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    Mean serum doxycycline concentrations in healthy Beagles (n = 5/group) after oral administration of a 1, 2, 3, or 5 mg/kg dose of doxycycline hyclate. The horizontal dashed line represents 1 μg/mL.

  • View in gallery

    Photographs of electrophoretic gels showing gelatinolytic intensities of periodontal tissue samples obtained from the same site before (week 0) and 4 weeks after (week 4) daily oral administration of a placebo (A) or 1 (B) or 2 (C) mg of doxycycline/kg in Beagles with periodontitis.

  • 1.

    Kinane DF. Regulators of tissue destruction and homeostasis as diagnostic aids in periodontology. Periodontology 2000; 24: 215225.

  • 2.

    Golub LMRamamurthy NSMcNamara TF, et al. Tetracyclines inhibit connective tissue breakdown: new therapeutic implications for an old family of drugs. Crit Rev Oral Biol Med 1991; 2: 297321.

    • Search Google Scholar
    • Export Citation
  • 3.

    Choi DHMoon ISChoi BK, et al. Effects of sub-antimicrobial dose doxycycline therapy on crevicular fluid MMP-8, and gingival tissue MMP-9, TIMP-1 and IL-6 levels in chronic periodontitis. J Periodontal Res 2004; 39: 2026.

    • Search Google Scholar
    • Export Citation
  • 4.

    Emingil GAtilla GSorsa T, et al. The effect of adjunctive low-dose doxycycline therapy on clinical parameters and gingival crevicular fluid matrix metalloproteinase-8 levels in chronic periodontitis. J Periodontol 2004; 75: 106115.

    • Search Google Scholar
    • Export Citation
  • 5.

    Emingil GAtilla GSorsa T, et al. Effectiveness of adjunctive low-dose doxycycline therapy on clinical parameters and gingival crevicular fluid laminin-5 gamma2 chain levels in chronic periodontitis. J Periodontol 2004; 75: 13871396.

    • Search Google Scholar
    • Export Citation
  • 6.

    Golub LMSorsa TLee HM, et al. Doxycycline inhibits neutrophil (PMN)-type matrix metalloproteinases in human adult periodontitis gingiva. J Clin Periodontol 1995; 22: 100109.

    • Search Google Scholar
    • Export Citation
  • 7.

    Caton JG. Evaluation of Periostat for patient management. Compend Contin Educ Dent 1999; 20: 451463.

  • 8.

    Polson AMSouthard GLDunn RL, et al. Periodontal pocket treatment in Beagle dogs using subgingival doxycycline from a biodegradable system. I. Initial clinical responses. J Periodontol 1996; 67: 11761184.

    • Search Google Scholar
    • Export Citation
  • 9.

    Zetner KRothmueller G. Treatment of periodontal pockets with doxycycline in Beagles. Vet Ther 2002; 3: 441452.

  • 10.

    Bennett JVBrodie JLBenner EJ, et al. Simplified, accurate method for antibiotic assay of clinical specimens. Appl Microbiol 1966; 14: 170177.

    • Search Google Scholar
    • Export Citation
  • 11.

    Kleibeuker WZhou XCentlivre M, et al. A sensitive cell-based assay to measure the doxycycline concentration in biological samples. Hum Gene Ther 2009; 20: 524530.

    • Search Google Scholar
    • Export Citation
  • 12.

    Pijpers ASchoevers EJHaagsma N, et al. Plasma levels of oxytetracycline, doxycycline, and minocycline in pigs after oral administration in feed. J Anim Sci 1991; 69: 45124522.

    • Search Google Scholar
    • Export Citation
  • 13.

    Ruz NZabala MKramer MG, et al. Rapid and simple determination of doxycycline in serum by high-performance liquid chromatography. Application to particulate drug delivery systems. J Chromatogr A 2004; 1031: 295301.

    • Search Google Scholar
    • Export Citation
  • 14.

    Wilson RCKemp DTKitzman JV, et al. Pharmacokinetics of doxycycline in dogs. Can J Vet Res 1988; 52: 1214.

  • 15.

    Yu ZLeung MKRamamurthy NS, et al. HPLC determination of a chemically modified nonantimicrobial tetracycline: biological implications. Biochem Med Metab Biol 1992; 47: 1020.

    • Search Google Scholar
    • Export Citation
  • 16.

    Wennström JLNewman HNMacNeill SR, et al. Utilisation of locally delivered doxycycline in non-surgical treatment of chronic periodontitis. A comparative multi-centre trial of 2 treatment approaches. J Clin Periodontol 2001; 28: 753761.

    • Search Google Scholar
    • Export Citation
  • 17.

    Lovrien RMatulis D. Assays for total protein. Curr Protoc Microbiol 2005; Appendix 3: Appendix 3A.

  • 18.

    Chandler HLGemensky-Metzler AJBras ID, et al. In vivo effects of adjunctive tetracycline treatment on refractory corneal ulcers in dogs. J Am Vet Med Assoc 2010; 237: 378386.

    • Search Google Scholar
    • Export Citation
  • 19.

    TeKoppele JMBeekman BVerzijl N, et al. Doxycycline inhibits collagen synthesis by differentiated articular chondrocytes. Adv Dent Res 1998; 12: 6367.

    • Search Google Scholar
    • Export Citation
  • 20.

    Yu LP JrBurr DBBrandt KD, et al. Effects of oral doxycycline administration on histomorphometry and dynamics of subchondral bone in a canine model of osteoarthritis. J Rheumatol 1996; 23: 137142.

    • Search Google Scholar
    • Export Citation
  • 21.

