Antimicrobial Activity of Eluates obtained from the Gingival Flow

In vitro drug release experiments are often carried out in constant volumes, which may be exchanged during the observed release period. This is also valid for the simulation

125 µg/ml 250 µg/ml 500 µg/ml

Log10 cfu

**p<0.01 vs. Control

A

B

C

arb Uarb U

Results and Discussion 55 of periodontal applications (126,127) (3.4.4). The aim of this experiment was to create conditions, which are closer to the clinical situation within the gingival pocket, through the simulation of the gingival crevicular flow (CGF). The initial volume of 23.5 µl was chosen to resemble the volume within a corrupted gingival pocket, and subsequently the exchanged volumes were decreased, due to the reduction of CGF during periodontal therapy (55). In view of the protein composition of the crevicular fluid, a resemblance to diluted serum can be discussed (128). Hence, a buffered saline solution paired with serum albumin was chosen as medium, and the obtained eluates were investigated towards their antimicrobial activity up to 42 days (Figure 28).

Figure 28: Minimal inhibitory concentrations (maximum dilution) of the eluates of PLGA-MLC formulations obtained over 42 days during simulation of the gingival fluid flow on S. gordonii ATCC 10558 (A) and P. gingivalis ATCC 33277 (B); PLGA-MLC formulation

As indicated by the prior experiments, all minocycline containing formulations possessed antimicrobial activity against S. gordonii ATC 10558 and P. gingivalis ATC33277 in the initial phase of the testing. In the beginning, the Arestin^® microspheres demonstrated an especially high activity just like the pure substance with active concentrations above 16 mg/ml (Table 13). As expected, the activity decreased during the release period. Especially within the first 24 h a vast reduction was

Time t

Time t

MIC (Dilution)MIC (Dilution)

PLGA₅₀₂-MLC Extrudate PLGA₅₀₃-MLC Extrudate PLGA₅₀₂-Placebo Extrudate Arestin^®microspheres

Minocycline

Streptococcus gordonii

Porphyromonas gingivalis

Results and Discussion 56 detectable into the µg/ml concentration range. Within 21 days the activity of the pure API vanquished. During the first three weeks, the microspheres demonstrated a higher activity compared to the pure substance on a near level to the PLGA-MLC extrudates.

But they were not able to extend the release beyond this period.

In contrast, the PLGA-MLC extrudates started off with initial lower active concentrations around 2 – 8 mg/ml, except for the placebo extrudates, which were not able to inhibit bacterial growth at any time point. From t = 24 h until day 7 their activity decrease was almost equal to the Arestin^® microspheres, with a slightly higher activity of the PLGA503 -MLC extrudates. The decisive difference between both systems is that the activity of the extrudates did not end after 21, but after 42 days. Active concentrations of 4 µg/ml were observed at the end of the experiment. Hence, they offered a controlled release with an up to twice the duration compared to the commercial drug delivery system. So, these results align with the findings of the in vitro testing from 4.4.2.

Table 13: Calculated concentrations of minocycline (µg/ml) containing formulations presented in Figure 28; Calculation based on maximum dilution multiplicated with the respective MIC

Minocycline Arestin^® PLGA502-MLC PLGA503-MLC

1 h >16000 >16000 2000 8000

2 h 4000 4000 1000 2000

4 h 1000 1000 1000 2000

24 h 250 1000 500 1000

2 d 62.5 125 125 250

7 d 31.3 31.3 31.3 125

14 d <2 8 15.6 31.3

21 d <1 <1 15.6 31.3

28 d <1 <1 15.6 15.6

35 d <1 <1 15.6 8

42 d <1 <1 4 4

Also, eluates of defined time points were anew tested towards their activity on the formation of biofilms (Figure 29). At day 1 all test formulations were capable of reducing the cfu count by 2 Log10 steps. This situation remained mainly unchanged for the first week. After two weeks however, only the microspheres and the PLGA-MLC compositions could maintain a reduction of more than 1 Log10 step. At day 28, only the eluates of PLGA503-MLC were capable of diminishing the cfu count with a reduction of 1.9 log steps. Also noteworthy is the antibacterial effect of the eluates obtained from

Results and Discussion 57 the placebo extrudate. On day 14 and 21 they reduced the cfu count by 1.4 Log10 and 1.1 Log10 steps, and therefore mirror the results from 4.5.2.

