Periodontology 2000, Vol. 48, 2008, 92–110 2008 The Author. Printed in Singapore. All rights reserved Journal compilation 2008 Blackwell Munksgaard PERIODONTOLOGY 2000 Host response modulation in periodontics

PHILIP M. PRESHAW

Host response modulation (or host modulation) is a future will see a range of host modulation therapies term that has been introduced to the dental profes- developed that target different aspects of the sion relatively recently. In the periodontal context, inflammatory pathogenic processes which occur in and in very simple terms, it means modifying or the diseased periodontium. modulating destructive or damaging aspects of the inflammatory host response that develops in the periodontal tissues as a result of the chronic chal- Periodontal pathogenesis lenge presented by the subgingival bacterial plaque. Host response modulation is routinely practised by To a large extent, the emergence of host response our medical colleagues, who use host modulation modulation as a treatment concept has resulted from strategies in the treatment of disorders such as our improved understanding of the pathogenesis of rheumatoid arthritis and osteoporosis. And while the periodontal disease. A common observation in peri- term host modulation has only recently started to be odontal practice is that while gingivitis and mild widely used in general dentistry, the concept was first periodontitis are relatively common in the popula- introduced to the research community in the late tion, severe periodontitis is less prevalent, despite 1980s and early 1990s (34, 107). Indeed, in 1990 plaque being a common finding in a majority of Williams (107) stated that Ôthere are compelling data people. Thus, advanced periodontal disease is now from studies in animals and human trials indicating considered to affect approximately 8–15% of adults that pharmacologic agents that modulate the host (3, 9, 48), which is considerably less than that re- responses believed to be involved in the pathogenesis ported in epidemiological studies conducted in ear- of periodontal destruction may be efficacious in lier decades (63). Certain individuals appear to be slowing the progression of diseaseÕ. Over the last two more susceptible to periodontal disease, and this decades, a variety of pharmacological agents have increased susceptibility is largely determined by the been studied for a possible role as host modulators immune-inflammatory response that develops in the in the management of periodontal disease. These periodontal tissues following chronic exposure to include nonsteroidal anti-inflammatory drugs, bis- bacterial plaque. phosphonates and the tetracycline family of com- Periodontal pathogenesis has been extensively re- pounds (and their chemically modified analogues). viewed by a number of authors (52, 54, 73) and it is Newer agents that have the potential to be of benefit not the purpose of this paper to cover this ground in periodontal treatment include anti-cytokine drugs again. Suffice to say, the microbial challenge pre- (which have successfully been used in the treatment sented by subgingival plaque results in an upregu- of rheumatoid arthritis), soluble cytokine blockers lated host immune-inflammatory response in the and lipoxins. To date, only one systemic medication periodontal tissues that is characterized by the has been licensed specifically as a host response excessive production of inflammatory cytokines (e.g. modulator for the treatment of periodontal disease, interleukins, tumor necrosis factor-a), prostanoids and that is subantimicrobial dose doxycycline (a fo- (e.g. prostaglandin E2) and enzymes [including the cus in this review). The inclusion of host response matrix metalloproteinases (MMPs)]. These pro- modulation into periodontal management strategies inflammatory mediators are responsible for the is an exciting development with significant potential majority of periodontal breakdown that occurs, for improving treatment outcomes. It is likely that the leading to the clinical signs and symptoms of disease.

92 Host response modulation in periodontics

Perhaps more important than the levels of any single titis. This treatment approach has changed little over inflammatory mediator in the periodontal tissues is the years, notwithstanding considerable debate about the relative balance between pro-inflammatory and issues such as the merits of manual vs. ultrasonic anti-inflammatory cytokines and enzymes. Thus, instrumentation, or the degree of root surface pro-inflammatory mediators, such as prostaglandins smoothness ⁄ hardness to be achieved. Traditionally, and many cytokines, are balanced by anti-inflam- this process has been referred to as Ôroot planingÕ, matory cytokines and lipoxins (102). The destructive although the term Ôroot surface instrumentationÕ is activities of MMPs are balanced by their inhibitors, now preferred as it is felt that ÔplaningÕ places too the tissue inhibitors of metalloproteinases. Imbal- much of an emphasis on the removal of cementum ances between pro-inflammatory and anti-inflam- and dentine from the root to create a smooth, hard matory activities in the periodontal tissues are a surface, which has now been demonstrated as major determinant of periodontal destruction. unnecessary for periodontal healing (15). The aim of The immune-inflammatory response against bac- root surface instrumentation is to disrupt physically terial plaque can thus be viewed as a Ôtwo-edged the subgingival biofilm and reduce the bacterial swordÕ. That is, the response is protective by intent, bioburden, while removing plaque and calculus to as and provides antibodies and polymorphonuclear large an extent as is achievable. The objective of this neutrophils that are responsible for the control of the treatment is to reduce the chronic challenge pre- bacterial infection. However, the inflammatory re- sented by the subgingival plaque bacteria, such that sponse, in certain individuals, results in the local inflammatory responses in the periodontal tissues production of excessive quantities of destructive en- are reduced, leading to resolution of inflammation zymes and inflammatory mediators that result in the and shrinkage of the gingival tissues. Resulting shal- tissue destruction which is observed clinically. It is lower pockets (with further gains in attachment paradoxical (although not unique in human diseases) possibly arising from the formation of a long junc- that the inflammatory response to the bacterial tional epithelium) are easier to maintain for both the challenge is primarily responsible for the breakdown patient and the clinician, and favour a less patho- of the periodontal hard and soft tissues. Periodontal genic microflora. disease is characterized by high concentrations of The outcomes that can be expected following MMPs, cytokines and prostanoids in the periodontal nonsurgical periodontal therapy are remarkably tissues, whereas periodontal health is characterized consistent. For example, for those pockets initially 4– by the opposite (75). 6 mm deep, mean probing depth reductions of Plaque bacteria therefore initiate the disease pro- approximately 1.0–1.5 mm and mean attachment cess, and bacterial antigens that cross the junctional gains of 0.5–1.0 mm can be expected (15). For dee- epithelium into the underlying connective tissues per pockets (7 mm or greater), mean probing depth drive the inflammatory response. Bacteria are there- reductions of 2.0–2.5 mm and mean attachment fore a necessary prerequisite for disease to develop but gains of 1.0–1.5 mm can be expected (15). In many are insufficient to cause periodontal disease alone. For patients, nonsurgical management alone (compris- periodontal disease to develop, a susceptible host is ing oral hygiene instruction, root surface instru- also required, in other words a host in which excessive mentation and periodontal maintenance care) may production of destructive enzymes (such as MMPs) be sufficient to result in clinical improvements and and inflammatory mediators (e.g. interleukins and control of periodontal disease. However, there are prostaglandins) are released during the cascade of many patients in whom treatment responses fol- destructive inflammatory events that occur as part of lowing conventional treatment are more limited and the inflammatory response (76). The purpose of host both patient and clinician may ask if anything fur- modulatory therapy is to restore balance between, on ther can be done. Our improved understanding of the one hand, pro-inflammatory mediators and the pathogenesis of periodontal disease has led to destructive enzymes, and, on the other hand, anti- the development of host modulation as a treatment inflammatory mediators and enzyme inhibitors. strategy that can be used in addition to conventional treatment approaches. Thus, a combination of therapeutic approaches may offer the best chance Conventional treatment strategies for clinical improvements (82), and this could in- clude: The mechanical removal of plaque and calculus is • reduction in the bacterial burden (by root surface considered as the standard treatment for periodon- instrumentation and hygiene therapy).

