1.1 Abstracts

      NEW BIOERODIBLE SYSTEM FOR THE LOCAL DELIVERY OF ANTIMICROBIALS TO PERIODONTAL POCKETS

      Clinical application of poly(ortho ester) as vehicle of antimicrobial agents for the treatment of periodontal pockets has been tested for the first time. Retention of the active agent tetracycline was measured and tolerance to the polymer was examined. The compound has the potential to remain in the periodontal pockets for up to 11 days. Retention was, however, variable and depended on clinical conditions at injection. Tetracycline concentrations in positive sites were up to 100 times higher than the MIC. Tolerance was excellent. Further studies are needed to improve the adhesive properties of the polymer.

      NOUVEAU SYSTEME RESORBABLE POUR LA LIBERATION LOCALE D'AGENTS ANTIMICROBIENS DANS LES POCHES PARODONTALES

      L'application clinique de poly(ortho ester) comme véhicule d'agents antimicrobiens pour le traitement local des maladies parodontales a été testée pour la première fois. La tolérance au polymère a été examinée et la rétention de son principe actif, la tétracycline, a été mesurée. Le produit a été retrouvé jusqu'à 11 jours après son administration, mais la rétention dans les poches parodontales n'est pas constante et dépend des conditions cliniques lors de l'injection. Dans les sites positifs, la concentration de tétracycline dépasse de 100 fois la CMI. La tolérance s'est révélée excellente. Des développements sont nécessaires pour améliorer les propriétés adhésives du polymère.

1.2 Résumé en français

2.1 Local Delivery Devices

      As irrigation of pockets can achieve therapeutic drug levels for only short periods of time (Pitcher et al. 1980, Eakle et al. 1986, Oosterwaal et al. 1990), controlled delivery systems have been proposed to extend the time of action of locally applied antimicrobial agents. The use of such systems has been advocated particularly in the therapy of sites difficult to reach by mechanical means, for example furcation defects (Nordland et al. 1987), and sites not responding to conventional mechanical therapy (see Greenstein & Polson 1998 for review). Although numerous local controlled delivery systems have been designed in the past two decades, only few have been tested extensively, and even fewer have become commercially available for broad use by dentists (for review see Mombelli & Tonetti 2001).

      Two multi-center controlled clinical trials have shown the efficacy a 2% minocycline ointment (Dentomycin, Lederle, Wayne, USA) to improve clinical parameters, if applied repeatedly. In the first study, subjects were treated 4 times in addition to scaling and root planing with minocycline or placebo ointment (van Steenberghe et al. 1993). The second study evaluated a seven-time repeated application of the drug over a 15-months period (van Steenberghe et al. 1993). The repeated subgingival administration of minocycline ointment yielded an adjunctive improvement after subgingival instrumentation in both clinical and microbiologic variables.

      Another product contains metronidazole as active principle (25%) in glyceryl-mono-oleate and sesame oil as carriers (Elyzol^®, Dumex, Copenhagen, Denmark). This degradable compound can be injected in the form of a gel with a syringe directly into the pocket. Although the gel increases its viscosity in the pocket due to the presence of water, about half of the product is lost immediately following placement, and in the hours thereafter the concentration of metronidazole decreases exponentially (Stoltze 1992, Stoltze 1995). The manufacturer therefore recommends two applications of the gel within one week. In clinical studies no significant difference in periodontal parameters was demonstrated between Elyzol^® and conventional SRP (Ainamo et al. 1992, Grossi et al. 1995, Pihlstrom et al. 1995); these results were interpreted to suggest equivalence of the two treatments.

      In one of the first clinical trials testing the controlled local administration of a drug into a periodontal pocket, delivery of tetracycline was attempted by placing hollow dialysis tubes filled with the active agent into the diseased sites (Goodson et al. 1979). Subsequent developments resulted in the Actisite^® tetracycline fiber (Alza, Palo Alto, USA), consisting of a monolithic ethylene-vinyl-acetate copolymer loaded with tetracycline hydrochloride crystals (Goodson et al. 1979). The nondegradable carrier assumes the form of a fiber with a diameter of 0.5 mm and a length of 23 cm, and contains 12.7 mg of the antimicrobial agent. The device is densely packed into the periodontal pocket and left in situ during 7 to 10 days. Measurements in humans have shown very high and constant concentrations of drug at the treated site during the entire time of application (Tonetti et al. 1990). In several multi-center studies, the tetracycline fiber has proved to be effective. Used as an adjunct to scaling and root planing (SRP), it improved probing pocket depth (PPD) and clinical attachment level (CAL) significantly better than mechanical treatment alone (Newman et al. 1994, Michalowicz et al. 1995). Despite of its proven efficacy, the device has been withdrawn from the market in early 2002, and is presently no longer available commercially.