    Caton JRyan ME. Clinical studies on the management of periodontal diseases utilizing subantimicrobial dose doxycycline (SDD). Pharmacol Res 2011; 63: 114120.

    • Search Google Scholar
    • Export Citation
  • 22.

    Thomas JWalker CBradshaw M. Long-term use of subantimicrobial dose doxycycline does not lead to changes in antimicrobial susceptibility. J Periodontol 2000; 71: 14721483.

    • Search Google Scholar
    • Export Citation
  • 23.

    Walker CThomas JNango S, et al. Long-term treatment with subantimicrobial dose doxycycline exerts no antibacterial effect on the subgingival microflora associated with adult periodontitis. J Periodontol 2000; 71: 14651471.

    • Search Google Scholar
    • Export Citation
  • 24.

    Dahlén G. Role of suspected periodontopathogens in microbiological monitoring of periodontitis. Adv Dent Res 1993; 7: 163174.

  • 25.

    Radice MMartino PAReiter AM. Evaluation of subgingival bacteria in the dog and susceptibility to commonly used antibiotics. J Vet Dent 2006; 23: 219224.

    • Search Google Scholar
    • Export Citation
  • 26.

    Hardham JDreier JWong J, et al. Pigmented-anaerobic bacteria associated with canine periodontitis. Vet Microbiol 2005; 106: 119128.

    • Search Google Scholar
    • Export Citation
  • 27.

    Michel GMosser JFauran F. Serum kinetics of doxycycline polyphosphate in dogs. Eur J Drug Metab Pharmacokinet 1979; 4: 4348.

  • 28.

    Preshaw PMNovak MJMellonig J, et al. Modified-release subantimicrobial dose doxycycline enhances scaling and root planing in subjects with periodontal disease. J Periodontol 2008; 79: 440452.

    • Search Google Scholar
    • Export Citation
  • 29.

    Lee HMCiancio SGTuter G, et al. Subantimicrobial dose doxycycline efficacy as a matrix metalloproteinase inhibitor in chronic periodontitis patients is enhanced when combined with a non-steroidal anti-inflammatory drug. J Periodontol 2004; 75: 453463.

    • Search Google Scholar
    • Export Citation
  • 30.

    Dong WXiang JLi C, et al. Increased expression of extracellular matrix metalloproteinase inducer is associated with matrix metalloproteinase-1 and −2 in gingival tissues from patients with periodontitis. J Periodontal Res 2009; 44: 125132.

    • Search Google Scholar
    • Export Citation
  • 31.

    Snoek-van Beurden PAVon den Hoff JW. Zymographic techniques for the analysis of matrix metalloproteinases and their inhibitors. Biotechniques 2005; 38: 7383.

    • Search Google Scholar
    • Export Citation
  • 32.

    Golub LMCiancio SRamamurthy NS, et al. Low-dose doxycycline therapy: effect on gingival and crevicular fluid collagenase activity in humans. J Periodontal Res 1990; 25: 321330.

    • Search Google Scholar
    • Export Citation

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Experimental determination of a subantimicrobial dosage of doxycycline hyclate for treatment of periodontitis in Beagles

Se Eun Kim DVM1, Soohee Kim DVM2, Manbok Jeong DVM, PhD3, Yesran Lee DVM4, Jeong Taek Ahn DVM5, Young Woo Park DVM6, Jae Sang Ahn DVM7, Euiri Lee DVM8, Doug-Young Ryu DVM, PhD9, and Kangmoon Seo DVM, PhD10
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  • 1 Department of Veterinary Surgery and Ophthalmology, College of Veterinary Medicine and BK21 Program for Veterinary Science, Seoul National University, 599 Gwanak-ro, Gwanak-gu, Seoul 151-742, Korea.
  • | 2 Laboratory of Environmental Health, College of Veterinary Medicine and BK21 Program for Veterinary Science, Seoul National University, 599 Gwanak-ro, Gwanak-gu, Seoul 151-742, Korea.
  • | 3 Department of Veterinary Surgery and Ophthalmology, College of Veterinary Medicine and BK21 Program for Veterinary Science, Seoul National University, 599 Gwanak-ro, Gwanak-gu, Seoul 151-742, Korea.
  • | 4 Department of Veterinary Surgery and Ophthalmology, College of Veterinary Medicine and BK21 Program for Veterinary Science, Seoul National University, 599 Gwanak-ro, Gwanak-gu, Seoul 151-742, Korea.
  • | 5 Department of Veterinary Surgery and Ophthalmology, College of Veterinary Medicine and BK21 Program for Veterinary Science, Seoul National University, 599 Gwanak-ro, Gwanak-gu, Seoul 151-742, Korea.
  • | 6 Department of Veterinary Surgery and Ophthalmology, College of Veterinary Medicine and BK21 Program for Veterinary Science, Seoul National University, 599 Gwanak-ro, Gwanak-gu, Seoul 151-742, Korea.
  • | 7 Department of Veterinary Surgery and Ophthalmology, College of Veterinary Medicine and BK21 Program for Veterinary Science, Seoul National University, 599 Gwanak-ro, Gwanak-gu, Seoul 151-742, Korea.
  • | 8 Department of Veterinary Surgery and Ophthalmology, College of Veterinary Medicine and BK21 Program for Veterinary Science, Seoul National University, 599 Gwanak-ro, Gwanak-gu, Seoul 151-742, Korea.
  • | 9 Laboratory of Environmental Health, College of Veterinary Medicine and BK21 Program for Veterinary Science, Seoul National University, 599 Gwanak-ro, Gwanak-gu, Seoul 151-742, Korea.
  • | 10 Department of Veterinary Surgery and Ophthalmology, College of Veterinary Medicine and BK21 Program for Veterinary Science, Seoul National University, 599 Gwanak-ro, Gwanak-gu, Seoul 151-742, Korea.