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• risk factor modification (by smoking cessation and tory destructive events in the tissues, and (ii) reso- improved diabetes control). lution of inflammation and downregulation of • host response modulation. destructive processes. Reducing the bacterial bio- As healthcare professionals, we should strive to burden by root surface instrumentation targets one continually improve patient choice and enhance the aspect of the pathogenic process by reducing the patientÕs experience as they progress through a antigenic challenge that drives the inflammatory re- course of treatment under our care. Central to this is sponse in the tissues. However, complete elimination the concept of patient education and involvement in of all subgingival bacteria is not achievable (or even decision-making about treatment strategies. A desirable), and recolonisation by putative pathogens paternalistic stance, in which the clinician makes all occurs. Host response modulation therefore offers decisions for the patient based on their superior the potential for downregulating destructive aspects knowledge, is no longer appropriate. Thus, patients of the host response so that, in combination with must become partners with the clinician and assume conventional treatments to reduce the bacterial a degree of responsibility for their care. The impor- burden, the balance between health (characterized tance of this in periodontal treatment is quite clear, by resolution of inflammation and wound healing) given that patient compliance (with oral hygiene, and disease progression (characterized by continued periodontal maintenance and smoking cessation) has pro-inflammatory, destructive events) is tipped in the a profound effect on treatment outcomes. This is not direction of a healing response. to say that responsibility for a good treatment out- In periodontitis, the host is responsible for most of come can be devolved entirely to patients. Rather, it the tissue breakdown that occurs, leading to the is our responsibility to make sure that patients are clinical signs of disease. Host response modulators aware of the importance of their own self-manage- offer the potential for modulating or reducing this ment, and we as clinicians facilitate this through destruction by ameliorating excessive or pathologi- education, motivation, empowerment and, of course, cally elevated inflammatory processes to enhance the provision of excellent clinical care. These con- opportunities for would healing and periodontal cepts fit well with management protocols in which stability. A variety of drug classes have been evalu- adjunctive host modulation is combined with con- ated as host response modulators, including the ventional treatment strategies. Anecdotally, host nonsteroidal anti-inflammatory drugs, bisphospho- modulation therapy is typically welcomed by the nates, and tetracyclines. patient once they have been informed of the rationale for such an approach. Nonsteroidal anti-inflammatory drugs Nonsteroidal anti-inflammatory drugs inhibit the Host modulation agents: historical formation of prostaglandins, including prostaglandin perspective E2, which is produced by a variety of resident and infiltrating cell types in the periodontium (including Host modulatory therapy is a treatment concept that neutrophils, macrophages, fibroblasts and epithelial aims to reduce tissue destruction and stabilize the cells) in response to lipopolysaccharide. Prostaglan- periodontium by downregulating or modifying din E2 is a key inflammatory mediator in periodontal destructive aspects and ⁄ or upregulating protective disease as it upregulates osteoclastic bone resorption or regenerative components of the host response. (45, 74), and prostaglandin E2 levels are significantly Host modulatory therapies could include systemi- increased in the tissue and gingival crevicular fluid of cally or locally delivered pharmaceuticals that are patients with periodontal disease compared to heal- prescribed as adjuncts to other forms of periodontal thy controls (37, 74). Nonsteroidal anti-inflammatory treatment. Host modulatory therapies offer the drugs inhibit the formation of prostaglandins by opportunity to move periodontal treatment strategies blocking the cyclo-oxygenase pathway of arachidonic to a new level. Historically, periodontal treatment has acid metabolism. They are used to reduce tissue focused on reducing the bacterial challenge by root inflammation and pain, and are indicated in a variety surface instrumentation. However, the outcomes of chronic inflammatory diseases. The ability of after conventional treatment of this chronic disease nonsteroidal anti-inflammatory drugs to block pros- are not always optimal, predictable or stable. Peri- taglandin E2 production, thereby reducing inflam- odontal disease can be viewed as a balance between mation and inhibiting osteoclast activity, has been (i) a persisting bacterial burden and pro-inflamma- investigated in patients with periodontal disease.

94 Host response modulation in periodontics

Thus, studies have shown that systemic flurbiprofen tissues was modified (103). However, the selective (109), indomethacin (108), naproxen (44) and others cyclo-oxygenase-2 inhibitors were later identified to (43), administered daily for periods of up to 3 years, be associated with significant and life-threatening significantly slowed the rate of alveolar bone loss adverse events, resulting in several of these drugs compared to patients treated with placebo. It is being withdrawn from the market (21). noteworthy that in one of these early papers pub- In summary, nonsteroidal anti-inflammatory drugs lished in 1993, the authors commented that Ôresearch have been extensively reviewed as potential host re- into nonsteroidal anti-inflammatory drugs in peri- sponse modulators for the treatment of periodontal odontal therapy may ultimately prove to have only disease (90), but unwanted effects (including the opened the door to research into host modulation as serious adverse effects of the selective cyclo-oxygen- an additional, but exciting, approach to periodontal ase-2 inhibitors) preclude their use as adjuncts to disease prevention and treatmentÕ (45). periodontal treatment. However, the nonsteroidal anti-inflammatory drugs suffer from some serious disadvantages that Bisphosphonates essentially preclude their use as an adjunctive treat- ment for periodontal disease. Daily administration The bisphosphonates disrupt osteoclastic activity and for extended periods of time (years rather than thereby inhibit bone resorption. Bisphosphonates months) is necessary for periodontal benefits to represent a class of chemical compounds structurally become apparent, and the nonsteroidal anti-inflam- related to pyrophosphate, which regulates minerali- matory drugs are associated with significant zation by binding to hydroxyapatite crystals, but is unwanted effects, including gastrointestinal prob- not stable in vivo, undergoing hydrolysis of its P-O-P lems, hemorrhage (as a result of decreased platelet bond as a result of pyrophosphatase activity (90). The aggregation), and renal and hepatic impairment. replacement of the oxygen atom with a carbon atom Also, once patients cease taking nonsteroidal (creating a P-C-P bond) results in the formation of a anti-inflammatory drugs, a return to, or even accel- bisphosphonate molecule that is chemically stable eration of, the rate of bone loss seen prior to drug and resists hydrolysis via pyrophosphatase and therapy occurs, sometimes referred to as a Ôrebound alkaline phosphatase. Thus, bisphosphonates bind to effectÕ. hydroxyapatite crystals and prevent their dissolution. The selective cyclo-oxygenase-2 inhibitors were They also increase osteoblast differentiation and in- investigated in the anticipation that they could offer hibit osteoclast activation, and are used extensively in potential in the treatment of periodontitis. The en- the management of osteoporosis and other bone- zyme cyclo-oxygenase, which metabolizes arachi- resorptive conditions. donic acid, exists in two functionally distinct iso- Given these properties, it is not surprising that forms: cyclo-oxygenase-1 (which is constitutively bisphosphonates have been investigated as adjuncts expressed and has antithrombogenic and cytopro- in the treatment of periodontal disease, and in a tective functions); and cyclo-oxygenase-2 (which is study in dogs of naturally occurring periodontitis, the induced after stimulation with various cytokines, use of alendronate was associated with a significant growth factors and lipopolysaccharide). Inhibition of increase in bone density compared with placebo (85). cyclo-oxygenase-1 by nonselective nonsteroidal anti- In experimentally induced periodontitis in animal inflammatory drugs results in the majority of the studies, bisphosphonates reduced alveolar bone unwanted effects associated with nonsteroidal anti- resorption (92, 106), and in human studies, bis- inflammatory drug use, such as gastrointestinal phosphonates increased alveolar bone density (22). ulceration and impaired hemostasis. Induction of In randomized, placebo-controlled clinical trials in cyclo-oxygenase-2 results in the production of ele- humans with periodontal disease, bisphosphonate vated quantities of prostaglandins, and therefore use resulted in statistically significant reductions in inhibition of cyclo-oxygenase-2 by selective inhibi- the proportion of teeth demonstrating alveolar bone tors results in a reduction of inflammation without loss after 9 months (84) and statistically significant the unwanted effects commonly seen after long-term improvements in alveolar bone height (87). However, nonsteroidal anti-inflammatory drug use. Pre- a recent systematic review concluded that the small liminary studies in animal models showed that number of studies that have been published, each selective cyclo-oxygenase-2 inhibitors slowed alveo- containing few subjects and investigating different lar bone loss (5, 42), and human studies confirmed outcome measures, prevented meaningful conclu- that prostaglandin production in the periodontal sions from being drawn about the benefits of bis-

