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A.放线菌和牙龈卟啉单胞菌。这些病原体侵入上皮细胞和吞噬细胞的能力使得它们难以单独通过常规刮治和根面平整来消除。然而，全身性抗生素的使用与洁治和根面平整的结合显著提高了临床和微生物治疗的结果。虽然没有被牙周病医生广泛使用，但阿奇霉素和克拉霉素是潜在有用的，因为它们被宿主细胞吸收并具有良好的抗微生物活性。这两种药物在牙龈中的水平可能高于血清。初步研究表明，主动运输在这一过程中起着重要作用。培养的牙龈成纤维细胞、上皮细胞和中性粒细胞样HL-60细胞似乎通过集中和饱和转运机制摄取克拉霉素。成纤维细胞对克拉霉素的摄取具有温度依赖性，并表现出Michaelis-Menten动力学，表明涉及能量依赖性转运系统。我们推测牙龈成纤维细胞、上皮细胞和中性粒细胞具有积累大环内酯类药物的活性转运蛋白。这些转运蛋白通过增强牙周袋软组织壁、吞噬细胞和龈沟液中的大环内酯水平发挥有益作用。由于成纤维细胞是牙龈的相对较大的细胞室，大环内酯转运蛋白可以允许这些细胞作为药物储库，提高和维持牙龈结缔组织和龈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增强细胞内大环内酯的积累来促进侵袭性病原体的消除。本提案的目的是研究大环内酯类药物在牙龈中被宿主细胞转运和浓缩的机制。具体目标1是表征运输系统，牙龈成纤维细胞，上皮细胞和中性粒细胞采取这些代理商，使用放射性标记的大环内酯类测定运输活性。[具体目标2是评估宿主细胞内大环内酯蓄积的药理学益处（包括对药物分布的影响）。具体目标3是评估细胞内A.在培养的中性粒细胞和牙龈上皮细胞中，大环内酯类药物对伴放线菌和牙龈卟啉单胞菌的作用]。这项工作的主要目标是为选择最有效的牙周抗菌化疗药物提供一个合理的基础。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类牙龈细胞中转运和浓缩的机制。利用细胞积累的大环内酯类药物，可以提高消除侵入性牙周病原体，这是难以治疗的传统规模和根面平整。通过为牙周抗菌化疗选择抗生素提供合理的依据，这项工作最终将有助于减少牙齿脱落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.