none
70 Moxi-2 D83G none E85K, S81F none
K137N, S79F
S. pneumoniae can acquire many fluoroquinolone resistance mutations. On this slide we show up to seven that were obtained by repeated challenge of two clinical isolates (15). The point is that as long as we require the bacteria to obtain only one more mutation to be resistant, the bacteria will readily make new fluoroquinolones obsolete. This is why we must force them to attain at least two mutations for resistant growth.

17. Applications to Staphylococcus aureus
Now let’s consider some potential applications with S. aureus. Remember that the general idea here is to try to make clinical predictions using in vitro data. We will have to wait for the clinical test of the idea.

18. Mutant Selection Window and Fluoroquinolone Pharmacokinetics: S. aureus
Ciprofloxacin
Garenoxacin (BMS284756) 10 1 2 3 4 5 6 7 2 4 6 8 10 12 14 16
MPC90 30
MPC90
cipS
cipR
[Drug] (mg/ml) 1
MIC90
We will consider the effects of a new quinolone called garenoxacin (see slide 33 for structure). Recall that with S. aureus ciprofloxacin concentrations fall right in the center of the selection window (panel A) and resistance developed readily. Garenoxacin is much more active, and it is well above the window with ciprofloxacin-susceptible (wild-type strains, panel B). The compound is so good that some authors have expressed optimism about using it with ciprofloxacin-resistant MRSA. If you only consider the MIC, that is a reasonable statement. However, when you determine MPC for cipro-resistant MRSA, you see that the curve looks just like the one for ciprofloxacin and wild-type cells. Thus garenoxacin is likely to readily select resistant mutants if used with ciprofloxacin-resistant strains (20). Getting the most out of garenoxacin will require careful attention to the quinolone-resistance status of the strain to be treated.
MPC90
MIC90
0.03
MIC90
0.2 2 4 6 8 5 10 15 20 25 5 10 15 20 25
Time post-administration (hr)

19. MPC-based Potencywith S. aureus
Dose above MPC
Serum or tissue drug con.
MPC-based Potencywith S. aureus
Time post-administration
Compound MPC (mg/ml) Cmax (mg/ml) half-life (hr)
 Norfloxacin
Tobramycin
Chloramphenicol
Rifampicin
Penicillin G
Vancomycin
Moxifloxacin
Let’s next consider some compounds other than the fluoroquinolones. We have measured MPC with S. aureus and then obtained a value for Cmax from the literature. What you see is that the Cmax for some compounds exceeds MPC and for others it does not. And with some for which Cmax exceeds MPC the half life is so short that it would be difficult to maintain drug concentrations above MPC throughout therapy. How should one use these compounds to avoid selecting resistant mutants? Our current dogma is that if you cannot maintain concentrations above MPC, you should resort to dual-drug therapy.

20. Mutant Selection Window with S. aureus
Narrow the window
Time post-administration
Serum or tissue drug con.
Mutant Selection Window with S. aureus
Compound MPC (mg/ml) MIC99 (mg/ml) MPC/MIC99
Norfloxacin
Tobramycin
Chloramphenicol
Rifampicin ,000
Penicillin G
Vancomycin
Moxifloxacin
Now let’s take a different point of view and see whether there are existing compounds that have a very narrow selection window. To get at this we measured both MIC and MPC. Then we determined the ratio of MPC to MIC (a ratio of 1 would mean that the window is closed). As you can see, no compound tested has a value of 1. Indeed, some compounds, such as rifampicin, have such wide selection windows that no drug concentration will block mutant growth. This is the result you would expect to see with a population of bacteria that contained a small subpopulation of plasmid-containing cells that are highly resistant. For such situations multidrug therapy is needed to close the selection window.

21. Is the Mutant Selection Window Restricted to Fluoroquinolones?
While the fluoroquinolones have been the most thoroughly studied compounds with respect to MPC, it is likely that some, but not all, compounds will behave in a similar way. We have initiated a survey.

22. Mutant Selection with Erythromycin and Penicillin
M. smegmatis
S. aureus
10-1
Fraction of input cfu recovered
10-3
10-5
10-7
The feature to watch for is an increasing steepness to the slope of selection curves as the drug concentration gets high. You see that this is true for the two examples shown (panel A is erythromycin treatment of S. aureus (solid) and M. smegmatis (open); panel B is penicillin treatment of the same two organisms). We emphasize that increasing steepness does not always occur and that for many cases MPC is not below the maximum serum level. Thus being able to dose above MPC may be an unusual situation. That would mean that combination therapy would be required for most pathogens.
10-9
0.1 1 10
100
1000
0.01
0.1 1 10
100
1000
[Erythromycin] (mg/ml)
[Penicillin] (mg/ml)

23. Mutant Selection with Yeast (Canadia glabrata)
clinical
resistant
isolate 1
10-2
10-4
wild-ytpe
Fraction of input CFU recovered
10-6
10-8
The general principles seen with antimicrobial agents and bacteria are also observed with eukaryotic cells. The slide shows a mutant selection curve with the yeast Candida glabrata applied to miconazole-containing agar plates. In this experiment colonies were recovered from drug-containing agar, restreaked on drug-free agar, and then retested on drug-containing agar to establish that the mutants were stable. Notice that the mutant recovery frequency (plateau) is very high and that the selection window is very wide. These features are expected to cause resistant mutants to be readily enriched by miconazole treatment. As expected, the selection curve was shifted by the presence of resistance mutations, which in this case are undefined. Similar results were obtained with Candida albicans. The slide shows unpublished work by Wang et al., Public Health Research Institute.
0.001
0.01
0.1 1 10
100
[Miconazole] (mg/ml)

24. Why is the Selection Window
Hypothesis Useful?
Now let's consider the utility of the mutant selection window hypothesis. First, it helps us decide whether a particular agent can be used with a particular organism as monotherapy or whether combination therapy is required. Several examples were illustrated. Second, the window idea helps us think in a different way about surveillance studies. We consider this in the next slide.

25. Gradual Increase of Resistance
breakpoint
Number of isolates
Surveillance work has generally let us know whether a particular compound is still good for curing disease. The answer is yes as long as a very high percentage of the isolates are considered susceptible. The data can also be used to warn us of upcoming resistance, but the system is not very good for that. There are two reasons. First, when one uses susceptibility breakpoints to determine whether a strain is resistant or susceptible, a strain can have many resistance mutations and still be considered susceptible. If those mutations accelerate the development of resistance, the surveillance work will give you a false sense of security. Second, the primary data for surveillance is generally derived only from the dominant members of the infecting population. This is because clinical labs generally only look at well-isolated colonies. Consequently, the growth of mutant subpopulations will not be observed. The slide shows what is called creeping resistance. With time the curve shifts to higher concentrations. The result is that resistance is thought to appear suddenly when it actually develops gradually.
MIC

26. Problems for implementing direct attack of mutants
No animal or clinical studies done
Currently requires altruism (dosing higher than needed for cure)
With S. pneumoniae time is running out (cross-resistance)
Although using the mutant selection window hypothesis to restrict the development of de novo resistance is conceptually straightforward, it is politically difficult. This is because we must generally use drug concentrations that are higher than necessary to cure; thus patient altruism is required. We cannot ask someone to take more personal risk for the sake of people as a whole if no clinical studies have been done to support the idea. With S. pneumoniae we are running out of time, since resistance to one quinolone predisposes the bacterium to developing resistance to other quinolones.The next step is to conduct animal studies to relate the in vitro dimensions of the selection window to those occurring in vivo.

27. Literature Cited LITERATURE CITED
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