Abstract

Objective—To identify a subantimicrobial dose of doxycycline hyclate (SDD) and for the treatment of periodontitis in dogs.

Animals—20 healthy Beagles for measurement of serum doxycycline concentration and 15 Beagles with periodontitis for evaluation of the efficacy of the SDD.

Procedures—5 dogs each received doxycycline hyclate PO at a dose of 1, 2, 3, or 5 mg/kg. Blood samples were collected before and after administration, and serum concentrations of doxycycline were measured via high-performance liquid chromatography. Mean serum doxycycline concentrations were calculated, and SDDs were identified. In a separate trial, the identified SDDs (1 or 2 mg/kg) were administered PO once a day for 1 month to dogs with periodontitis (n = 5/group) and a control group (5) was fed vehicle only during the same period. Degree of gingival attachment and bleeding on probing (present or absent) were recorded. Gingival samples were collected before and after the 1-month period from the same anatomic sites. Degree of matrix metalloproteinase inhibition in gingival samples was determined via gelatin zymography and compared among treatment groups.

Results—Mean serum doxycycline concentrations in healthy dogs that received 1 or 2 mg of doxycycline/kg were consistently significantly lower than the minimal inhibitory doxycycline concentration for treatment of periodontitis throughout the 24-hour posttreatment period. Zymographic intensities were lower in dogs given 1 and 2 mg/kg than in the control dogs, and the degree of gingival attachment and bleeding significantly improved in dogs given 2 mg/kg, compared with in the control dogs and dogs given 1 mg of doxycycline/kg.

Conclusions and Clinical Relevance—A doxycycline dosage of 2 mg/kg daily appeared to be an appropriate subantimicrobial regimen for dogs with periodontitis. Furthermore, this dosage may be suitable for long-term treatment of gelatinolytic inflammatory diseases such as periodontitis in this species.

Abstract

Objective—To identify a subantimicrobial dose of doxycycline hyclate (SDD) and for the treatment of periodontitis in dogs.

Animals—20 healthy Beagles for measurement of serum doxycycline concentration and 15 Beagles with periodontitis for evaluation of the efficacy of the SDD.

Procedures—5 dogs each received doxycycline hyclate PO at a dose of 1, 2, 3, or 5 mg/kg. Blood samples were collected before and after administration, and serum concentrations of doxycycline were measured via high-performance liquid chromatography. Mean serum doxycycline concentrations were calculated, and SDDs were identified. In a separate trial, the identified SDDs (1 or 2 mg/kg) were administered PO once a day for 1 month to dogs with periodontitis (n = 5/group) and a control group (5) was fed vehicle only during the same period. Degree of gingival attachment and bleeding on probing (present or absent) were recorded. Gingival samples were collected before and after the 1-month period from the same anatomic sites. Degree of matrix metalloproteinase inhibition in gingival samples was determined via gelatin zymography and compared among treatment groups.

Results—Mean serum doxycycline concentrations in healthy dogs that received 1 or 2 mg of doxycycline/kg were consistently significantly lower than the minimal inhibitory doxycycline concentration for treatment of periodontitis throughout the 24-hour posttreatment period. Zymographic intensities were lower in dogs given 1 and 2 mg/kg than in the control dogs, and the degree of gingival attachment and bleeding significantly improved in dogs given 2 mg/kg, compared with in the control dogs and dogs given 1 mg of doxycycline/kg.

Conclusions and Clinical Relevance—A doxycycline dosage of 2 mg/kg daily appeared to be an appropriate subantimicrobial regimen for dogs with periodontitis. Furthermore, this dosage may be suitable for long-term treatment of gelatinolytic inflammatory diseases such as periodontitis in this species.

Periodontal disease involves multiple interactions between bacterial biofilms and inflammatory responses of the periodontal connective tissue. Several MMPs play important roles in periodontal tissue breakdown by initiating extracellular matrix degradation.1 Tetracyclines can have therapeutic value by improving periodontal status through inhibition of MMP activity, independent of any antimicrobial properties.2 For this reason, SDDs have been intensively investigated for the treatment of chronic periodontitis in human medicine.3–6 The targeted plasma concentration for an SDD is recommended to be < 1 μg/mL, which is the MIC in humans.7

In canine dentistry, topical administration of doxycycline can resolve periodontal inflammation and improve periodontal status.8,9 A commercial topical doxycycline preparation in the form of a polymer can bind to the tooth surface. The subsequent slow release of doxycycline from the polymer matrix exerts a therapeutic effect consisting of antibiosis and local inhibition of collagenase activity.2,9 However, to date, no reports exist of an appropriate SDD for oral treatment of periodontitis in dogs. The inhibitory effect of long-term SDD treatment on MMP activity in periodontal tissue has not been determined in dogs to the authors’ knowledge.

Several analytic methods have been developed for the measurement of serum concentrations of antimicrobials in various species of animals.10–15 High-performance liquid chromatography is rapid, simple, and sufficiently sensitive to determine this concentration in microsamples of serum and has been successfully used in experimental studies.13,15 The purpose of the study reported here was to identify the optimal SDD of doxycycline for treatment of periodontitis in dogs. The desired dosing regimen would not result in a serum doxycycline concentration that exceeded the MIC but would result in clinical improvement through a hypothesized inhibitory effect on MMP activity in inflamed periodontal tissues.