95 Preshaw phosphonates when used as an adjunctive treatment or collagenase-1) and are concerned with the main- in periodontal disease (84). Following this, a more tenance of the periodontal connective tissues. Tran- recently published study of a randomized clinical scription of genes coding for MMPs is upregulated by trial of 335 patients with periodontal disease who inflammatory cytokines such as interleukin-1b and received either alendronate or placebo, reported that tumor necrosis factor-a (61). Regulation of MMP after 2 years of therapy there were no differences in activity involves specific, endogenous tissue inhibi- alveolar bone level or bone density between the two tors of metalloproteinases and a-macroglobulins, treatment groups (47). However, in a subgroup of which form complexes with active MMPs and their patients with low mandibular bone mineral density, latent precursors (86, 88). Tissue inhibitors of alendronate significantly reduced bone loss com- metalloproteinases are produced by fibroblasts, pared with placebo. macrophages, keratinocytes and endothelial cells (7). A recent development has been the publication of In healthy tissues, collagen homeostasis is a con- several case reports of avascular necrosis of the jaws, trolled process that is mediated extracellularly by particularly the mandible, following bisphosphonate MMP-1 (fibroblast collagenase) and intracellularly by therapy, with an increased risk of bone necrosis fol- a variety of lysosomal acid-dependent enzymes. lowing dental extractions. This has been termed Excessive quantities of MMPs are secreted in in- bisphosphonate-associated osteonecrosis and is a flamed periodontal tissues, and the balance between significant and clinically serious complication of MMPs and their inhibitors is disrupted, resulting in bisphosphonate therapy (55). Current guidance is to breakdown of the connective tissue matrix (6, 97). avoid extractions in patients who have received long- Neutrophils are key infiltrating cells in periodontitis term bisphosphonate therapy and seek expert opinion that accumulate in large numbers in inflamed peri- (55). At the present time, there are no bisphosphonate odontal tissues. Neutrophils have evolved to respond drugs that are approved and indicated for use as rapidly and aggressively to external stimuli and they adjuncts in the treatment of periodontal disease. release large quantities of destructive enzymes very rapidly (6). The predominant MMPs in periodontitis – MMP-8 and MMP-9 – are secreted by neutrophils Subantimicrobial dose doxycycline (33) and are very effective in degrading type 1 colla- gen, the most abundant type of collagen in the peri- Subantimicrobial dose doxycycline remains, at pres- odontal ligament (62). MMP-8 and MMP-9 levels ent, the only systemic host response modulator spe- increase with increasing severity of periodontal dis- cifically indicated as an adjunctive treatment for ease and decrease after treatment (28, 33). The periodontitis and will therefore be a focus in this excessive release of large quantities of MMPs in the review. Subantimicrobial dose doxycycline is ap- periodontium leads to significant breakdown of proved by the US Food and Drug Administration, the structural components of the connective tissues, UK Medicines and Healthcare products Regulatory contributing to the clinical signs of disease. Agency, and by similar agencies in other countries The rationale for using doxycycline at subantimi- throughout the world, and was introduced under the crobial doses as a host response modulator is that it trade name Periostat (CollaGenex Pharmaceuticals inhibits the activity of MMPs by a variety of syner- Inc., Newtown, PA). It is a 20-mg dose of doxycycline gistic mechanisms independent of any antibiotic hyclate that is taken twice daily for periods of 3– properties (summarized in Table 1). Early studies on 9 months as an adjunct to root surface instrumen- the use of tetracyclines to inhibit MMPs identified, tation in the treatment of periodontitis. interestingly, that those metalloproteinases which Doxycycline, similarly to other members of the were produced in excessive quantities in inflamed tetracycline family, has the ability to down-regulate periodontal tissues were more sensitive to inhibition MMPs, a family of zinc-dependent enzymes that by tetracyclines than those MMPs that were consti- are capable of degrading a variety of extracellular tutively expressed. Thus, MMP-13 is more sensitive to matrix molecules, including collagens (6, 88). MMPs tetracycline inhibition, with an inhibitory concen- play a key role in the tissue destruction observed tration50 (IC50)of<1lM (i.e. the concentration of in periodontal disease and are secreted by the tetracycline required to reduce the MMP concentra- majority of cell types in the periodontium, including tion by 50%), than MMP-8 (IC50  30 lM), and MMP- macrophages, neutrophils, fibroblasts, keratinocytes, 1 (fibroblast collagenase) is the least sensitive endothelial cells and osteoclasts. In healthy tissues, (IC50 > 200 lM) to inhibition by tetracyclines in vitro MMPs are produced primarily by fibroblasts (MMP-1 (31). Furthermore, doxycycline was shown to be more

96 Host response modulation in periodontics

with chronic periodontitis (28). Subsequent studies of Table 1. Downregulation of destructive events in the relatively short duration (1–3 months) indicated that periodontium by doxycycline results from modula- tion of a variety of different pro-inflammatory path- this dosage regimen could prevent periodontitis ways [adapted from Golub et al. (31)] progression without the emergence of doxycycline- Effect of doxycycline resistant organisms or other typical antibiotic side- • Direct inhibition of active MMPs by cation chelation effects (36). These initial studies paved the way for (dependent on Ca2+- and Zn2+-binding properties) progressively larger and longer clinical trials of the • Inhibits oxidative activation of latent MMPs efficacy of subantimicrobial dose doxycycline in the (independent of cation-binding properties) management of periodontal disease. • Downregulates expression of key inflammatory cytokines (interleukin-1, interleukin-6 and tumor

necrosis factor-a) and prostaglandin E2 Clinical studies of subantimicrobial dose • Scavenges and inhibits production of reactive oxygen doxycycline species produced by neutrophils One of the early clinical trials involved just 14 pa- • Inhibits MMPs and reactive oxygen species thereby tients with chronic periodontitis who, after removal protecting a1-proteinase inhibitor, and thus indirectly reducing tissue proteinase activity of subgingival plaque and calculus, were randomized • Stimulates fibroblast collagen production to either 20 mg of doxycycline twice daily or placebo • Reduces osteoclast activity and bone resorption for 2 months, then no study medications for • Inhibits osteoclast MMPs 2 months, then 20 mg of doxycycline twice daily or MMP, matrix metalloproteinase. placebo again for 2 months (16). The doxycycline regimen resulted in significant improvements in effective than other tetracyclines in reducing colla- probing depths and attachment levels, but did not genase activity in the gingival crevicular fluid of affect plaque or gingival inflammation. Furthermore, chronic periodontitis patients (35). Doxycycline was crevicular fluid collagenase levels were significantly also confirmed as being a more effective inhibitor of reduced in the doxycycline group, as was a1-pro- MMPs than either minocycline or tetracycline; teinase inhibitor degradation. In a clinical study of 75 doxycycline has a much lower inhibitory concentra- patients assigned to one of five treatment groups tion (IC50 =15lM) than minocycline (IC50 = 190 lM) (including various dosage regimens of low-dose or tetracycline (IC50 = 350 lM), indicating that a doxycycline and placebo) for 12 weeks after initial much lower dose of doxycycline is necessary to re- scaling and prophylaxis, followed by 12 weeks of no duce a given collagenase level by 50% compared with drug then a second episode of scaling and prophy- minocycline or tetracycline (10, 29). Furthermore, laxis and 12 more weeks of drug therapy, those doxycycline has also been found to be more effective patients who received subantimicrobial dose doxy- in blocking neutrophil collagenase activity (MMP-8) cycline (20 mg twice daily) demonstrated significant than fibroblast collagenase activity (MMP-1) (33, 94), reductions in crevicular fluid collagenase levels suggesting that doxycycline can provide a safe compared with the placebo group (32). In another method of reducing pathologically elevated collage- small study by the same research group, subanti- nase levels without interfering with normal connec- microbial dose doxycycline was given to 12 patients tive tissue turnover. with chronic periodontitis for 2 months following The ability of doxycycline to downregulate MMP a course of subgingival instrumentation and six activity was recognized as representing a novel patients were prescribed placebo (30). After 2 months treatment strategy for the management of periodon- of subantimicrobial dose doxycycline, there were titis. However, a major concern with the long-term significant decreases in the crevicular fluid con- administration of doxycycline was the possibility of centrations of MMP-8 and MMP-9 and carboxy- development of antibiotic resistance. Indeed, when terminal peptide (a pyridinoline-containing fragment high (antibiotic) doses of tetracycline (250 mg daily of type 1 collagen) compared with placebo. This was for 2–7 years) had previously been given as treatment the first study to show that subantimicrobial dose for refractory periodontitis, up to 77% of the patientsÕ doxycycline resulted in simultaneous reductions in culturable subgingival microflora exhibited tetracy- MMP activity together with a concomitant reduction cline resistance (53). Therefore, a low dose of 20 mg in the levels of collagen degradation products. Of twice daily was introduced, which was shown, after interest, root surface instrumentation alone has no 2 weeks, to inhibit collagenase activity by 60–80% in effect on crevicular fluid carboxyterminal peptide the gingival tissues and crevicular fluid of patients levels (1).