Figure 29: Antimicrobial activity of the eluates obtained during the gingival fluid flow simulation against the formation of biofilms at different time points (cfu count after 6 h)

Local drug delivery systems can achieve high concentrations in the gingival sulcus, and therefore unfold strong effects. Non-biodegradable tetracycline fibers (Actisite^®) accomplished concentrations above 1000 µg/ml over a few days (129). Also, doxycycline in situ forming depots were capable of realizing local concentrations of 1000 µg/ml within the first two hours of application (130). Depending on the drug load of either 8.5% or 14%, the concentration lowered to 8 µg/ml or respectively to 19 µg/ml after 12 days. Contrary, an oral administration of tetracycline analogues was found to result in gingival crevicular concentrations below 1 µg/ml in approximately 50% of the cases (131). This underlines again the significance of local drug delivery also for periodontitis treatment. Hence, the achieved concentration levels of the tested extrudates are in range of tetracycline derivative containing systems described in the literature. But it has to be considered, that the results described in the literature were observed in vivo, and the acquired data about the extrudates represents in vitro data, so far.

AAAA A

**p<0.01 vs. Control

**p<0.01 vs. PLGA₅₀₂-MLC

**p<0.01 vs. PLGA₅₀₃-MLC

Log10 cfuLog10 cfuLog10 cfu Log10 cfuLog10 cfu

Results and Discussion 58 Despite the positive findings on the performance of the extrudates during this experiment, this in vitro-assay has limitations, which need to be labeled. The simulation of the GCF effectively mimics the clinical situation with a limited release medium and varying flow rates. But active transport processes into epithelial cells (132), uptake by fibroblasts (133) and immunologic active cells cannot be depicted. Nevertheless, through the simultaneous examination of approved systems like Arestin^®, data can be extracted for evaluating the suitability for clinical applications. Thus, the observed long lasting and high antimicrobial activity suggest promising potential for the transfer of the extrudates to in vivo applications.

Regarding both tested extrudates in comparison to each other, the eluates of PLGA503 -MLC prototypes possessed a higher inhibitory effect on the bacterial growth as well as on the formation of biofilms. Their higher molecular weight presumes a theoretically slower release rate compared to PLGA502, as described in 4.4.2. Thus, a higher antimicrobial activity of PLGA503-MLC stands in contrast to the results of the in vitro release at first sight. But section 4.4.2 covers extrudates with a defined length, and therefore varying masses depending on the diameter, which was not equal, due to the viscoelastic properties of PLGA503. Also, their relative release rate was defined as

% [m/m] minocycline, as illustrated in Figure 22 (page 45). During the simulation of the CGF however, all samples were weighed equally to 1 mg minocycline content. Hence, the larger diameter and therefore larger surface of the sample probably had an impact on the initial faster release. For instance, an extrudate with a diameter of 600 µm and a length of 400 µm possesses a surface of 1.319 mm² and a volume of 0.113 mm³. Under the assumption of an equal density, an extrudate with the same volume and a diameter of 800 µm then offers a surface of 1.571 mm². Even though the extrudates are micronized within this assay, the inner surface of these samples could be larger as well. Thus, this circumstance could have contributed to the release of higher API amounts.

Also noteworthy is the observed inhibition of the placebo extrudate on the formation of new biofilms, during the direct contact as well as for the eluates. The composition of the extrudate matrix, consisting of PLGA and magnesium stearate, does not suggest such an interior activity. PLGA served as release controlling matrix for several local antibiotic drug delivery systems for dental applications (69,134). Still, an antimicrobial activity of PLGA had not been described so far (69,135). This is also confirmed by the report of unloaded PLGA-nanoparticles, which were not able to inhibit the formation of biofilms of Pseudomonas aeruginosa (135,136). Hence, a connection between magnesium and the disturbed biofilm formations can be discussed. As implant material,

Results and Discussion 59 magnesium demonstrated encouraging results with antibacterial activity against planktonic Staphylococcus epidermidis, Staphylococcus aureus, and Escherichia coli, and also suppressed biofilm formation of the mentioned species (137). Also, magnesium oxide nanoparticles had a strong antimicrobial effect on Escherichia coli, Pseudomonas aeruginosa and Candida ssp. (138).

In conclusion, the PLGA-MLC extrudates exhibited suitable antimicrobial properties to serve as a serious alternative to commonly applied systems, especially the PLGA503 -MLC extrudates. With the stabilized API incorporated in an easily applicable application form, which efficiently acts over a prolonged time period, first successes could be recorded. But for these extrudates adjustments were still necessary. Their reproducible manufacturing and their mechanical properties were not yet adequate enough.

Therefore, further development was necessary, and the second generation was introduced, as described below.