Materials and Methods

Animals—Twenty healthy Beagles (approx 1.5 years old) were used to identify an SDD. Fifteen 3- to 5-year-old Beagles with moderate to severe periodontitis were used to assess the efficacy of the identified SDD. Dogs with periodontitis were confirmed to be otherwise healthy through a physical examination. Dogs were excluded from either portion of the study if they had been treated systemically with any medication during the 2 weeks prior to the study. The study protocol was approved by the Institutional Animal Care and Use Committee of Seoul National University.

SDD identification—The 20 healthy Beagles were randomly allocated to receive doxycycline hyclatea at a dose of 1, 2, 3, or 5 mg/kg (n = 5 dogs/group). Doxycycline was contained in a gelatin capsule and was administrated orally after 6 hours of food withholding. Blood samples (1.7 mL) were collected via a 23-gauge needle from a jugular vein before doxycycline administration and 0.5, 1, 1.5, 2, 3, 4, 5, 6, 8, 12, and 24 hours after drug administration. The blood was allowed to clot in a serum-separating tube,b and serum was harvested by centrifugation at 1,300 × g for 10 minutes.

A stock solution of doxycycline for standards preparation was made by dissolving 10 mg of doxycycline in 10 mL of untreated canine serum. Five standard solutions were prepared by further dilution of the stock solution with untreated serum to produce solutions containing 0.49, 0.98, 1.95, 3.91, and 7.81 μg of doxycycline/mL The serum and standard solutions were stored at −20°C until analyzed.

Frozen samples and standard solutions were thawed for 60 minutes at 4°C, and 200 μL of each sample was transferred to a 1.5-mL microcentrifuge tubec and mixed with the same volume of acetonitrile. Tube contents were mixed with a vortex device for 1 minute and centrifuged at 13,440 × g and 4°C for 10 minutes. After centrifugation, the supernatants were diluted with the same volume of methanold–acetic acidd mixture (1:1 [vol/vol]) and transferred to autosampler vials.e

The HPLC systemf was coupled with a UV detector set at 347 nm that was run by means of computer software.g The technique used for HPLC analysis was a modification of a described method.13 Serum samples were analyzed with a silica reversed-phase columnh (150 × 4.6 mm, C18, 5 μm). The mobile phase was made by mixing 5% acetic acid, acetonitrile, and methanol (55:25:20 [vol/vol/vol]). Isocreatic solvent elution was performed at a flow rate of 1.0 mL/min.

Clinical and biochemical effects of SDD on periodontitis—In each of the 15 healthy dogs, 1 to 3 premolars and molars were selected for examination on the basis of existence of CAL (≥ 1 mm/tooth) or BoP (≥ 0.33/tooth). Degree of CAL was evaluated by measurement of the distance between the cementoenamel junction and the bottom of probeable pocket.16 Bleeding on probing was scored as absent or present within 10 seconds after probing.16

The dogs were randomly allocated to 3 groups of 5 dogs (10 teeth) each: administration of doxycycline at a dosage of 1 or 2 mg/kg/d or administration of vehicle only (control group). All treatments were administrated PO once a day, 30 minutes after every morning meal. The clinical condition of each dog was ascertained daily. Periodontal status including CAL and BoP was evaluated 4 weeks after doxycycline administration began at the mesial-buccal, buccal, and distal-buccal gingival margins of each tooth. All measurements were made by an experienced clinician (SEK), who used a Williams periodontal probe.i

For evaluation of the regional MMP inhibitory effect by the systemic administration of doxycycline, gelatin zymography was performed with full-thickness gingival tissue. Tissue sample collection was performed at 1 to 3 oral sites in each dog. Before and 4 weeks after daily doxycycline administration began, tissue samples (approx 1 × 2 mm/sample) were obtained from the buccal gingival margin. Samples were washed immediately with cold distilled water (4°C) to remove blood and debris and stored at −80°C until analyzed. Thawed gingival tissue samples were weighed, and protein was extracted from the tissues at 4°C with lysis bufferj blended with protein cocktailk (10 mg [wet weight] of gingival tissue/100 μL of buffer). Protein in the gingival extracts was quantified with the Bradford method.17 Twenty-five micrograms of extracted tissue proteins was mixed with the same volume of 2× zymography sample bufferl without heat denaturation.

Electroporesis was performed with a 10% zymogram gel containing 0.1% gelatin at 125 V for 95 minutes. After electrophoresis, the gels were incubated in 1× renaturating bufferm for 30 minutes at room temperature (approx 20°C) with gentle agitation and equilibrated in 1× developing buffern in the same condition. The gels were incubated in fresh developing buffer at 37°C for 8 hours. Following incubation, gels were stained with 0.1% Coomassie brilliant blue R250o and destained with 10% acetic acid in 40% methanol. Prestained protein markersp were run on each gel to identify the molecular size of gelatinase included in the samples. The intensities of the destained bands were determined with a software programq following the gel-scanning process with a luminescent image analyzer.r

Statistical analysis—Serum doxycycline concentrations in each group are reported as mean ± SD. Statistical analyses were performed with a commercial software program.s To evaluate differences in serum doxycycline concentration change within a treatment, data were analyzed via repeated-measures ANOVA. The correlation between the amount of orally administered doxycycline and the maximum doxycycline concentration in serum was assessed by calculation of the Pearson correlation coefficient (r).

To evaluate the clinical effect of SDD, changes in periodontal status from weeks 0 through 4 in the same group were assessed via a paired t test. One-way ANOVA was used for the intergroup comparison of the periodontal values at weeks 0 and 4 and the variance of gelatinolytic activity between weeks 0 and 4. Baseline gelatinolytic values (week 0) were also compared via 1-way ANOVA to evaluate the difference among the 3 treatment groups. The Tukey method was used as a post hoc test, and 95% confidence intervals or values of P < 0.05 were used to indicate significant differences.