      A more recently developed system contains 8.5% doxycycline hyclate as active agent. Polylactic acid is the biodegradable vehicle and N-methyl-2-pyrrolidone (NMP) the solvent (Atridox, Atrix Laboratories, Ft.Collins, USA). Two separate syringes are coupled and mixed just prior to use. As soon as the compound is injected into the pocket, the NMP is replaced by water, which causes the polymer to return to its solid state (Stoller et al. 1998). On the short term, clinical studies have demonstrated that chemical therapy alone is at least as effective as SRP in improving clinical parameters (Garrett et al. 1999, Garrett et al. 2000). In one study, even better results were obtained in a group treated with the drug during a single session of scaling, compared to those obtained in subjects treated in four appointments with subgingival mechanical therapy (Wennstrom et al 2001).

      The potential usefulness of microcapsules as local delivery device for periodontal therapy has also been investigated. In 1994, (Jones et al.) described the use of minocycline HCl microencapsulated in poly(lactic acid-co-glycolic acid) (PLGA) for the treatment of chronic periodontitis. The biodegradable polymer containing the active agent had the form of a powder and was administered into the periodontal pocket with a syringe. Maybe due to the small number of subjects and/or insufficient plaque control, clinical and microbiological parameters failed to demonstrate superiority in comparison to control or mechanical treatment alone (Jones et al. 1994). A few years later, Yeom (1997) presented a new bioabsorbable polymeric system consisting of chitosan-encapsulated alginate microspheres loaded with 10% minocycline. The injectable polymeric compound was tested in 15 patients as adjunct to supragingival scaling compared to SRP alone. The six-week observation period permitted to conclude that BoP was better reduced by antimicrobial therapy than by SRP. Microbiological indicators of periodontal health tended to be better in minocycline sites than SRP sites (Yeom et al. 1997). A multicentric study, including nearly 750 subjects in 18 centers, demonstrated the superiority of locally delivered minocycline (in PLGA) over nine months upon SRP alone, or SRP plus vehicle (Williams et al. 2001).

2.2 Poly (ortho esters)

      Because their physico-chemical properties can be varied to a large extent, polymers attract much attention for their potential use as biomaterials in various fields of medicine. A wide range of applications are currently explored in orthopedics, ophthalmology, or dentistry, including their potential use as carriers of active agents for controlled local drug delivery (Sintzel et al. 1996, Peter et al. 1998).

      Poly(ortho esters) (POE) are synthetic biodegradable hydrophobic polymers that allow the liberation of incorporated agents by surface erosion. This mechanism has been validated in animal models for applications in ophthalmology (Einmahl et al. 2000, Einmahl et al. 2001a, Merkli et al. 1994, Zignani et al. 1998, Zignani et al. 2000b). The use of semi-solid POEs is expected to lead to significant advances in glaucoma filtering surgery, and may make drug delivery to various parts of the human eye possible (Zignani et al. 2000a, Einmahl et al. 2002). Investigations are currently in progress in several other fields of veterinary and human medicine (for review see Einmahl et al. 2001b). POEs may also become suitable candidates for local controlled drug delivery in periodontal pockets. Preliminary laboratory investigations have been presented (Roskos et al. 1995.

      The polymer described in the present work is a low molecular weight auto-catalyzed POE containing 30 mol% of lactic acid units (POE₇₀LA₃₀). It was recently synthesized, characterized and optimized (Schwach-Abdellaoui et al. 1999, Schwach-Abdellaoui et al. 2001). Synthesis reaction is an acid-catalyzed condensation of 3,9-diethylidene-2,4,8,10-tetraoxaspiro[5,5]undecane (DETOSU) with 1,10decanediol and 1,10decanediol-dilactate. ptoluenesufonic acid (p-TSA) is added as catalyst and tetrahydrofuran (THF) is the solvent (Figure 1).

