Effect of azithromycin on gingival overgrowth induced by cyclosporine A + nifedipine combination therapy: A morphometric analysis in rats.

BACKGROUND: Drug-induced gingival overgrowth (DIGO) is a well-known adverse effect of cyclosporine A (CsA) and nifedipine (Nf) therapy. The aim of the present morphometric study was to evaluate the effect of azithromycin (Azi) on the combined GO in rats induced by CsA + Nf combination.
MATERIALS AND METHODS: Thirty Sprague-Dawley male rats were randomly divided equally into three groups. Group 1 (control) received olive oil only; Group 2 received a combination of CsA and Nf in olive oil throughout the study period; Group 3 received CsA + Nf combination therapy, and Azi was added for 1 week in the 5th week. All the drugs were delivered by oral route. Impressions of the mandibular central incisal regions were taken, and study models were prepared at baseline and biweekly up to the 8 weeks. Statistical analysis was done by one-way analysis of variance and intergroup comparisons were made using Tukey's post hoc analysis.
RESULTS: Significant GO was evident in Group 2 and Group 3 rats when compared to Group 1. However, in Group 3 (Azi), GO was observed up to the 4th week, but a significant decrease in GO was noticed during 6-8th week after the administration of Azi in 5th week.
CONCLUSION: Azi is an effective drug in the remission of DIGO induced by combined therapy of CsA + Nf and thereby can be considered as a useful therapeutic regimen in minimizing the DIGO in transplant patients.

INTRODUCTION

Cyclosporine A (CsA) is the most frequently used powerful immunosuppressant in organ transplantation cases to prevent graft rejection since its miracle discovery by Borel[1] CsA has also been effective in the treatment of a number of autoimmune diseases. However, the widespread clinical use of CsA has been questioned for its several inherent side effects such as nephrotoxicity, hepatotoxicity, hypertension, and gingival overgrowth (GO). CsA-induced GO was first observed by Calne et al.[2] and documented by Rateitschak-Plüss et al.[3] in renal transplant patients.

Nifedipine (Nf) is frequently administered in many organ transplant patients receiving cyclosporine to control CsA-induced hypertension and nephrotoxicity. Nf is a calcium channel blocker and drug of choice for cardiac insufficiencies. However, this is also known to induce the GO as its adverse effect.[4] GO is potentiated when CsA and Nf are combined.[567] The prevalence and severity of GO were found much higher when the patients were taking both the drugs, CsA and Nf concurrently than CsA or Nf as monotherapy.[7]

Since GO interferes with esthetics, function, phonetics, and oral hygiene maintenance, various nonsurgical and surgical methods are being carried out to treat the drug-induced GO (DIGO). However, the prevention and management of DIGO in organ transplant patients are more challenging.

Azithromycin (Azi), a macrolide antibiotic of the azalide subclass, has been reported as bearing an inhibitory effect on CsA-induced GO in human[89] and animal studies.[10] Gómez et al.[11] have suggested favorable effect of Azi on GO in the patients receiving CsA + Nf combination therapy. Although the adjuvant beneficial effect of Azi (both as local drug delivery[12] and systemic administration[13]) has been documented in the management of periodontal infections, its effect on GO-induced by the CsA + Nf combination drug therapy has not been reported in dental literature. Therefore, the present study was undertaken to evaluate the role of Azi in CsA + Nf combination induced GO in experimental animal model, i.e., in Sprague-Dawley (SD) rats.

MATERIALS AND METHODS

Experimental animals

Thirty male SD rats (6-week old and weighing approximately 150–220 g) were procured from Venkateshwara Enterprises, Bengaluru and were shifted to the animal house of Bapuji Pharmacy College, Davangere. All the animals were housed in plastic cages using husk as their bedding. Normal atmospheric temperature (21°C) and 12/12 h light-dark illumination cycle were maintained. These animals were allowed free access to water ad libitum and standard laboratory pellets.

Study design and drug administration

All the 30 rats were randomly distributed into three equal groups of ten animals each. Group 1 (control) received olive oil (Sasso, Nestle, Milan, Italy) for the 8 weeks. Group 2 and Group 3 received a combination of CsA (PanimunBioral™ Panacea, Biotec, India) (30 mg/kg body weight) and Nf (Nifedipine SR 20, Nicholas Piramal, India) (50 mg/kg body weight) in olive oil for 8 weeks. In Group 3 rats, Azi (Azithral® Kid tab, Alembic, India) (10 mg/kg body weight) was added to this regimen, in the 5th week. The total study period was 8 weeks. Ethical clearance was obtained from the Institutional Animal Care Committee, J.J.M. Medical College, Davangere, Karnataka state.