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Following on from these small-scale clinical studies, full-mouth subgingival debridement and oral hygiene a variety of larger clinical trials have been conducted instruction each week for 4 weeks plus 6 months of to investigate the efficacy of subantimicrobial dose adjunctive subantimicrobial dose doxycycline or doxycycline when used as an adjunct to nonsurgical placebo, with follow-up for approximately 9 months therapy, and the clinical data from these studies are (70). Ten subjects completed the study in each group, summarized in Table 2. The data in this table dem- and the mean reduction in probing depths at deep onstrate that clinical outcomes are improved when sites (baseline probing depths of ‡7 mm) in the adjunctive subantimicrobial dose doxycycline is pre- doxycycline group was 3.02 mm compared with scribed and these benefits are particularly clear in the 1.42 mm in the placebo group. In the doxycycline larger studies of longer duration. Thus, in the largest group, 38% of pockets of ‡7 mm were reduced by clinical trials to date (12, 83), mean attachment gains ‡4 mm (representing a huge clinical improvement) and probing depth reductions were statistically sig- compared with <10% of pockets in the placebo nificantly greater in patients treated with adjunctive group. Furthermore, in the doxycycline group, more subantimicrobial dose doxycycline compared with than double the number of pockets initially ‡7mm placebo. Mean changes in probing depth or attach- improved by ‡3 mm when compared with the pla- ment level (whether stratified by baseline probing cebo group (55% vs. 24%, respectively). These data depth or calculated for the full mouth) are not partic- demonstrate a very clear, clinically significant benefit ularly useful for revealing, in a meaningful way, the of subantimicrobial dose doxycycline when com- benefits, or not, of a particular treatment, however bined with appropriate and effective conventional (46), and as clinicians we never calculate mean prob- therapy. ing depths (for example) when we are evaluating the In a 9-month, randomized, placebo-controlled outcomes of the treatment we have provided. Rather, study of 24 institutionalized geriatric patients we focus on individual sites; thus, threshold change (‡65 years of age), patients treated with root surface data, in which we calculate the proportion of sites instrumentation and subantimicrobial dose doxycy- demonstrating probing reductions of ‡2or‡3 mm are cline for 9 months demonstrated significantly greater particularly useful. probing depth reductions and attachment gains These data are presented for some of the studies in compared to patients treated with root surface Table 2, and reveal very clearly the clinical benefit of instrumentation and placebo (66). Indeed, in deep subantimicrobial dose doxycycline. Thus, in the sites (baseline probing depths ‡7 mm), the mean studies by Caton et al. (12) and Preshaw et al. (83), probing depth reduction in the subantimicrobial approximately one-third more sites demonstrated dose doxycycline group was more than three times probing depth reductions of ‡2 mm in the suban- greater than that observed in the placebo group timicrobial dose doxycycline groups compared with (Table 2). Furthermore, at month 9, no sites in the the placebo groups, and nearly twice as many sites subantimicrobial dose doxycycline group demon- demonstrated probing depth reductions of ‡3mmin strated attachment loss or probing depth increases of the doxycycline groups compared with the placebo ‡2or‡3 mm from baseline. However, in the placebo groups. These changes represent tangible and clini- group at month 9, 47 sites (17.4%) demonstrated cally significant benefits for the patients treated with attachment loss of ‡2 mm, 30 sites (11.1%) demon- subantimicrobial dose doxycycline compared with strated attachment loss of ‡3 mm, 13 sites (4.8%) those treated by root surface instrumentation alone. demonstrated probing depth increases of ‡2 mm and While it is not possible to predict with certainty the five sites (1.9%) demonstrated probing depth in- likely clinical benefits of using adjunctive subantim- creases of ‡3 mm. The authors concluded that su- icrobial dose doxycycline in an individual patient or bantimicrobial dose doxycycline prevented disease at a particular periodontal site, these data do support progression, which may be particularly important in that, on balance, within a patient population, signif- elderly patients. The clinical benefits observed in the icantly more periodontal sites will experience clini- doxycycline group were particularly impressive in cally significant improvements in probing depths and this study, which may reflect (i) that this was an attachment levels (of ‡2or‡3 mm) compared with institutionalized population over whom complete patients treated by root surface instrumentation control was exercised regarding compliance with alone. plaque control and with taking the study medication, In a study of 30 subjects, £45 years of age, with (ii) study medication was prescribed for a full severe, generalized chronic periodontitis, the subjects 9 months, and (iii) this was a population of non- received intensive periodontal therapy comprising smokers.

98 Table 2. Clinical data reported in randomized clinical trials of subantimicrobial dose doxycycline used as adjunctive treatment for chronic periodontitis Year Author Study duration Study groups n1 Mean CAL gain (mm)2 Mean Percentage of Percentage of (drug duration) PD sites with CAL sites with PD reduction (mm)2 gain3 reduction3

4–6 mm ‡7mm 4–6 mm ‡7mm pockets pockets pockets pockets ‡2mm ‡3mm ‡2mm ‡3mm

2000 Caton et al. Study: 9 months SRP + SDD 90 1.03* 1.55* 0.95** 1.68** 46 22 47* 22* (12) (drug: 9 months) SRP + placebo 93 0.86 1.17 0.69 1.20 38 16 35 13 2002 Novak et al. Study: 9 months SRP + SDD 10 1.00 1.78 1.20 3.02* 29 15 48 26** (70) (drug: 6 months) SRP + placebo 10 0.56 1.24 0.97 1.42 21 11 21 6 2004 Preshaw Study: 9 months SRP + SDD 107 1.27** 2.09* 1.29** 2.31** 58* 33** 62** 37** et al. (83) (drug: 9 months) SRP + placebo 102 0.94 1.60 0.96 1.77 44 20 45 21 2004 Emingil Study: 12 months SRP + SDD 10 0.21 (all sites) 1.59* (all sites) et al. (23) (drug: 3 months) SRP + placebo 10 0.05 (all sites) 1.32 (all sites) 2004 Lee et al. Study: 9 months SRP + SDD 24 1.56* (all sites) 1.63* (all sites) (57) (drug: 9 months) SRP + placebo 17 0.80 (all sites) 1.19 (all sites) 2004 Choi et al. Study: 120 days SRP + SDD 15 2.2* (test sites) 1.6* (test sites) (14) (drug: 120 days) SRP + placebo 17 0.6 (test sites) 1.1 (test sites) 2005 Gurkan Study: 6 months SRP + SDD 13 1.12 2.15 1.80 3.38 et al. (40) (drug: 3 months) SRP + placebo 13 0.78 1.76 1.46 2.57 2005 Preshaw Study: 9 months SRP + SDD, nonsmokers 116 1.23* 1.89** 1.22** 2.16* 59** 33** 63*** 37*** 4 et al. (81) (drug: 9 months) SRP + SDD, smokers 81 1.03 1.71 1.01 1.80 44 21 45** 20 SRP + placebo, nonsmokers 135 0.96 1.43 0.88 1.53 43 19 44 18 SRP + placebo, smokers 60 0.85 1.58 0.80 1.62 37 15 31 13 2005 Mohammad Study: 9 months SRP + SDD 12 2.14*** 3.18* 1.57*** 3.22*** et al. (66) (drug: 9 months) SRP + placebo 12 0.02 0.25 0.63 0.98 otrsos ouaini periodontics in modulation response Host 2006 Gorska & Study: 3 months SRP + SDD 33 0.33* (all sites) 0.29* (all sites) Nedzi-Gora (drug: 3 months) SRP + placebo 33 0.04 (all sites) 0.08 (all sites) (38) 2007 Needleman Study: 6 months SRP + SDD (all smokers) 18 0.65 (all sites) 1.40 (all sites) et al. (67) (drug: 3 months) SRP + placebo (all smokers) 16 0.40 (all sites) 0.98 (all sites) CAL, clinical attachment level; PD, probing depth; SDD, subantimicrobial dose doxycycline; SRP, scaling and root planing – the term used to describe the treatment in the original publications. *P < 0.05, **P < 0.01 and ***P < 0.001 compared with placebo. 1n (subject numbers) are based on subjects completing the study or the intent-to-treat population, according to the original publication. 2Mean clinical attachment level gains and probing depth reductions were calculated either within categories of baseline probing depth, 4–6 mm or ‡ 7 mm as indicated (12, 40, 66, 70, 81, 83), or for all sites (23, 38, 57, 67) or for test sites (defined as the deepest site in the treated sextant) (14). 3Threshold change data are based on sites with probing depth ‡6 mm at baseline. These data were not reported in the original publications by Caton et al. (12) and Novak et al. (70), but were calculated from the raw data. Threshold change data were determined using the Generalised Estimating Equations model with adjustment for within-subject correlations among sites. 4Smoking data for Preshaw et al. (81) are combined data from Caton et al. (12) and Preshaw et al. (83). P values for mean attachment gain and probing depth reduction data indicate significantly greater improvements in