      By modifying the proportion of lactic acid in the polymer, different degradation rates can be obtained (see 2.3) and the physicochemical properties of the compound (POE_xLA_y) can be varied. For example, POE₇₀LA₃₀ shows a low viscosity and, therefore, is suitable for injectable preparations (Schwach-Abdellaoui et al. 2001).

2.3 Previous experiments

      Degradation of auto-catalyzed POE occurs mostly by surface erosion. An incorporated drug could therefore be released for days to weeks (Ng et al. 1997, Schwach-Abdellaoui et al. 1998, Sintzel et al. 1998a, Sintzel et al. 1998b, Schwach-Abdellaoui et al. 1999). Delivery of tetracycline incorporated in POE₇₀LA₃₀ has been previously studied in vitro in thermostated cells at 37°C, circulated with phosphate buffer (Schwach-Abdellaoui et al. 2001). The released amount of drug was measured by high-performance liquid chromatography (HPLC). Figure 2 shows, for 10% or 20% tetracycline load, that the delivery lasted two weeks at an almost constant rate. The complete degradation of the polymeric matrix was simultaneous.

2.4 Purpose

      The aim of this phase I in vivo study was to explore the clinical potential of poly(ortho esters) as carriers of antimicrobial agents for the local treatment of periodontal disease. Observations focused on the retention of the material in periodontal pockets and the tolerance to the product. Additionally, the release of tetracycline incorporated in the polymer was measured. The study was subdivided in three parts. The specific purpose of part I was to preliminarily determine the potential for retention and the possible bioavailability of tetracycline after treatment of single rooted teeth and molars with class two furcation (Hamp et al. 1975) defects. The purpose of part II was to explore if retention could be improved by modifying the clinical conditions at application time to minimize the presence of fluid on the root surface. The purpose of part III was to determine whether a small and controlled amount of polymer could be better retained within the pocket than an uncontrolled quantity overfilling the pocket.

3.1 Subjects

      Eighteen systemically healthy volunteers with chronic periodontitis (4 females and 14 males, mean age 54, range 36-77), with periodontal pockets ³ 6 mm, participated in this three-part study. Each part was carried out in six persons. Subjects had not received any periodontal treatment during the year preceding the study and had neither taken antibiotics nor long-term anti-inflammatory medication during the previous six months. The general health history and oral health status were registered for each patient. Subjects with a medical condition (diabetes, rheumatic fever, blood dyscrasia, immunologic anomalies) or allergic to tetracycline or poly(ortho esters) were not included. Pregnant or lactating females were also not considered (see Patient Record Form in Appendix). The study was conducted according to the principles of the Declaration of Helsinki and was approved by the ethical board of the School of Dental Medicine, University of Geneva. The participants were informed on the aim of the study, the possible side effects, and gave their consent in written form.

3.2 Formulations

      Self-catalyzed poly(ortho ester) (POE) of low molecular weight, containing 30 mol% of lactic acid (LA) units in the polymeric backbone, was used as vehicle (POE₇₀LA₃₀). The compound was manufactured by the laboratory of Pharmaceutics and Biopharmaceutics of the University of Geneva. Drug loading was of 10% (w/w) or 20% (w/w) tetracycline base (TB₁₀, TB₂₀ ; Sigma, Steinheim, Germany). Synthesis and degradation of these polymers have been presented in sections 2.2 and 2.3. Both formulations were prepared by mixing the polymer with the corresponding quantity of tetracycline. All samples were g-irradiated under argon at -70°C with a total of 2 Mrad, a dose validated in previous studies (Sintzel et al. 1998c). The final products had the consistency of a highly viscous paste.

3.3 Clinical protocol

      Patients were instructed in proper plaque control methods to reach a good level of oral hygiene before the experiments were carried out. Hard and soft deposits were removed mechanically from all supraginigival tooth surfaces by scaling and polishing.