Impression making and stone models

Impressions of mandibular anterior region were made to record the sequence of gingival changes at baseline, using customized acrylic trays and vinyl polysiloxane (3M ESPE, Express STD, Putty Consistency, 3M Dental Products, St. Paul, MN, USA) (rubber base) impression material. Stone models were poured using dental stone (Labstone, Dentsply, New Delhi, India). The procedure was repeated at 2 weeks interval till the end of 8th week. Gingival dimensions were measured on the cast using Boley's gauge.

Measurement of gingival overgrowth (morphometry)

The dimensions were measured at the interdental and keratinized gingival level in the region of mandibular incisors and recorded as buccolingual (BL) dimension, mesiodistal (MD) dimension, and vertical height (VH). The dimensions were designated as BLi, MDi, and VHi in interdental region and BLk, MDk, and VHk in the region of keratinized gingiva as described in the previous studies [Figure 1].[1415]

Figure 1

(a) Lingual view and (b) lateral view. Schematic image of parameters for gingival macroscopic examination at interdental (i) tissue level representing as buccolingual (BLi), mesiodistal (MDi), and vertical height (VHi) and at keratinized (k) tissue level as buccolingual (BLk), mesiodistal (MDk), and vertical height (VHk). (c) Impression making with acrylic trays and rubber base impression material. (d) Dental stone model of mandibular anterior region of Sprague-Dawley rats

Statistical analysis

Statistical analysis was carried out by one-way analysis of variance. Simultaneous intergroup comparisons were made using Tukey's post hoc test. P < 0.05 was considered for statistical significance.

Clinical image of rat mandibular incisal region. (a) Group 1 (control) at baseline; (b) at 4 weeks; (c) at 8 weeks. (d) Group 2 (cyclosporine A + nifedipine) at baseline; (e) at 4 weeks; (f) at 8 weeks. (g) Group 3 (cyclosporine A + nifedipine + azithromycin) at baseline; (h) at 4 weeks, 3; (i) at 8 weeks

RESULTS

Observations from 0 week (baseline) to 4 weeks [Figure 2a, b, d, e, g, h and Tables 1, 2]

At the baseline (week 0), no significant difference was appreciated in the gingival dimensions of the three groups indicating all the measured gingival dimensions in rats of Group 1, Group 2, and Group 3 are almost equal.

Changes in BLi and BLk

BLi in Group 2 (1.76 ± 0.15) and Group 3 (1.78 ± 0.12) was increased significantly in comparison to the Group 1 (1.04 ± 0.13) at the end of the 4th week. BLk in Group 2 (3.99 ± 0.33) and Group 3 (3.97 ± 0.25) was increased significantly in comparison to the Group 1 (2.11 ± 0.13) at the end of the 4th week [Figure 3a and b].

Figure 3

Graphical representation of macroscopic parameters in all the groups at baseline, 2 weeks, 4 weeks and 8 weeks. (a) Changes in the BLi. (b) Changes in BLk. (c) Changes in the MDi. (d) Changes in the MDk. (e) Changes in the VHi. (f) Changes in the VHk

Changes in MDi and MDk

MDi in Group 2 (1.19 ± 0.27) and Group 3 (1.22 ± 0.14) was increased significantly in comparison to the Group 1 (0.62 ± 0.06) at the end of the 4th week. MDk in Group 2 (4.20 + 0.51) and Group 3 (4.15 ± 0.28) was increased significantly in comparison to the Group 1 (2.28 ± 0.18) at the end of the 4th week [Figure 3c and d].

Changes in VHi and VHk

VHi in Group 2 (1.06 ± 0.13) and Group 3 (1.05 ± 0.18) was increased significantly in comparison to the Group 1 (0.70 ± 0.08) at the end of the 4th week. VHk in Group 2 (4.03 + 0.25) and Group 3 (4.01 ± 0.26) was increased significantly in comparison to the Group 1 (1.88 ± 0.09) at the end of the 4th week [Figure 3e and f].

The BL dimensions (BLi and BLk), MD dimensions (MDk) and vertical dimensions (VHi and VHk) were significantly increased (P < 0.05) at the end of the 4th week in Groups 2 and 3. Intergroup comparison showed no significant difference between Groups 2 and 3, which indicated almost equal GO in both the groups at the end of 4 weeks.

Observations at 6th and 8th week [Figure 2b, c, e, f, h, i and Tables 1, 2]

There was no significant change in gingival dimensions of Group 1 at BLi, MDi, VHi, BLk, MDk, and VHk level.

Group 2 showed increased BL gingival dimensions for both interdental (from 2.1 ± 0.16 mm at 6th week to 2.16 ± 0.12 mm at the end of the 8th week) as well as keratinized tissue (from 4.25 ± 0.34 mm to 4.49 ± 0.37 mm). On the contrary, Group 3 was showed decreased BL gingival dimensions, when compared to the 4th week values, i.e. BLi decreased from 1.5 ± 0.12 mm to 1.12 ± 0.18 mm where as BLk decreased from 3.54 ± 0.21 mm to 3.27 ± 0.39 mm [Figure 3a and b].

