Conventional periodontal therapy, with its focus on mechanical débridement of bacterial plaque, usually prevents the progression of periodontal breakdown. In some cases, however, the disease progresses despite periodontal débridement. These types of periodontal infections usually are related to increased levels of subgingival bacteria.^4,5 The use of antibiotics as an adjunctive therapy has been of particular interest to clinicians and researchers in the treatment of periodontal diseases.^6–10 It is important that the clinician use an appropriate antibacterial agent when treating patients with periodontal disease. In addition, it may be useful for him or her to determine the local (that is, in the gingival crevicular fluid, or GCF) and systemic concentrations of antibiotics in patients with the disease to eliminate these infections in clinical practice.^11,12

In periodontal and other types of bacterial infections, polymorphonuclear leukocytes, or PMNs, migrate to the infection site, phagocytose the bacteria and attempt to kill them with reactive oxygen metabolites and microbicidal proteins.^13,14 Although PMNs are highly effective in defending against bacterial infections, some pathogens are difficult to kill.

For example, A. actinomycetemcomitans, an important pathogen that has been implicated in aggressive periodontitis, chronic periodontitis and refractory periodontitis, resists phagocytic killing by PMNs.^3,13,15,16 PMNs’ oxidative killing mechanisms are not completely effective under anaerobic conditions. Therefore, antimicrobial agents can help the clinician control these types of infections. Ciprofloxacin, the most active form of fluoroquinolones, accumulates and remains active inside PMNs (usually fourfold to eightfold higher levels than those in the extracellular area), and retains its bactericidal activity inside PMNs by enhancing intracellular destruction of susceptible bacteria.^5,13,17,18

To eliminate the risk of salivary contamination, the clinician chose sampling areas among maxillary teeth.

Although tetracycline and nitroimidazole families have been studied widely in periodontal research, we could not find any reports in the literature regarding ciprofloxacin concentrations in GCF of patients with periodontitis. Because the inflamed periodontium is densely infiltrated by PMNs, we hypothesized that systemic ciprofloxacin may reach higher levels in GCF than in serum. Thus, the aim of this clinical study was to compare the serum and GCF concentrations of orally administered ciprofloxacin in patients with chronic periodontitis.

	SUBJECTS, MATERIALS AND METHODS

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Study design. Subjects consisted of five women and five men (between the ages of 20 and 52 years) who were in good general health and had no allergies. They had untreated, generalized, chronic periodontitis characterized by periodontal probing depths greater than 5 mm, 4 to 5 mm of clinical attachment loss and moderate-to-severe alveolar bone loss. We excluded from the study pregnant women and patients taking anti-inflammatory agents or antibiotics during the previous six months. All patients received the treatment protocol and signed an informed consent form. The Ethical Committee of Hacettepe University, Ankara, Turkey, gave its approval for our study protocol.

Clinical sampling. One week before sampling, one of us (A.Y.) measured full-mouth periodontal parameters. The mean (± standard deviation) probing depth was 5.77 ± 0.35 mm; gingival index, 1.55 ± 0.11; and plaque index, 1.41 ± 0.16.^19,20

We gave subjects oral ciprofloxacin (500 mg) to be taken twice daily for five days to establish steady-state tissue levels of the agent.⁵ Two of us (A.Y. and F.Ç.) began collecting serum and GCF samples immediately after patients received the first dose of the drug (baseline = 0 hours). We gave patients detailed information about the importance of taking the drug every 12 hours.

Serum samples were collected at the following time points after baseline sampling: 0.5, one, 1.5, two, 2.5, four, five, seven, 24 and 120 hours. Atici and colleagues²¹ suggested that the crevicular area serves as a reservoir, and the components of GCF (for example, enzymes) and the concentration of the materials analyzed in GCF (ciprofloxacin in this case) would be affected if the area was physically irritated often. Thus, the flow rate of the GCF and the concentration of the drug would be affected less if the number of irritations (brought on by sampling repetitions) was kept to a minimum. Therefore, we preferred to use fewer repetitions of GCF sampling compared with serum sampling. We collected GCF samples at one, two, four, five, seven, 24 and 120 hours after baseline sampling.

To eliminate the risk of salivary contamination, the clinician (A.Y.) chose sampling areas among maxillary teeth. She placed cotton rolls into the vestibular area of the sampled teeth and air-dried the surrounding soft tissues. The orifice method was used to collect the GCF, in which standardized paper strips (Periopapers, Ora Flow, Plainview, N.Y.) were placed at the orifice of the gingival sulcus at periodontally involved teeth and held for 30 seconds.²² The clinician manipulated the paper strips gently during sampling to avoid contamination with blood.

We measured GCF volume with an electronic instrument (Periotron 8000, Ora Flow) that had been calibrated by an established method. We then used a software program (MLCONVERT.EXE, Ora Flow) to convert the measurements to microliters.²³ After sampling, we placed the paper strips into sterilized plastic tubes. Using the venipuncture technique, we also drew approximately 5 mL of venous blood from subjects.

All GCF and serum samples were transferred immediately to the Hacettepe University Department of Pharmacology Laboratories and processed with minimal delay. After clinical sampling, all patients received periodontal therapy, including scaling and root planing, followed by conventional flap surgical procedures.