      At day 0, PPD, BoP and gingival recession (REC) were recorded from all study sites. The selected teeth were then scaled and planed subgingivally under local anesthesia, using ultrasonic scalers and hand curettes. Subgingival treatment was not limited in time and lasted approximately 5 to 10 minutes per tooth. Prior to the placement of the compound into the pocket, each site was isolated from saliva with cotton rolls and dried with gauze. The prepared teeth then received the trial medication and were followed up according to the protocols outlined below (see 3.3.1.-3.3.3, parts I-III). In all subjects the polymer was placed into the periodontal sites with a syringe designed for intraligamentar anesthesia (Ligmaject^®, Henke-Sass Wolf, Tuttlingen, Germany), fitted with a canula 0.8 mm in diameter. At each follow-up session the GCF was assayed. The tolerance to the product was evaluated by visual inspection of the gingiva at each site. Changes in texture and color as well as presence or absence of suppuration were recorded. Bad taste, discomfort, or pain were also assessed. For the collection of GCF, teeth were first isolated and dried by a gentle stream of compressed air. Three minutes later, paper strips (Periopaper^®, Oraflow, Plainview, NY, USA) were placed for 30 seconds at the gingival margin of the test sites without entering the pocket. As a general rule, if no active agent could be detected at the first and second visits following placement in any given site of a subject, no further GCF samples were taken thereafter in this individual.

      At the completion of the observation period, PPD, BoP and REC were recorded again from all selected study sites. Patients received additional periodontal therapy according to their needs.

3.4 Laboratory procedures

      The retention time of the formulations was determined indirectly by measuring the concentration of tetracycline in gingival crevicular fluid (GCF) with a bioassay (Gordon et al. 1980, Goodson et al.1983, Tonetti et al. 1990). The volume of GCF was determined by a fluid analyzer (Periotron^®8000, Oraflow, Plainview, NY, USA). A standard curve was obtained for each of the three parts by recording fixed volumes of serum with the fluid analyzer (see Appendix), and by fitting a 4^th order polynomial plot to the data points as recommended by the manufacturer (www.oraflow.com). To determine the amount of tetracycline, strips were placed onto the surface of Müller-Hinton 2 agar (OXOID, Basingstoke, UK) previously inoculated with a strain of Bacillus cereus (ATCC 11778). After 24h incubation at 37°C, the inhibition diameter around the strip was measured and the quantity of tetracycline determined according to a standard curve (Goodson et al. 1983, Tonetti et al. 1990). The concentration of tetracycline was computed by dividing the quantity of antibiotic by the volume of GCF on the strip.

4.1 Part I

4.2 Part II

      From the six patients enrolled in this second part (subjects 2.1 to 2.6), five completed the study; subject 2.5 missed the last appointment (day 11) because of sickness. Placement of the retraction chords took 2 to 5 minutes. Four to 12 cm of chord were used. Clinical inspection of the sites after the removal of the chords indicated that 22 out of 24 sites (92%) were dry.

4.2.2 Retention

      In this second part of the study, a better retention of the polymer was observed: as shown in Figure 4, 5 out of 6 subjects showed evidence for tetracycline release in at least one site, at least once. Seven out of 24 treated surfaces (29%) were positive on day 4, eight on day 7 (33%), and two on day 11 (8%). The tetracycline concentration in positive sites ranged from 0.0004 mg/ml to 281 mg/ml of GCF. Three out of the six patients showed positive pockets on day 4 (subjects 2.1, 2.2, 2.3), and four on day 7 (subjects 2.1, 2.2, 2.4, 2.5). In two patients (2.1, 2.2), tetracycline was also detected on day 11 (Figure 4).

      

Figure 4 : Tetracycline concentrations in the six patients of the part II.

4.3 Part III

4.4 Release of tetracycline

      As mentioned above, retention of POE could be demonstrated only in a few samples taken in part one, but in positive specimens high tetracycline concentrations were recorded (Fig. 3). The positive samples obtained in part II are enumerated in Figure 5. Here, low concentrations were detected in many specimens. In 1/3 of the positive samples the concentration did not reach 1 mg/ml.

      An overall tendency for concentrations to decrease with time was noted. Figure 6 shows the average concentrations found in positive sites at day 4, day 7 and day 11. An exponential decrease can be observed.

      In Part III, tetracycline concentrations were extremely low in the three positive sites, ranging from 0.001 mg/ml to 0.003 mg/ml.