99 nonsmokers who received SDD compared with other groups. P values for threshold analyses indicate a significant difference from placebo within the smoking category. Preshaw

Some of the studies described in Table 2 evaluated crevicular fluid concentrations of transforming whether subantimicrobial dose doxycycline had any growth factor-b1 were significantly higher in the effect on biochemical markers of inflammation, in subantimicrobial dose doxycycline group than in the addition to studying clinical outcomes. Thus, Emingil placebo group, indicating that subantimicrobial et al. (23) identified that after root surface instru- doxycycline may contribute to connective tissue mentation followed by 3 months of treatment with healing and collagen matrix formation via an increase either subantimicrobial dose doxycycline or placebo, in transforming growth factor-b1. with monitoring for a further 9 months, reductions in The concentrations of MMP-8, MMP-9 and tissue crevicular fluid MMP-8 levels were identified in both inhibitor of metalloproteinase-1 were measured in groups as a result of treatment, and these reductions saliva and peripheral blood before and after suban- were significantly greater in the doxycycline group timicrobial dose doxycycline therapy for 3 months in than in the placebo group at 6 months. Similarly, the study by Gorska & Nedzi-Gora (38). Thirty-three laminin-5 c2 chain fragment levels, which are asso- patients were prescribed subantimicrobial dose ciated with the migration of epithelial cells during doxycycline for 3 months after root surface instru- periodontal pocket formation, were significantly mentation and 33 patients were prescribed placebo. reduced in the subantimicrobial dose doxycycline Statistically significantly greater attachment gains group compared with the placebo group (24). Lee and and probing depth reductions were observed in the co-workers (57) also studied the effect of subantimi- subantimicrobial dose doxycycline group, as shown crobial dose doxycycline on the crevicular fluid levels in Table 2, but there was no apparent effect of of MMP-8 and MMP-9. The significantly greater doxycycline therapy on serum or saliva levels of the improvements in attachment levels and probing MMPs or tissue inhibitor of metalloproteinase-1 depths that they identified at all time-points in the compared with the placebo group. doxycycline group were accompanied by significantly In a randomized, single-blind study to determine greater reductions in MMP-8 and MMP-9 levels in the relative effectiveness of various systemic the doxycycline group compared with the placebo adjunctive therapies, 92 patients with chronic peri- group over the course of the study. odontitis were assigned to receive root surface In the study by Choi et al. (14), crevicular fluid instrumentation alone (n = 23), or combined with concentrations of MMP-8 also demonstrated statis- 500 mg of azithromycin per day for 3 days (n = 25), tically significantly greater reductions following su- 250 mg of metronidazole three times per day for bantimicrobial dose doxycycline therapy compared 14 days (n = 24), or subantimicrobial dose doxycy- with placebo, although there were no differences cline 20 mg twice per day for 3 months (n = 20) (41). between the groups with regard to MMP-9 concen- The patients were then monitored for 12 months. trations. Interestingly, this study also showed that The majority of patients in each treatment group following treatment, crevicular fluid concentrations demonstrated improvements in probing depths and of tissue inhibitor of metalloproteinase-1 significantly attachment levels, and the patients receiving increased in both groups, supporting the effect of adjunctive treatments exhibited greater improve- treatment on reducing inflammation in the perio- ments overall in probing depths (although this did dontium. not achieve statistical significance). Sites with initial In the study by Gurkan and colleagues (40), pa- probing depths of >6 mm demonstrated sig- tients with severe, generalized chronic periodontitis nificantly greater probing depth reductions and were randomized to full-mouth supragingival attachment level gains in the subjects receiving instrumentation at baseline followed by 3 months of metronidazole or azithromycin compared with the subantimicrobial dose doxycycline or placebo. At other groups, and the authors concluded that the use 6 months, clinical improvements were evident in of adjunctive systemic antibiotics or adjunctive su- both groups, although there were no statistically bantimicrobial dose doxycycline should be limited to significant differences in mean probing depth those subjects who have more potential to receive the reductions or attachment gains between the groups. maximum benefit from these agents (i.e. the subjects However, significantly more sites (73% of sites) in the with the most advanced periodontal disease) (41). subantimicrobial dose doxycycline group that had These authors also identified an increase in mean probing depths of ‡7 mm at baseline demonstrated probing depths and attachment levels at the 6-month probing reductions of ‡3 mm compared with the recall period in the subantimicrobial doxycycline placebo group (50% of sites) (P = 0.011). Further- group (i.e. 3 months after completion of 3 months of more, at the end of the 3-month medication period, drug therapy), possibly indicating a relapse after