High-performance liquid chromatography, or HPLC. Using a slightly modified method of Jim and colleagues,²⁴ we used HPLC to determine the concentrations of ciprofloxacin in GCF and serum samples. The separation was performed on a carbon 18, or C18, cartridge column (100 x 8–millimeter internal diameter, with a particle size of 10 µm) (Radial-Pak, Waters, Milford, Mass.) and precolumn (C18). We monitored the sample elution with a fluorescence detector, using excitation and emission wavelengths of 280 and 455 nanometers, respectively.

The mobile phase consisted of acetonitrile and 0.1 mol/L of sodium dihydrogen phosphate (2:8 volume per volume). We used phosphoric acid to adjust the pH of the mobile phase to 3.9. The mobile phase was delivered at a flow rate of 2 mL/minute^–1. Quinine sulfate was used as an internal standard (2 µg/mL^–1). We used a mixture of methanol and water (50:50 vol/vol) to extract the ciprofloxacin from the GCF samples, and a mixture of acetonitrile and diethylether (50:50 vol/vol) to extract the drug from the serum samples.

Statistical analysis. We used the Friedman test to compare changes in drug concentration in serum and GCF for each time point. The differences between serum and GCF concentrations across time were compared with the Wilcoxon signed rank test.

	RESULTS

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None of the subjects reported any adverse effects during the five days of ciprofloxacin therapy. The measurements from the paper strips loaded with GCF during sampling were converted immediately to microliter values, and the volume of GCF collected from periodontally involved sites ranged between 0.03 µL and 0.68 µL. The results of this study demonstrated that ciprofloxacin concentrations were higher in GCF than in serum. The table shows the ciprofloxacin levels in GCF and serum of subjects; GCF concentrations were significantly higher than serum concentrations at two, four, seven, 24 and 120 hours.

The ciprofloxacin concentrations in GCF and serum at day 5 (120 hours) were 2.22 µg/mL and 0.25 µg/mL, respectively (P = .017). The GCF concentration at day 1 was significantly higher than the serum concentration at day 1 (P = .005).

	DISCUSSION

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Conventional periodontal therapy may not be effective enough to débride bacteria from infected periodontal sites, and the soft-tissue wall of a periodontal pocket still may act as a harbor for periodontal pathogens.^15,25,26 An antimicrobial approach to periodontal therapy targets the sub-gingival biofilm, permitting recolonization of a microbiota that is compatible with periodontal health.²⁷ Tetracyclines and nitroimidazoles (for example, metronidazole) have been studied extensively and serve as useful adjuncts in reducing the levels of periodontal pathogens.^12,28–31 Tetracycline is a widely accepted antibiotic owing to its ability to reach high concentrations in GCF.²⁸ The most significant drawback to tetracycline is that some bacteria have been shown to be resistant to the drug³²; consequently, other antibacterial agents, such as ciprofloxacin, may be an alternative adjunctive therapy.^10,13

The study results demonstrated a twofold-to-threefold higher ciprofloxacin level in gingival crevicular fluid compared with that in serum.

In our study, we found that ciprofloxacin concentrations in serum and GCF were high at all time points. The results also demonstrated a twofold-to-threefold higher ciprofloxacin level in GCF compared with that in serum during the entire sampling period. These findings may be similar to those in other in vitro studies,^10,13,33 in which PMNs enhanced the distribution of the drug to inflamed sites, PMNs enhanced local concentrations of the drug, and fluoroquinolones were shown to be a promising candidate for adjunctive, systemic, antibiotic therapy compared with penicillin/sulbactam, macrolides and nitroimidazole.

Cacchillo and Walters¹³ demonstrated that PMNs loaded with ciprofloxacin maintained therapeutic levels of the agent and killed A. actinomycetemcomitans more rapidly than did unloaded PMNs. In addition, Holm and colleagues³⁴ noted that laboratory strains of this organism appeared to be more susceptible to killing by PMNs than were fresh isolates. Therefore, ciprofloxacin may have a greater impact on A. actinomycetemcomitans in vivo than it has had in laboratory studies. We speculate that the higher levels of ciprofloxacin in GCF compared with serum levels may be due to higher levels of PMNs in infected periodontal sites than elsewhere; the PMNs may help transport the drug from these sites into the gingival crevice.

In addition to its effect on A. actinomycetemcomitans, ciprofloxacin is active against enteric rods and pseudomonads associated with chronic periodontitis.^10,13,35,36 This antibiotic also has been used in combination with metronidazole to treat mixed infections of anaerobic bacteria, enteric rods and A. actinomycetemcomitans.^35,37 Like other studies of bodily fluids (including urine, prostatic fluid, bronchial secretions and saliva),^33,38–40 this follow-up study elicited information about ciprofloxacin’s ability to reach infected periodontal sites via GCF.

	CONCLUSION

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The results of our clinical study show that ciprofloxacin is found in GCF, and its concentration in GCF is significantly higher than it is in serum. Consequently, it may be an effective adjunctive treatment for infected periodontal sites. Further studies are needed to determine the effectiveness of ciprofloxacin, as well as the necessary concentration of the drug, in patients with aggressive or refractory periodontitis.