5.1 Retention

      The initial series of treatments (part I) yielded disappointing results with regard to the retention of poly(ortho ester). No differences could be observed between the single- and multi-rooted teeth subgroups. It was, however, encouraging to note that in those sites where the material was retained, drug was available during several days at considerable concentrations, suggesting the establishment of a stable subgingival drug reservoir, fulfilling the requirements for successful local chemotherapy (Mombelli 1997). In fact, the tooth with a swelling of the gingiva a few millimeters apically to the site of injection (see 4.1.1) also showed the highest sustained levels of tetracycline in GCF, indicating that a physically stable reservoir had been formed.

      In part two, a somewhat higher number of pockets were found to harbor the antibiotic system. Some of these positive sites demonstrated bacterial inhibition until day 11. Thus, changing the clinical conditions by "drying" the pocket at application time improved the number of positive sites and the retention time of the product as well. Shen et al. (1997) used a retraction cord to improve the effectiveness of SRP. Packed periodontal pockets gave enhanced access and visibility. Significantly more calculus was removed from the root surfaces of the test group compared to pockets where no cord was used (Shen et al. 1997). In both cases, the retraction cord gave a benefit for the treatment outcome but the clinical relevance of using such a device is questionable. Insertion of the cord into periodontal pockets and hemostasis requires attention and is time consuming.

      The loss of the local delivery device in several sites could be explained by an increase of tissue tonus resulting from a favorable response to treatment. Shrinkage of the gingiva could have pushed the compound out of the pocket. The retention of the device could also be compromised by the flushing action of the GCF. It has been estimated that in a periodontal pocket the GCF is replaced as much as 40 times per hour (Goodson et al. 1979). A relatively large reservoir of the drug seems to be necessary to maintain sufficiently high levels of an active agent in GCF over a prolonged period. Part III demonstrated that a small quantity of compound was insufficient to obtain positive results after one week.

5.2 Bioavailability

      Tetracyclines are broad-spectrum antimicrobials affecting anaerobes and facultative microorganisms. After systemic administration, such antibiotics are found in the periodontium at bacteriostatic concentration; 3 to 8 mg/ml were reported in GCF (Gordon et al. 1981, Sutter et al. 1983). Local delivery to periodontal pockets produces much higher, bactericidal, concentrations. By using tetracycline fibers, Tonetti et al. (1990) were able to demonstrate sustained concentrations of 1500 mg/ml in the GCF for ten days. Stoller et al. (1998) showed levels of doxycycline in excess of 150 mg/ml for 7 days with a degradable injectable system. Our results compare favorably with the latter, showing GCF levels of tetracycline base ranging from 70 to 325 mg/ml for positive sites in part 1 (Fig. 3). This is about 2 log over the MIC, which is 1-2 mg/ml for most of the periodontal pathogens (Seymour & Heasman 1995 Kleinfelder et al. 1999) but may be as high as 8 mg/ml for some organisms (Walker et al. 1981).

      In Part II, maximal concentrations were comparable to those found in the first part, but a higher proportion of sites showed low concentrations (Fig. 5). 2/3 of the antibiotic concentrations recorded in positive samples were above the MIC. In the other positive sites, inhibition zones on the bioassays were very small, reaching the detection limit of the assay, and indicating minimal amounts of retained tetracycline. This phenomenon was observed again in part III, where tetracycline quantities were extremely low in all three positive sites. It should be noted that close to the detection limit of the assay tetracycline concentration could not be determined precisely.

5.3 Release kinetics

      An overall tendency for concentrations to decrease with time was noted, suggesting that the compound was eliminated exponentially. Figure 8 compares the mean concentrations found with the poly(ortho ester)/tetracycline device as reported in section 4.4, to the pharmacokinetics of three commercially available drug delivery systems. Metronidazole gel (MET) showed rapidly decreasing concentrations, resulting in low values after the first day (Stoltze 1992). The release profile of tetracycline from monolithic, non-resorbable ethylene-vinyl-acetate fibers (THF) has been characterized as zero-order for ten days by Tonetti et al. (1990). The release profile of doxycycline from biodegradable (DOX) was characterized by Stoller et al. (1998). Doxycycline level decreased with time but concentrations were above 150 mg/ml for 7 days. The release kinetics of the poly(ortho ester)/tetracycline compound can be compared to the doxycycline system. Both have similar profiles, which differ from the solid device on one hand and the gel on the other.