Macrolide Accumulation by Host Cells in the Gingiva

描述(申请人提供)：侵袭性和复发性牙周炎与放线菌和牙龈假单胞菌的感染有关。这些病原体入侵上皮细胞和吞噬细胞的能力使它们很难通过常规的刮除和根面平整来消除。然而，全身抗生素的使用与洁治和根面平整联合使用显著提高了临床和微生物治疗结果。虽然牙周科医生没有广泛开出阿奇霉素和克拉霉素的处方，但它们有潜在的用途，因为它们被宿主细胞吸收，具有良好的抗菌活性。这两种药物都有可能在牙龈中达到比血清更高的水平。初步研究表明，主动转运在这一过程中起着重要作用。培养的牙龈成纤维细胞、上皮细胞和类PMN的HL-60细胞似乎通过一种集中和饱和的转运机制摄取克拉霉素。成纤维细胞对克拉霉素的摄取是温度依赖的，符合Michaelis-Menten动力学，这表明能量依赖的转运系统参与其中。我们假设牙龈成纤维细胞、上皮细胞和中性粒细胞具有积聚大环内酯类化合物的活性转运蛋白。这些转运蛋白通过提高牙周袋软组织壁、吞噬细胞和牙周沟液中大环内酯类物质的水平而发挥有益的作用。由于成纤维细胞是牙龈中一个相对较大的细胞室，大环内酯转运体可以允许这些细胞作为药物储存库，提高和维持牙龈结缔组织和牙周沟液中的大环内酯水平。在上皮细胞和中性粒细胞中，这些转运蛋白可以通过促进细胞内大环内内酯的积累来促进侵袭性病原体的清除。这项建议的目的是研究大环内酯类化合物在牙龈中由宿主细胞运输和浓缩的机制。具体目的1是利用放射性标记的大环内酯类化合物来检测转运活性，以确定牙龈成纤维细胞、上皮细胞和中性粒细胞吸收这些药物的转运系统。[具体目标2是评估宿主细胞在细胞内积累大环内酯的药理学益处(包括对药物分布的影响)。具体目标3是评估大环内酯类化合物对培养的中性粒细胞和牙龈上皮细胞内伴生放线菌和牙龈假单胞菌的杀灭作用]。这项工作的总体目标是为选择最有效的牙周抗菌化疗药物提供合理的依据。最终，这项工作可能有助于减少牙齿脱落和治疗牙周炎的成本负担。 公共卫生相关性：这项提案将研究大环内酯类抗生素在人牙龈细胞中运输和浓缩的机制。利用大环内酯类化合物的细胞积聚，可以增强对牙周侵袭性病原体的清除，而这些病原体很难用传统的洁治和根面平整来治疗。通过为牙周抗菌化疗选择抗生素提供合理的依据，这项工作最终可能有助于减少牙齿丢失和治疗破坏性牙周炎的成本负担。

DESCRIPTION (provided by applicant): Aggressive and recurrent forms of periodontitis are associated with infections by A. actinomycetemomitans and P. gingivalis. The ability of these pathogens to invade epithelial cells and phagocytes makes them difficult to eliminate by conventional scaling and root planing alone. However, the use of systemic antibiotics in conjunction with scaling and root planing significantly enhances clinical and microbiological treatment outcomes. While not widely prescribed by periodontists, azithromycin and clarithromycin are potentially useful because they are taken up by host cells and have favorable antimicrobial activity. Both agents can potentially attain higher levels in gingiva than in serum. Preliminary studies suggest that active transport plays an important role in this process. Cultured gingival fibroblasts, epithelial cells and PMN-like HL-60 cells appear to take up clarithromycin by a concentrative and saturable transport mechanism. Clarithromycin uptake by fibroblasts is temperature-dependent and exhibits Michaelis-Menten kinetics, suggesting the involvement of an energy-dependent transport system. We hypothesize that gingival fibroblasts, epithelial cells and PMNs possess active transporters that accumulate macrolides. These transporters play a beneficial role by enhancing macrolide levels in the soft tissue wall of the periodontal pocket, in phagocytes and in gingival crevicular fluid. Since fibroblasts are a relatively large cellular compartment of the gingiva, macrolide transporters could permit these cells to function as drug reservoirs that enhance and sustain macrolide levels in gingival connective tissue and in gingival crevicular fluid. In epithelial cells and PMNs, these transporters could facilitate elimination of invasive pathogens by enhancing intracellular macrolide accumulation. The objective of this proposal is to study mechanisms by which macrolides are transported and concentrated by host cells in the gingiva. Specific aim 1 is to characterize transport systems by which gingival fibroblasts, epithelial cells and PMNs take up these agents, using radiolabeled macrolides to assay transport activity. [Specific aim 2 is to assess the pharmacological benefits of intracellular macrolide accumulation by host cells (including effects on drug distribution). Specific Aim 3 is to assess the killing of intracellular A. actinomycetemcomitans and P. gingivalis by macrolides in cultured PMNs and gingival epithelial cells]. The broad goal of this work is to provide a rational basis for selecting the most effective agents for periodontal antimicrobial chemotherapy. Ultimately, this work could contribute to reductions in tooth loss and the cost burden of treating periodontitis. PUBLIC HEALTH RELEVANCE: This proposal will study mechanisms by which macrolide antibiotics are transported and concentrated by cells in human gingiva. Exploiting cellular accumulation of macrolides could enhance the elimination of invasive periodontal pathogens that are difficult to treat with conventional scaling and root planing. By providing a rational basis for selecting an antibiotic for periodontal antimicrobial chemotherapy, this work could ultimately contribute to reductions in tooth loss and the cost burden of treating destructive periodontitis.