Results

SDD identification—Changes in the mean serum doxycycline concentration in healthy dogs before and after doxycycline hyclate administration were summarized (Table 1). The mean ± SD HPLC retention time for doxycycline was 2.94 ± 0.19 minutes. Serum doxycycline concentrations reached a maximum 3 to 4 hours after administration in all groups and remained lower than the doxycycline MIC for treatment of periodontitis for 24 hours in dogs that received the 1 or 2 mg/kg doses (Figure 1). Repeated-measures ANOVA revealed significant differences in the mean serum doxycycline concentration within each group according to the time flow. The correlation between dose administered and detectable serum concentration was significant (r = 0.72).

Figure 1—
Figure 1—

Mean serum doxycycline concentrations in healthy Beagles (n = 5/group) after oral administration of a 1, 2, 3, or 5 mg/kg dose of doxycycline hyclate. The horizontal dashed line represents 1 μg/mL.

Citation: American Journal of Veterinary Research 74, 1; 10.2460/ajvr.74.1.130

Table 1—

Mean ± SD serum doxycycline concentrations (μg/mL) in healthy Beagles (n = 5/group) before and at various points after administration of 1 dose of doxycycline hyclate at 1, 2, 3, or 5 mg/kg, PO.

Measurement point (h)1 mg/kg2 mg/kg3 mg/kg5 mg/kg
00.00 ± 0.000.00 ± 0.000.00 ± 0.000.00 ± 0.00
0.50.26 ± 0.440.35 ± 0.640.30 ± 0.681.65 ± 1.77
10.31 ± 0.560.48 ± 0.721.46 ± 0.666.59 ± 5.71
1.50.33 ± 0.490.53 ± 0.792.08 ± 0.469.88 ± 8.60
20.30 ± 0.470.64 ± 0.732.71 ± 0.9610.95 ± 9.30
30.32 ± 0.530.70 ± 0.643.13 ± 1.3512.14 ± 10.11
40.32 ± 0.500.79 ± 0.672.95 ± 1.5511.24 ± 8.24
50.28 ± 0.430.76 ± 0.672.44 ± 1.3210.77 ± 6.97
60.27 ± 0.420.66 ± 0.572.33 ± 1.2010.93 ± 7.45
80.24 ± 0.370.59 ± 0.611.99 ± 0.998.41 ± 5.47
120.27 ± 0.370.47 ± 0.481.94 ± 0.896.04 ± 4.98
240.08 ± 0.190.37 ± 0.560.85 ± 1.002.41 ± 1.39

Clinical and biochemical effects of SDD on periodontitis—As a result of findings in the preliminary portion of the study, the SDD chosen for further evaluation was 1 and 2 mg/kg, once a day. Values of periodontal variables, including CAL and BoP, were not significantly different among the groups at week 0 (before treatment began). After the 4-week medication period, dogs that received doxycycline at a dosage of 2 mg/kg/d had a significantly lower degree of CAL, compared with the value at week 0 and that for dogs that received a placebo or a 1 mg/kg/d dosage (Table 2). On week 4, the BoP score of dogs that received 2 mg/kg/d was significantly lower than at week 0, whereas those for other groups were higher but not significantly so.

Table 2—

Mean ± SD values of clinical variables in Beagles with periodontitis (n = 5/group) that received a placebo, 1 mg of doxycycline/kg, or 2 mg of doxycycline/kg once a day for 30 days.

VariablePlacebo1 mg/kg/d2 mg/kg/d
Week 0
 CAL2.67 ± 1.452.67 ± 1.352.23 ± 1.33
 BoP0.80 ± 0.410.80 ± 0.410.70 ± 0.47
Week 4
 CAL2.53 ± 1.11a2.53 ± 1.25a1.83 ± 0.83*b
 BoP0.93 ± 0.25a0.86 ± 0.34a0.56 ± 0.50b

Value is significantly (P < 0.05) lower than that at week 0 in same group.

Within a row, values with different superscript letters are significantly (P < 0.05) different.

In the zymographic evaluation, gelatinolytic bands were evident in extracts from all gingival samples at the 92- and < 72-kDa areas (Figure 2). Gelatinolytic intensities were not significantly different among the groups at week 0. However, the intensities at both molecular weights increased in the control group after 4 weeks of treatment, whereas in the other groups, a decrease in intensity was evident after treatment concluded. The variation was significantly different in the control group, compared with in the other groups (Table 3).

Figure 2—
Figure 2—

Photographs of electrophoretic gels showing gelatinolytic intensities of periodontal tissue samples obtained from the same site before (week 0) and 4 weeks after (week 4) daily oral administration of a placebo (A) or 1 (B) or 2 (C) mg of doxycycline/kg in Beagles with periodontitis.

Citation: American Journal of Veterinary Research 74, 1; 10.2460/ajvr.74.1.130

Table 3—

Mean ± SD gelatinolytic intensities and relative variations over 4 weeks in the dogs represented in Table 2.

VariablePlacebo1 mg/kg/d2 mg/kg/d
MMP-9 (92 kDa)
 Week 0 (AU)162,908.02 ± 96,988.48105,017.01 ± 89,518.88150,371.73 ± 60,799.43
 Week 4 (AU)198,439.90 ± 119,743.49a88,782.09 ± 86,515.17b132,924.08 ± 61,878.73a,b
 Variation in zymographic intensity (fold change)1.29 ± 0.35a0.82 ± 0.27b0.85 ± 0.16b
MMP-2 (< 72 kDa)
 Week 0 (AU)96,676.43 ± 35,103.6259,774.34 ± 34,235.3467,280.69 ± 35,243.54
 Week 4 (AU)124,537.10 ± 66,853.04a41,648.80 ± 32,858.28b42,457.11 ± 56,361.93b
 Variation in zymographic intensity (fold change)1.31 ± 0.55a0.65 ± 0.34b0.66 ± 0.58b

Across each row, values with different superscript letters are significantly (P < 0.05) different.