100 Host response modulation in periodontics cessation of doxycycline. This is in contrast to timicrobial dose doxycycline for the first 6 months another study in which no evidence for relapse was demonstrated greater reductions in probing depths of identified 3 months after patients finished a 9-month surgically treated sites of ‡ 6 mm compared with the course of subantimicrobial dose doxycycline (13). placebo group, and greater reductions in carboxy- This may indicate that a 9-month regimen of terminal peptide, with a rebound in carboxyterminal subantimicrobial dose doxycycline is more effective peptide levels when the study medication was stop- at maintaining the initial benefits observed after ped. Additionally, there were no significant differ- 3 months of medication, supporting prolonged dos- ences in the subgingival microflora between the ing for 9 months to achieve maximum benefit with doxycycline and control groups at any time-point. less chance of relapse upon stopping the medication. Also, methodological differences between the two Subantimicrobial dose doxycycline in studies may have contributed to these different out- smokers comes: in the study by Caton et al. (12), data analyses included only those sites with baseline probing Most periodontists would consider that the patient depths of > 3 mm; and when only sites with initially group which is most difficult to manage is the deep pockets were analysed in the study of Haffajee smokers. Smokers tend to have more advanced et al. (41), an increase in clinical parameters at the periodontal disease than nonsmokers and more 6-month time-point was not seen. limited outcomes following treatment (50). However, Taken collectively, the clinical studies summarized research has shown that if smokers stop smoking, in Table 2 reveal a clear clinical benefit of suban- treatment outcomes following nonsurgical therapy timicrobial dose doxycycline when used as an ad- are improved (80). Two studies to date have focussed junct to root surface instrumentation, supporting the specifically on the use of subantimicrobial dose findings of a recent systematic review which con- doxycycline in smokers with periodontal disease. The cluded that subantimicrobial dose doxycycline used first of these (81) was a meta-analysis of two previ- as an adjunct to nonsurgical therapy is beneficial in ously published clinical trials (12, 83). This meta- the management of chronic periodontitis over analysis of 392 patients (intent-to-treat population) 12 months (90). The clinical benefits are particularly was stratified into four subgroups depending on apparent with longer dosing periods (e.g. 9 months smoking status (current smoker or nonsmoker) and rather than 3 months) and especially in patients with whether subantimicrobial dose doxycycline was more advanced disease. The clinical significance of prescribed (doxycycline or placebo) (Table 2). Non- the improved outcomes following subantimicrobial smokers included both ex-smokers and never-smok- dose doxycycline is most notable when considering ers, and it was not possible to identify from the data the proportions of sites demonstrating clinically sig- how long ago the ex-smokers had stopped smoking. nificant probing depth reductions and attachment A hierarchical treatment response was observed gains (e.g. ‡2or‡3 mm) rather than when consider- following 9 months of adjunctive subantimicrobial ing mean probing depth and attachment changes. dose doxycycline, such that nonsmokers who re- This make sense, and, as clinicians, we use outcomes ceived subantimicrobial dose doxycycline demon- such as changes in the proportions of deep pockets strated the greatest attachment gains and probing when we evaluate our patients as part of routine depth reductions (81). Smokers who received placebo clinical care (rather than calculating mouth mean demonstrated the smallest clinical improvements probing depth changes, which have limited use in following treatment. Smokers who received sub- assessing a patientÕs response to treatment). As was antimicrobial dose doxycycline demonstrated an concluded by Haffajee and co-workers (41), not all intermediate treatment response that was broadly patients assigned to receive a specific adjunctive equivalent to that seen in nonsmokers who received treatment respond clinically in the same manner, and placebo. In sites with baseline probing depths of this reinforces the importance of determining the 4–6 mm, month-9 attachment gains were 19–45% factors that influence treatment outcome so that the better in nonsmokers who received subantimicrobial most appropriate therapy can be provided to indi- dose doxycycline compared with all other subgroups vidual patients. (P < 0.05) and were 21% greater in smokers who re- Subantimicrobial dose doxycycline was also used ceived subantimicrobial dose doxycycline compared as an adjunct to surgical management in a pilot study with smokers who received placebo (P < 0.05). Fur- of 24 patients undergoing access flap surgery (27). thermore, month-9 probing depth reductions were Over 12 months, those patients treated with suban- 21–53% greater in nonsmokers who received sub-

101 Preshaw antimicrobial dose doxycycline compared with all no evidence of a benefit of using subantimicrobial other subgroups (P < 0.01) and were 26% greater in dose doxycycline as an adjunctive treatment in smokers who received subantimicrobial dose doxy- smokers, which is in contrast to the findings of the cycline compared with smokers who received pla- meta-analysis discussed previously (81). cebo (P < 0.05). In sites with a baseline probing depth Clearly, further research is required to answer the of ‡7 mm, month-9 attachment gains were signifi- question of whether subantimicrobial dose doxycy- cantly greater (range 20–32% greater) in nonsmokers cline confers any clinical benefit in smokers. The who received subantimicrobial dose doxycycline than meta-analysis by Preshaw and co-workers (81) in- in either of the placebo groups (smokers or non- volved a large number of subjects (n = 392) enrolled smokers) (P < 0.01) (Table 2). into two separately conducted multicentre clinical This meta-analysis also considered the number of trials, and involved 9 months of drug therapy. How- periodontal sites that achieved clinically significant ever, the recruitment of subjects was not undertaken thresholds of attachment gain and probing depth specifically to involve smokers; the creation of the reduction (‡2or‡3 mm) (81). As seen in Table 2, a smoking and nonsmoking subgroups was performed hierarchical treatment response was again observed, retrospectively. The study by Needleman and col- such that nonsmokers who received subantimicrobial leagues (67) was a small, single-centre study, with dose doxycycline demonstrated the greatest number just 30 patients completing the study. Subjects of sites achieving these thresholds of change. The received study medication for only 3 months and smallest number of sites achieving these thresholds were followed-up for 6 months. The authors recog- was observed in the smokers who received placebo. nized that this study was under-powered as a result Smokers who received adjunctive subantimicrobial of the treatment effects being smaller than originally dose doxycycline, and nonsmokers who received anticipated when powering the project. However, the placebo, demonstrated a similar, and intermediate, advantage of this study was that smokers were number of sites achieving these thresholds of change. specifically recruited, thereby avoiding all bias, but When considering sites that at baseline had probing whether this advantage in a small number of patients depths of ‡6 mm, subantimicrobial dose doxycycline (n = 34) truly outweighs the disadvantages of possi- in smokers resulted in significantly more sites that ble bias in the retrospective analysis of a much larger demonstrated probing depth reductions of ‡2mm subject population (n = 392), in the study of Preshaw (45% of sites) compared with smokers who received et al. (81), seems doubtful. Clearly, further research is placebo (31% of sites) (P < 0.01). required, but additional support for the benefits of A criticism that could be levelled at this meta- subantimicrobial dose doxycycline in smokers can analysis (81) is that the comparisons between be derived from, for example, the clinical study by smokers and nonsmokers were not direct compari- Novak and co-workers (70), in which almost 50% of sons based on the original randomization scheme subjects were current smokers and 70% had a history and therefore could be subject to bias, as was sug- of tobacco use, and yet hugely improved clinical gested by the authors of a later study that prospec- outcomes were reported in the subantimicrobial dose tively recruited smokers only to a randomized clinical doxycycline group compared with the placebo group. trial of subantimicrobial dose doxycycline (67). In The mechanism by which subantimicrobial dose this investigation of 34 patients, 16 patients in the test doxycycline may be of clinical benefit in smokers group received nonsurgical therapy plus 3 months of with periodontal disease has not been clearly iden- subantimicrobial dose doxycycline, and 18 patients tified, but presumably relates to the ability of doxy- in the control group received nonsurgical therapy cycline to downregulate MMPs, together with other plus 3 months of placebo. The absolute changes in anti-inflammatory properties (as summarized in mean probing depth reductions and attachment Table 1). Smoking has been associated with increased gains at 6 months did not differ significantly between cytokine production, with peripheral blood mono- the two groups (but were greater in the subantimi- nuclear cells from smokers secreting significantly crobial dose doxycycline group) (Table 2). However, greater levels of interleukin-1b than cells from non- the velocity of change was significantly greater for the smokers upon exposure to cigarette smoke (89). subantimicrobial dose doxycycline group compared Smokers have also been reported to have significantly with the placebo group for clinical attachment higher crevicular fluid levels of tumor necrosis factor- gain (0.19 mm ⁄ 6 month greater, P < 0.05) and a than nonsmokers (8) and show suppressed levels of probing depth reduction (0.30 mm ⁄ 6 month greater, the crevicular fluid protease inhibitors a1-antitrypsin P < 0.001). These authors concluded that there was and a2-macroglobulin (77), suggesting that smoking

