In both normal and granulocytopenic animals, treatment with TR-701 resulted in a good dose-response curve. The difference between the two groups represented an improvement in the exposure response as a function of the presence of granulocytes. Examination of shows that, for the granulocytopenic animals, stasis was achieved with a human-equivalent dose exposure of slightly less than the 2,000, 2,100, and 2,300 mg administered daily for the 72-, 48-, and 24-h endpoints, respectively. In the normal animals, stasis was achieved at human-equivalent exposures of slightly more than 100 mg/day at the 24-h endpoint and less than 100 mg/day at the 48-h and 72-h endpoints. The maximal killing rate was achieved at 72 h, with the lowest dose (200 mg/day) providing an effect similar to that seen with the highest dose (3,200 mg/day); those regimens were associated with bacterial burdens in thigh muscles of 4.42 ± 0.433 log CFU/g and 4.49 + 0.264 log CFU/g, respectively, which represent bacterial killing levels that were 3.12 and 3.05 log CFU/g greater than the stasis response. Thus, after 72 h of therapy with TR-701, the lowest and the highest doses produced equivalent microbiological effects. On day 1, both achieved stasis. This suggests that human patients with ABSSSIs who are treated with 200 mg/day of TR-701 should have a successful treatment outcome, since it has been shown that the stasis response is predictive of clinical success in immunocompetent human patients with ABSSSIs (1
). A successful treatment outcome has recently been validated in a human clinical phase 2 study of cSSSI in which a clinical cure rate of 98.2% was found for clinically evaluable patients treated with 200 mg of TR-701 (10
The results showing the differences between the effects of treatment with TR-700 in the presence and absence of granulocytes are unprecedented. For β-lactams and quinolones, differences in efficacy of 2- to 4-fold are seen (2
). The stasis endpoint values corresponding to the effects of TR-700 treatment differed on the order of 20-fold. When looking at the maximal killing effect, it is best to compare the differences in numbers of log CFU per gram of organisms between the 200 and 3,200 mg/day human-equivalent doses at the 24-, 48-, and 72-h endpoints. For the granulocytopenic mice, the differences were 3.8 log CFU/g at 24 h, 5.34 log CFU/g at 48 h, and 6.05 log CFU/g at 72 h. In nongranulocyotopenic mice, the differences were muted, at 0.98 log CFU/g at 24 h, 0.99 log CFU/g at 48 h, and −0.07 log CFU/g at 72 h. Thus, there was a major, multilog difference between the values corresponding to the effects mediated by the smallest versus largest doses in neutropenic mice at all time points evaluated whereas in the nongranulocyotopenic mice the difference was on the order of 1.0 log CFU/g at 24 and 48 h and was nonexistent at 72 h.
The only difference between the groups was the lack of granulocytopenia in one group versus its presence in the other. While we are cognizant of the importance of granulocytes in defense against many bacterial infections, this circumstance is unique. The true importance of the presence of granulocytes alone (i.e., without drug) is found in the contrast in bacterial growth rates in neutropenic versus nonneutropenic mice in the absence of drug therapy (). Here, there was a net growth of 3.26 log CFU/g in the first 24 h in the neutropenic group. At 48 h, this difference had increased to 3.48 log CFU/g, and at 72 h, all control mice had died. In the nongranulocyotopenic mice, there was a net growth of 0.68 log CFU/g at 24 h, increasing at 48 and 72 h to 1.05 and 1.18 log CFU/g, respectively. All control mice survived to 72 h. Granulocytopenia allowed a rapid, multilog amplification in CFU per gram, resulting in animal death at 72 h, whereas the granulocyte-replete animals exhibited amplification limited to slightly greater than 1 log CFU/g. This is important, but it is clear that in the presence of granulocytes alone (i.e., without drug), net growth was observed. Use of the smallest dose of TR-701 is instructive (). From the baseline, the granulocytopenic group treated with the lowest human equivalent dose of 200 mg/day exhibited net growth of 2.81, 3.62, and 3.31 log CFU/g at 24, 48, and 72 h, respectively. Normal animals receiving a 200 mg/day human-equivalent dose of TR-701 exhibited net killing levels (relative to the normal, no-treatment control level) of 0.998, 2.545, and 4.3 log CFU/g at 24, 48, and 72 h, respectively. One way of measuring the effect of the drug mediated by the presence of granulocytes is to contrast these figures, as the direct drug effect was seen in the neutropenic animals receiving the drug (note that the net amplification from baseline was less than in the no-treatment controls that were granulocytopenic and, importantly, that animals survived to 72 h). The cumulative effects of the presence of granulocytes plus the direct drug effect plus the drug effect mediated through the granulocytes is seen in the normal cohort receiving drug. Taking the differences in the net growth/net killing values shows that the level seen with the normal cohort was down by 3.12 log CFU/g at 24 h, 5.12 log CFU/g at 48 h, and 6.43 log CFU/g at 72 h relative to that seen with the neutropenic cohort receiving a 200 mg/day human-equivalent dose of TR-701. The granulocytopenic cohort showed amplification from the baseline, whereas the normal cohort exhibited net killing from the baseline, and the difference in the two results compared to the baseline give the preceding numbers contrasting the two groups. The vast bulk of this difference was due to the effect of drug mediated through the presence of the granulocytes.
This difference can be seen in . First, the direct effect of TR-700 can be estimated as the difference in organism amplification/kill ratios between the no-treatment control and the granulocytopenic cohorts subjected to the 200 mg/day TR-701 human-equivalent treatment. At 24 and 48 h, these differences resulted in 0.45 log CFU/g less amplification in the TR-701-treated group. At 48 h, the TR-701-treated group actually exhibited greater amplification (0.14 log CFU/g). This difference could be evaluated at 24 and 48 h, but not at 72 h, as all no-treatment controls had succumbed by this time. For the 72-h data, we simply fixed the value to zero. The direct effect of granulocytes together with the effect of TR-700 treatment mediated through the granulocytes is estimated as the difference between the counts seen with the normal cohort treated with a 200 mg/day human-equivalent dose of TR-701 and those seen when the direct effect of TR-700 is subtracted. For 24, 48, and 72 h, the effects attributable to those two factors were seen as reductions of 2.67, 5.26, and 6.43 log CFU/g. Finally, the effect of the drug mediated through the presence of granulocytes was calculated as the difference between the total effect (determined for the normal cohort treated with 200 mg/day of TR-701) and the effect mediated by the granulocytes without therapy. For the 24-, 48-, and 72-h time points, the estimates of the effect due to TR-700 acting through granulocytes were seen as killing levels of 1.16, 3.53, and 4.70 log CFU/g, respectively. These differences are well reflected in the modeling.
Determination of the effect of tedizolid mediated through the presence of granulocytes
The mechanism behind this finding is unclear. Lemaire et al. (8
) demonstrated that TR-700 penetrates into macrophages better than linezolid in cell culture and, once there, kills intracellular staphylococci significantly better. The difference in our study is that we evaluated the interaction of TR-700 and granulocytes in an in vivo
system. It would not be surprising to find that, as with macrophages, granulocyte penetration by TR-700 is better than that seen with linezolid and, most importantly, that killing of staphylococci within granulocytes is also improved.
It is clear that TR-701 causes a multilog decline in S. aureus CFU per gram in the mouse thigh model and that it differs from many other antibiotics in that a considerable portion of the multilog decline is attributable to the effect of the drug mediated through the activity of effector cells, especially granulocytes. This work was performed with a single isolate, and more work needs to be performed with multiple isolates. Also, additional basic research needs to be done in this area to place these observations into perspective.