AU = Arbitrary units in the densitometer.q

Discussion

Tetracyclines are generally used for their bacteriostatic properties. However, tetracycline analogs additionally possess nonantimicrobial properties, namely the ability to inhibit MMP activity. Because of these host-modulatory effects, doxycycline has been used for the treatment of inflammatory diseases such as osteoarthritis and refractory corneal ulcers in dogs.18–20 The effects of topical doxycycline application for the treatment of periodontitis have been investigated, and a commercial preparation is available for use in dogs.8,9 However, the method might be a 1-time remedy administered with dogs anesthetized, with the effects lasting only several days or weeks.2,8,9 Furthermore, susceptibility of various microbes to antimicrobials could change with a long-term systemic administration of antimicrobial doses of doxycycline.

Studies21 have demonstrated the benefit of SDDs as a systemic host-modulating treatment of human patients with periodontal disease. The SDD for humans is 20 mg/person every 12 hours; this dosage does not exert a subgingival antimicrobial effect and does not lead to changes in antimicrobial susceptibility among microflora, even during long-term use.22,23 The SDD in humans is approximately a fifth of the general antimicrobial dose and was experimentally confirmed to yield serum doxycycline concentrations of 0.6 to 0.8 μg/mL, which are considerably lower than the MIC of subgingival microflora as determined in vitro.7,23 Additionally, the genera of canine subgingival pathogenic anaerobes are similar to those of humans (eg, Porphyromonas spp, Bacteroides spp, and Prevotella spp), although the specific bacterial species differ.24–26 Findings of the present study showed the mean serum doxycycline concentration of dogs that received 1 or 2 mg of doxycycline/d for 30 days was maintained at considerably < 1 μg/mL, which is the MIC for subgingival microflora.

Research concerning the pharmacokinetics of doxycycline polyphosphate in dogs shows that the maximum concentration time and the half-life of orally administrated doxycycline is 2.8 and 11.8 hours, respectively.27 The maximum concentration time was similar to that of the present study; however, the half-life tended to be extended to > 12 hours in all groups. Therefore, serum doxycycline concentration could exceed the MIC through periodic twice-daily administration even in dogs treated with 2 mg/kg/d. In general, gelatinolytic activity could not be maintained consistently in the once-daily dosing accompanied with the diurnal variation of serum doxycycline concentration. However, the serum concentration should remain below the MIC for the clinical use of SDDs in dogs to treat periodontitis, even though long-term medication might be continued.

Long-term oral administration of 40 mg of doxycycline once daily results in no antimicrobial resistance,28 and this dosage also improves periodontitis in humans. The doxycycline used for that long-term administration is in the form of a novel, sustained-release formulation designed to yield subantimicrobial blood concentrations of doxycycline for patient compliance because once-daily dosing is preferred to more frequent dosing.28 In the present study, twice-daily dosing at 1 mg/kg or once-daily dosing at 2 mg/kg could be suggested to avoid development of antimicrobial resistance in canine microflora; however, daily administration of doxycycline at 2 mg/kg would facilitate pet owner compliance.

Some limitations in the present study involved the poor detection of doxycycline in some blood samples obtained from dogs that received 1 mg of doxycycline/kg. Also, the number of dogs in each group was small, hindering generalizable conclusions. The confirmed concentration range for the HPLC method is reportedly 0.4 to 80 μg/mL for doxycycline quantification in serum.13 The mean maximum serum doxycycline concentration in dogs that received 1 mg/kg in our study did not reach 0.4 μg/mL Thus, the serum doxycycline concentration in that group might have been underestimated slightly. However, the amount of administered doxycycline was significantly correlated with the serum doxycycline concentration as shown by the Pearson correlation coefficient. Serum doxycycline concentrations differed significantly by dose and time when the repeated-measures ANOVA was performed. One can consequently conclude that the doxycycline concentrations of all treatment groups were reliably measured.

The 92-kDa form of gelatinase is the most prominent form in the gingival tissues of humans with chronic periodontitis.6,29 The degree of expression of MMP-2 in gingival tissue is correlated with pathological severity of periodontal disease.30 The 72- and 92-kDa species represent MMP-2 and MMP-9, respectively, and could be detected from diseased periodontium via gelatin zymography.6,29,31 Molecular weight standards of this molecular weight were used to determine the type of the gelatinase. In the zymographic results, a sole gelatinolytic band was identified at the < 72-kDa region, which could have been regarded as the active form of MMP-2. In contrast, the bands located around the 92-kDa region comprised double layers, which were considered a mix of pro–MMP-9 and activated MMP-9 on week 0.