102 Host response modulation in periodontics has a direct effect on the inflammatory response. seen in both the subantimicrobial dose doxycycline Nicotine has also been shown to alter gingival and placebo groups, indicating a return to a flora fibroblast function, resulting in decreased collagen more associated with periodontal health. No differ- formation and increased collagenase activity (100), ences in other microbiological parameters were de- and smokers have increased crevicular fluid neutro- tected between groups, with the exception that the phil elastase activity compared with nonsmokers (95). spirochetal proportions present in the subantimi- The exposure of coronary endothelial cells to ciga- crobial dose doxycycline group were significantly rette smoke condensate results in the up-regulation lower at certain time-points than in the placebo of genes involved in matrix degradation (MMP-1, group, and these time-points were preceded by a MMP-8 and MMP-9) and the increased production of significant decrease in the number of sites sampled cytokines, suggesting a complex pro-inflammatory that bled on probing. It was considered that the de- response to cigarette smoke that probably involves crease in spirochetal groups in the subantimicrobial the recruitment of leukocytes, cytokine signalling dose doxycycline group resulted from increasingly and MMP upregulation (68). Thus, based on what is aerobic conditions in the pockets in this group as a known of the effect of smoking on inflammatory result of the clinical improvements derived from the responses, subantimicrobial dose doxycycline would doxycycline therapy, as opposed to any effect of appear to offer the potential for improving clinical doxycycline on the spirochetes themselves. Because outcomes even in smokers, as confirmed in one of spirochetes are more sensitive to local oxygen tension the two studies to date that have specifically inves- than other pathogens and have relatively low redox tigated this issue (81), but not in another (67). requirements for growth (65), a reduction in the proportion of spirochetes is not unexpected in Microbiological and safety concerns pockets that exhibit reduced probing depths. The authors of this paper concluded that the microbial A concern expressed by many colleagues initially was differences observed were attributed to the anti- that the prolonged use of doxycycline, even at a dose collagenase and anti-inflammatory properties of of 20 mg twice daily, may lead to the development of subantimicrobial dose doxycycline and not to an antibiotic resistance. However, no evidence for anti- antimicrobial effect (105), and a consistent finding in biotic resistance has been identified in any of the these various studies has been that long-term studies that have investigated this issue to date. This subantimicrobial dose doxycycline does not alter or has been confirmed in several randomized studies in contribute to changes in antibiotic susceptibility of which subgingival plaque samples were collected the subgingival microflora compared with placebo from patients prior to and after 9–27 months of su- (98, 99, 105). bantimicrobial dose doxycycline therapy or placebo Similar findings were reported by Lee and co- (98, 99, 105). In these studies, susceptibility testing on workers (57) in a 9-month placebo-controlled study the most prevalent organisms was performed using of the use of subantimicrobial dose doxycycline in tetracycline, amoxicillin, doxycycline, minocycline, which subgingival plaque samples were collected at erythromycin and clindamycin, and 50% minimum baseline and at months 1, 3, 6 and 9. These authors inhibitory concentrations (MIC50) and 90% mini- identified a general tendency for the number of cocci, mum inhibitory concentrations (MIC90) values were nonmotile rods and aerobes to increase over time calculated for each antibiotic ⁄ organism combina- and for the number of spirochetes, motile rods and tion. A consistent finding in these studies was that anaerobes to decrease over time (both indicating a subantimicrobial dose doxycycline did not result in return to a flora more associated with health), but any overgrowth or replacement by opportunistic oral with no significant differences between the suban-

flora, there were no significant shifts in the MIC50 or timicrobial dose doxycycline and placebo groups.

MIC90 data, and there was no development of mul- In a later study, carried out to investigate whether tidrug resistance to the antibiotics. subantimicrobial dose doxycycline exerts any anti- In the study by Walker and colleagues (105), plaque microbial effects in other body compartments samples were collected at baseline and then every (specifically the intestinal or vaginal microflora), 3 months during 9 months of medication, and again 70 patients were randomized to receive either at 3 months after cessation of drug therapy. Relative subantimicrobial dose doxycycline or placebo for to baseline, statistically significant reductions in the 9 months, and fecal and vaginal samples were proportions of spirochetes and motile rods, and an collected at baseline and at months 3 and 9 (104). increase in the proportion of coccoid forms, were Samples were examined for total anaerobic counts,

103 Preshaw opportunistic pathogens and doxycycline resistance were randomly allocated to receive subantimicrobial (‡4 lg ⁄ ml). All isolates that survived subculture were dose doxycycline (20 mg twice daily), flurbiprofen identified and their susceptibilities were determined (50 mg four times per day), or a combination of the to tetracycline, amoxicillin, doxycycline, minocycline, two drugs, for 3 weeks (56). Gingival biopsies were erythromycin and clindamycin. No differences obtained from the planned surgery sites before between the treatment groups were detected at 3 or and after drug therapy and were analyzed for 9 months, either in the predominant bacterial taxa a1-proteinase inhibitor, its breakdown product, var- present or in their antibiotic susceptibilities, and ious MMPs and neutrophil elastase. Three weeks of these authors concluded that there was no evidence subantimicrobial dose doxycycline alone produced a that subantimicrobial doxycycline exerted an effect significant reduction in host-derived neutral pro- on the composition or doxycycline resistance level of teinases, whereas flurbiprofen alone produced no either the fecal or the vaginal microflora. reduction. However, the combination therapy pro- Regarding safety data, the plasma concentration duced a statistically significant synergistic reduction of doxycycline following a subantimicrobial dose of collagenase, gelatinase and serpinolytic (a1-pro- (20 mg) is low, with pharmacokinetic studies reveal- teinase inhibitor-degrading) activities (of 69, 69 and ing peak concentrations of 0.7–0.8 lg ⁄ ml and stea- 75%, respectively) and a lesser reduction of elastase dy-state concentrations of approximately 0.4 lg ⁄ ml activity (46%). These authors concluded that, con- (11), which are well below the concentrations of 3– sistent with other studies of chronic inflammatory 4 lg ⁄ ml that can be expected following antibiotic diseases (e.g. rheumatoid arthritis), the combination doses of 100–200 mg of doxycycline (105). Thus, un- of subantimicrobial dose doxycycline and the non- wanted effects are less likely to occur as a result of steroidal anti-inflammatory drug synergistically sup- treatment with subantimicrobial doses, compared pressed MMPs and other neutral proteinases in the with antibiotic doses, of doxycycline. Indeed, many gingiva of patients with chronic periodontitis. dermatologists now prescribe subantimicrobial doses A similar effect has also been described when of doxycycline for prolonged periods for the treat- chemically modified tetracyclines are administered ment of acne in preference to the antibiotic doses together with flurbiprofen in arthritic rats: the tetra- they were previously using, as the same clinical cycline levels were increased in the joints when improvements are achieved (presumably as a result flurbiprofen was administered even though serum of the anti-inflammatory properties of doxycycline as levels of the tetracycline were the same as when the opposed to any antibacterial effect) but without the tetracycline was administered alone (58). The mech- same frequency of adverse events, and without the anism by which flurbiprofen promoted the uptake of development of antibiotic resistance or any effect on the tetracycline in the local inflammatory lesion is the microflora of the skin (93). In the periodontal not known, but could relate to an improved local literature, randomized clinical trials (Table 2) have blood flow in the lesion (thus improving the local regularly reported that subantimicrobial dose delivery of the chemically modified tetracycline) as a doxycycline is well tolerated with no significant result of the flurbiprofen therapy. Similarly, CMT-8 (a differences in adverse event profiles compared with nonantimicrobial chemically modified doxycycline), placebo, and no evidence at all of any unwanted has been combined with a bisphosphonate (clodro- effects that could be attributed to any antimicrobial nate) in rats with experimental periodontitis (60). activity (12, 83). Overall adverse event rates of After 1 week of treatment with either CMT-8 alone or approximately 0.15% have been reported (82), and, the bisphosphonate alone, there were slight reduc- taken collectively, these studies support the safety of tions in the levels of MMP-8 and MMP-9 in the gin- using subantimicrobial dose doxycycline. gival tissues. However, combination of these agents ÔnormalizedÕ the pathologically elevated levels of these MMPs, indicating a synergistic inhibition in Combinations of host response this animal model (60). modulators Subantimicrobial dose doxycycline has also been combined with the locally delivered doxycycline gel Host modulating drugs that target different aspects of (10%; Atridox, CollaGenex Pharmaceuticals Inc., the pathogenic processes in periodontitis have been Newtown, PA), placed into pockets of ‡5 mm, in combined to maximize therapeutic outcomes. Thus, combination with root surface instrumentation in a in 19 patients with chronic periodontitis who were placebo-controlled study of 171 subjects (69). This scheduled for periodontal flap surgery, the patients combination therapy resulted in significantly greater