Although mean values before treatment in the groups were not exactly the same, gelatinolytic activity was not significantly different in the present study. Therefore, we used a method to detect differences in gelatinolytic activity at the same sample collection site before and after medication. Zymographic results showed a significant decrease in MMP-2 and -9 activity in dogs that received doxycycline at 1 and 2 mg/kg/d, but the control group that received no doxycycline had a greater degree of MMP expression after the 1-month administration of SDD. Studies3,32 have revealed that the activities of polymorphonuclear leukocyte–derived MMPs can be directly inhibited by SDD in humans. Furthermore, the activity of active-form MMPs can be directly inhibited in part by doxycycline.6 In the present study, the expression of both pro-MMP-9 and active MMP-9 was greater in the control group, whereas dogs treated with doxycycline at 1 and 2 mg/kg/d had decreases in the activated form of MMP-9 in addition to reduction in the pro form of MMP-9. These results suggest that the SDD we identified reduces the expression of pro–MMP-9 but also might inhibit the conversion of the pro form into the activated form in dogs. Although zymography revealed no significant difference, the values of clinical variables of dogs treated with doxycycline at 2 mg/kg/d were significantly different from those of the other groups after the medication period.

Considering these results, a regimen of 2 mg of doxycycline/kg once daily could be used for the clinical improvement of periodontal disease in dogs, given that it yielded MMP-2 and -9 inhibition without an antimicrobial effect. The SDD could be recommended for long-term treatment of gelatinolytic inflammatory diseases such as periodontitis and arthritis. Because removal of injurious bacteria should be performed prior to the treatment for periodontitis, the medication protocol of SDD treatment should include subgingival scaling before SDD administration. Before clinical application of SDDs in dogs, additional in vivo studies, including evaluation of other biomarkers such as other MMPs, tissue inhibitor of metalloproteinase-1, and interleukin-6 that might support clinical improvement, but also including researches of antimicrobial resistance, would be needed for long-term use.

a.

DongKoo Pharm Co Ltd, Seoul, Republic of Korea.

b.

SST Tube, BD, Franklin Lakes, NJ.

c.

Mallinckrodt Baker Inc, Phillipsburg, NJ.

d.

Yakuri Pure Chemicals Co Ltd, Osaka, Japan.

e.

Waters Co, Milford, Mass.

f.

Series 200 LC, PerkinEhlmer Inc, Shelton, Conn.

g.

TotalChrom Workstation, version 6.3.1, PerkinElmer Inc, Shelton, Conn.

h.

Alltima C18 5 μm, Grace Davison Discovery Science, Deerfield, Ill.

i.

XP23-W Williams Explorer-Probe, Osung, Gimpo, Korea.

j.

Tissue Extraction Reagent I, Invitrogen, Camarillo, Calif.

k.

Protease Inhibitor Cocktail III, GenDEPOT, Barke, Tex.

l.

Novex Tris-Glycine SDS Sample Buffer, Invitrogen, Carlsbad, Calif.

m.

Novex Zymogram Renaturating Buffer, Invitrogen, Carlsbad, Calif.

n.

Novex Zymogram Developing Buffer, Invitrogen, Carlsbad, Calif.

o.

Amresco, Solon, Ohio.

p.

Xpert Prestained Protein Marker, GenDEPOT, Barke, Tex.

q.

Multi-gauge, version.3.0, Fujifilm, Tokyo, Japan.

r.

LAS-3000, Fujifilm, Tokyo, Japan.

s.

PASW Statistics, version 18, SPSS Inc, Chicago, Ill.

References

  • 1.

    Kinane DF. Regulators of tissue destruction and homeostasis as diagnostic aids in periodontology. Periodontology 2000; 24: 215225.

  • 2.

    Golub LMRamamurthy NSMcNamara TF, et al. Tetracyclines inhibit connective tissue breakdown: new therapeutic implications for an old family of drugs. Crit Rev Oral Biol Med 1991; 2: 297321.

    • Search Google Scholar
    • Export Citation
  • 3.

    Choi DHMoon ISChoi BK, et al. Effects of sub-antimicrobial dose doxycycline therapy on crevicular fluid MMP-8, and gingival tissue MMP-9, TIMP-1 and IL-6 levels in chronic periodontitis. J Periodontal Res 2004; 39: 2026.

    • Search Google Scholar
    • Export Citation
  • 4.

    Emingil GAtilla GSorsa T, et al. The effect of adjunctive low-dose doxycycline therapy on clinical parameters and gingival crevicular fluid matrix metalloproteinase-8 levels in chronic periodontitis. J Periodontol 2004; 75: 106115.

    • Search Google Scholar
    • Export Citation
  • 5.

    Emingil GAtilla GSorsa T, et al. Effectiveness of adjunctive low-dose doxycycline therapy on clinical parameters and gingival crevicular fluid laminin-5 gamma2 chain levels in chronic periodontitis. J Periodontol 2004; 75: 13871396.

    • Search Google Scholar
    • Export Citation
  • 6.

    Golub LMSorsa TLee HM, et al. Doxycycline inhibits neutrophil (PMN)-type matrix metalloproteinases in human adult periodontitis gingiva. J Clin Periodontol 1995; 22: 100109.

    • Search Google Scholar
    • Export Citation
  • 7.

    Caton JG. Evaluation of Periostat for patient management. Compend Contin Educ Dent 1999; 20: 451463.

  • 8.

    Polson AMSouthard GLDunn RL, et al. Periodontal pocket treatment in Beagle dogs using subgingival doxycycline from a biodegradable system. I. Initial clinical responses. J Periodontol 1996; 67: 11761184.

    • Search Google Scholar
    • Export Citation
  • 9.

    Zetner KRothmueller G. Treatment of periodontal pockets with doxycycline in Beagles. Vet Ther 2002; 3: 441452.

  • 10.

    Bennett JVBrodie JLBenner EJ, et al. Simplified, accurate method for antibiotic assay of clinical specimens. Appl Microbiol 1966; 14: 170177.

    • Search Google Scholar
    • Export Citation
  • 11.

    Kleibeuker WZhou XCentlivre M, et al. A sensitive cell-based assay to measure the doxycycline concentration in biological samples. Hum Gene Ther 2009; 20: 524530.

    • Search Google Scholar
    • Export Citation
  • 12.

    Pijpers ASchoevers EJHaagsma N, et al. Plasma levels of oxytetracycline, doxycycline, and minocycline in pigs after oral administration in feed. J Anim Sci 1991; 69: 45124522.

    • Search Google Scholar
    • Export Citation
  • 13.

    Ruz NZabala MKramer MG, et al. Rapid and simple determination of doxycycline in serum by high-performance liquid chromatography. Application to particulate drug delivery systems. J Chromatogr A 2004; 1031: 295301.

    • Search Google Scholar
    • Export Citation
  • 14.

    Wilson RCKemp DTKitzman JV, et al. Pharmacokinetics of doxycycline in dogs. Can J Vet Res 1988; 52: 1214.

  • 15.

    Yu ZLeung MKRamamurthy NS, et al. HPLC determination of a chemically modified nonantimicrobial tetracycline: biological implications. Biochem Med Metab Biol 1992; 47: 1020.

    • Search Google Scholar
    • Export Citation
  • 16.

    Wennström JLNewman HNMacNeill SR, et al. Utilisation of locally delivered doxycycline in non-surgical treatment of chronic periodontitis. A comparative multi-centre trial of 2 treatment approaches. J Clin Periodontol 2001; 28: 753761.

    • Search Google Scholar
    • Export Citation
  • 17.

    Lovrien RMatulis D. Assays for total protein. Curr Protoc Microbiol 2005; Appendix 3: Appendix 3A.

  • 18.

    Chandler HLGemensky-Metzler AJBras ID, et al. In vivo effects of adjunctive tetracycline treatment on refractory corneal ulcers in dogs. J Am Vet Med Assoc 2010; 237: 378386.

    • Search Google Scholar
    • Export Citation
  • 19.

    TeKoppele JMBeekman BVerzijl N, et al. Doxycycline inhibits collagen synthesis by differentiated articular chondrocytes. Adv Dent Res 1998; 12: 6367.

    • Search Google Scholar
    • Export Citation
  • 20.

    Yu LP JrBurr DBBrandt KD, et al. Effects of oral doxycycline administration on histomorphometry and dynamics of subchondral bone in a canine model of osteoarthritis. J Rheumatol 1996; 23: 137142.

    • Search Google Scholar
    • Export Citation
  • 21.

    Caton JRyan ME. Clinical studies on the management of periodontal diseases utilizing subantimicrobial dose doxycycline (SDD). Pharmacol Res 2011; 63: 114120.

    • Search Google Scholar
    • Export Citation
  • 22.

    Thomas JWalker CBradshaw M. Long-term use of subantimicrobial dose doxycycline does not lead to changes in antimicrobial susceptibility. J Periodontol 2000; 71: 14721483.

    • Search Google Scholar
    • Export Citation
  • 23.

    Walker CThomas JNango S, et al. Long-term treatment with subantimicrobial dose doxycycline exerts no antibacterial effect on the subgingival microflora associated with adult periodontitis. J Periodontol 2000; 71: 14651471.

    • Search Google Scholar
    • Export Citation
  • 24.

    Dahlén G. Role of suspected periodontopathogens in microbiological monitoring of periodontitis. Adv Dent Res 1993; 7: 163174.

  • 25.

    Radice MMartino PAReiter AM. Evaluation of subgingival bacteria in the dog and susceptibility to commonly used antibiotics. J Vet Dent 2006; 23: 219224.

    • Search Google Scholar
    • Export Citation
  • 26.

    Hardham JDreier JWong J, et al. Pigmented-anaerobic bacteria associated with canine periodontitis. Vet Microbiol 2005; 106: 119128.

    • Search Google Scholar
    • Export Citation
  • 27.

    Michel GMosser JFauran F. Serum kinetics of doxycycline polyphosphate in dogs. Eur J Drug Metab Pharmacokinet 1979; 4: 4348.

  • 28.

    Preshaw PMNovak MJMellonig J, et al. Modified-release subantimicrobial dose doxycycline enhances scaling and root planing in subjects with periodontal disease. J Periodontol 2008; 79: 440452.

    • Search Google Scholar
    • Export Citation
  • 29.

    Lee HMCiancio SGTuter G, et al. Subantimicrobial dose doxycycline efficacy as a matrix metalloproteinase inhibitor in chronic periodontitis patients is enhanced when combined with a non-steroidal anti-inflammatory drug. J Periodontol 2004; 75: 453463.

    • Search Google Scholar
    • Export Citation
  • 30.

    Dong WXiang JLi C, et al. Increased expression of extracellular matrix metalloproteinase inducer is associated with matrix metalloproteinase-1 and −2 in gingival tissues from patients with periodontitis. J Periodontal Res 2009; 44: 125132.

    • Search Google Scholar
    • Export Citation
  • 31.

    Snoek-van Beurden PAVon den Hoff JW. Zymographic techniques for the analysis of matrix metalloproteinases and their inhibitors. Biotechniques 2005; 38: 7383.

    • Search Google Scholar
    • Export Citation
  • 32.

    Golub LMCiancio SRamamurthy NS, et al. Low-dose doxycycline therapy: effect on gingival and crevicular fluid collagenase activity in humans. J Periodontal Res 1990; 25: 321330.

    • Search Google Scholar
    • Export Citation

Contributor Notes

Supported by the BK21 Program for Veterinary Science and Research Institute for Veterinary Science, College of Veterinary Medicine, Seoul National University.

Address correspondence to Dr. Seo (kmseo@snu.ac.kr).