104 Host response modulation in periodontics clinical improvements than instrumentation alone Given the huge and ever-expanding range of over 6 months, with mean probing depth reductions pathogenic pathways that play a role in periodontal (in pockets that were ‡7 mm at baseline) of 2.4 mm tissue destruction [for example, the interleukin-1 in the combination group compared with 1.7 mm in cytokine family is now far more complex than pre- the control group (P < 0.01). viously realized (4)], it is inevitable that the future will see a range of different host response modulators developed. Furthermore, most biological responses The future of host response involve a range of different mechanisms, and there- modulation fore blocking one single inflammatory pathway may not achieve the desired outcome because receptor- Host response modulation has emerged as a valid mediated responses could be activated by alternate treatment concept for the management of peri- pathways. Thus, polypharmaceutical approaches odontal disease and represents a significant step may be developed that modify a number of different forward for clinicians and patients. To date, only pathways associated with inflammation and tissue subantimicrobial dose doxycycline has been ap- destruction. Alternatively, targeting of mediators that proved specifically as a host response modulator for play a particularly important role in periodontal the treatment of periodontitis and the majority of pathogenesis, such as interleukin-1b or tumor clinical trials of this drug have clearly demonstrated a necrosis factor-a, may constitute a rational thera- benefit. Most of these studies have been undertaken peutic strategy. Thus, cytokine antagonists, such as in secondary care settings with well-managed popu- interleukin-1 receptor antagonist or soluble tumor lations of patients and therefore probably represent necrosis factor-a receptors, which competitively in- the best that is achievable in terms of periodontal hibit receptor-mediated signal transduction, may care and treatment outcomes. Further research is offer potential in the treatment of periodontal disease necessary to evaluate the efficacy of subantimicrobial (20, 59). The effects of soluble receptors and receptor dose doxycycline in primary care, and also to focus antagonists of interleukin-1 and tumor necrosis fac- on very long-term outcomes, such as prevention of tor-a have been studied in experimental periodontitis tooth loss. The health economics of therapy should models in nonhuman primates (90), and collectively also be investigated: thus, if subantimicrobial dose demonstrate a reduction in the progression of the doxycycline confers significant clinical improve- inflammatory cell infiltrate towards the alveolar bone ments, the cost of medication may be offset by the crest, reduced recruitment of osteoclasts, and de- reduced need for additional treatment such as peri- creased attachment loss and alveolar bone loss (2, 18, odontal surgery. 19, 39, 64, 72). However, a degree of caution is re- Future developments in relation to subantimicro- quired, as, for example, the monoclonal antibody to bial dose doxycycline will include modified-release tumor necrosis factor (infliximab), which has been formulations that achieve sustained plasma concen- successfully used over recent years in the treatment trations of doxycycline over 24 h, but only require of rheumatoid arthritis, has also been associated with once per day dosing, thereby improving patient com- the re-emergence of latent tuberculosis infection in a pliance. Furthermore, the development of chemically small percentage of patients (71). Thus, we must modified tetracyclines is welcomed, as this will com- carefully evaluate new agents that, while modifying pletely eliminate all concerns about any possible inflammatory responses, may also have unexpected antimicrobial effects of these agents. Given the safety effects on host defences. of subantimicrobial dose doxycycline, it is likely that Interleukin-11 has anti-inflammatory effects host response modulators with similar safety profiles including inhibition of tumor necrosis factor-a (101) will be welcomed by practising clinicians if proven to and recombinant human interleukin-11 has been have a clinical benefit and minimal unwanted effects. shown to result in significant reductions in the rate of Such drugs that target relatively specific aspects of attachment and bone loss over an 8-week period in pathogenic processes (and therefore tend to have experimental periodontitis in dogs (64). Blockade of more predictable outcomes) might be preferred in cytokine receptors, soluble cytokine blockers and comparison to agents that have profound, and possi- anti-inflammatory cytokines therefore hold promise bly unpredictable, effects on human inflammatory for the future. Other agents that block transcriptional responses, such as evidenced in the recent, disastrous pathways (e.g. the nuclear factor-jB and mitogen- clinical trial of TGN1412 (the activating ÔsuperagonistÕ activated protein kinase pathways), such as the pro- anti-CD28 monoclonal antibody) (96). tein kinase inhibitors, may be useful because genes

105 Preshaw that are regulated by nuclear factor-jB (for example) topical application of 15-epi-lipoxin A4, enhanced include many cytokines, chemokines, cell-adhesion expression of 15-lipoxygenase as well as topical 15- molecules, acute-phase proteins and anti-apoptotic epi-lipoxin A4 significantly reduced bone loss and proteins (71). gingival inflammation (91). These results suggest that Vasoactive intestinal peptide has a role in immu- lipoxins can be targets for novel approaches in dis- noregulation and has been identified as a molecule eases such as periodontitis in which inflammation with therapeutically beneficial immunosuppressive and bone destruction occur. effects in inflammatory and autoimmune conditions To summarize, host response modulators must be (17). We have recently demonstrated that this viewed as comprising part of the overall manage- immunologically active peptide significantly reduces ment strategy for patients with periodontitis. Thus, tumor necrosis factor-a production in human they should form part of an integrated treatment monocytes stimulated with Porphyromonas gingiva- approach, together with hygiene therapy, plaque lis lipopolysaccharide and also inhibits nuclear control, root surface instrumentation, maintenance translocation of nuclear factor-jB and c-Jun (25). We care and risk factor modification. Periodontal disease have also shown that vasoactive intestinal peptide is an unfortunate and distressing condition, and inhibits lipopolysaccharide-induced differentiation many patients are relieved to realize that multifac- of monocytes with a concomitant reduction in the eted treatment strategies are possible, including, for expression of Toll-like receptor-2 and -4. It also example, root surface instrumentation, enzyme blocks lipopolysaccharide-induced differentiation of suppression and modification of local and systemic monocytes to macrophages, possibly via inhibition of risk factors. Thus, periodontal therapy in the 21st the transcription factor PU.1 (26). century should not only involve a high standard of However, no agents that block intracellular sig- clinical treatment and monitoring, but should also nalling pathways have been (as yet) the subject of focus on patient involvement and improving the clinical trials for the treatment of periodontal disease, patient experience. The future will see a range of but as the pilot data reviewed above indicate, they host response modulators developed as adjunctive may ultimately prove to be of benefit. Improved treatments for periodontitis. At present, subantimi- knowledge of pro-inflammatory signal transduction crobial dose doxycycline provides improvements in pathways has suggested new therapeutic targets, and probing depth reductions and attachment gains as these pathways are common to various cytokines, compared with root surface instrumentation alone their blockade may be more efficacious than target- (49), and is the only licensed and approved host re- ing specific cytokines (51). However, it should be sponse modulator available to dentists to date. Al- remembered that these pathways are important in though the use of nonsteroidal anti-inflammatory physiological processes and therefore their inhibition drugs has been associated with reduced alveolar could also result in adverse effects, such as increased bone loss, the unwanted effects of these drugs pre- susceptibility to infection, and the development and cludes their use. Similarly, although data supporting investigation of such agents require careful moni- the use of bisphosphonates to improve clinical toring. periodontal status have been published, given the Lipoxins are another group of compounds that may association with osteonecrosis, further studies are ultimately be of benefit in modifying inflammatory warranted to determine the risks and benefits of responses in periodontal tissues. These lipid-derived these drugs. Lipoxins and compounds that block mediators are released during inflammatory re- cytokine receptors have been shown to reduce gin- sponses and have the effect of damping inflamma- gival inflammation and bone loss in animal models tion and modulating resolution of inflammation and may represent the future of host response (102). Resolution of inflammation is now considered modulation for treating periodontal disease, al- an active process, and failure of resolution of peri- though this remains to be demonstrated in clinical odontal inflammation contributes to ongoing tissue trials in humans. breakdown. Lipoxins block interleukin-1b secretion from human neutrophils stimulated with tumor necrosis factor-a (79) and block neutrophil migration References following exposure to P. gingivalis in a murine air pouch model (78). In an experimental periodontitis 1. Al-Shammari KF, Giannobile WV, Aldredge WA, Iacono VJ, study of transgenic rabbits overexpressing 15-lipox- Eber RM, Wang HL, Oringer RJ. Effect of non-surgical periodontal therapy on C-telopeptide pyridinoline cross- ygenase and in nontransgenic animals receiving

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