The Group 2 showed increased MD gingival dimensions for both interdental (from 1.26 ± 0.22 mm at 6th week to 1.29 ± 0.2 mm at 8th week) as well as keratinized tissue (from 4.66 ± 0.40 mm at 6th week to 5.04 ± 0.44 mm at 8th week). On the contrary, Group 3 exhibited decreased MD gingival dimensions, when compared to the 4th week values, i.e. MDi decreased from 1.09 ± 0.16 mm of 6th week to 0.9 ± 0.13 mm at 8th week whereas MDk decreased from 3.85 ± 0.25 mm of 6th week to 3.44 ± 0.25 mm at 8th week [Figure 3c and d].

The Group 2 showed increased vertical gingival dimensions for both interdental (from 1.3 ± 0.21 mm at 6th week to 1.35 ± 0.25 mm at the end of the 8th week) as well as kertinized tissue (from 4.57 ± 0.29 mm to 4.81 ± 0.32 mm). On the contrary, Group 3 exhibited decreased gingival dimensions, when compared to the 4th week values, i.e., VHi decreased from 0.94 ± 0.23 mm to 0.75 ± 0.17 whereas VHk decreased from 3.60 ± 0.33 mm to 3.04 ± 0.3 mm [Figure 3e and f].

Group 2 showed a continuous and significant increase in gingival dimensions for both interdental as well as keratinized tissue (BLi, BLk, MDi, MDk, VHi, and VHk) whereas Group 3 exhibited decreased gingival dimensions during 6–8th week, in comparison to the 4th week values. The intergroup comparison at the end of the 8th week indicates that the values are significant between Groups 1 and 2 and Groups 2 and 3 [Tables 1 and 2].

Table 1

Gingival macroscopic dimensions of the mandibular incisal region at the interdental papilla level (in mm)

Table 2

Gingival macroscopic dimensions of the mandibular incisal region at the keratinized gingiva level (in mm)

DISCUSSION

Recently, Azi has been extensively evaluated in the management of chronic periodontitis,[13] aggressive periodontitis[16] and in periodontal infections associated with diabetics[17] and smokers.[1819] The results of various studies have shown Azi as an effective drug in controlling periodontitis as an adjuvant to the periodontal therapy.[1316171819] Although Azi has also been observed to be effective in improving DIGO but this conclusion still needs to be explored.[9101120] and therefore, the present study was undertaken to evaluate the efficacy of Azi in inhibiting the GO caused by CsA + Nf combination therapy in SD rat model.

DIGO is multifactorial in etiology and is influenced by many risk factors such as genetic susceptibility, age, gender, dose and the duration of medication.[21] Most of these parameters can be successfully controlled in animal studies. In the present study, the incidence of DIGO was found to be 100% both in Groups 2 and 3 in the first 4 weeks of GO induction, which could be appreciated in both the interdental as well as in keratinized gingiva. This observation is in accordance with the earlier studies.[10141522] In one of the recent experimental studies using the rat model, authors reported DIGO only after 24 weeks of administration of tacrolimus and Nf, alone or in combination.[23] Differences of drug doses were accounted as the possible reason for such variation in findings.

The pathogenesis of DIGO is an issue which could not be completely understood yet. Some researchers have considered complex interference of drugs with the balance between collagen synthesis and degradation as a mechanism for DIGO.[7] According to Kataoka et al.[24] combination of cyclosporine and Nf has a more disruptive effect on collagen degradation because it is accompanied by the augmented inhibitory effects of both the drugs on calcium ion-dependent collagenase. In one of the recent reviews on this topic, authors have extensively enlisted the presence of differential proportions of fibroblast subsets, role of various cytokines, and inflammatory mediators such as interleukin (IL)-6, IL-8, transforming growth factor β1, and prostaglandin E2; role of mast cells and matrix metalloproteinases (MMP) as the possible factors responsible in the etiopathogenesis of DIGO.[25]

In the present study, Group 3 rats were administered Azi (10 mg/kg of body weight) once daily for 7 days in the 5th week, similar to the drug regimen followed in the previous animal study.[26] During the follow-up period, i.e., in 6–8th week, Group 2 rats exhibited a continuous increase in all gingival dimensions. This finding may be attributed to the continuation of CsA + Nf combination therapy till the end of the study without any interference. On the other hand, Group 3 rats exhibited statistically significant (P < 0.05) decreased gingival dimensions for both interdental as well as keratinized tissue despite the continuous administration of CsA + Nf combination therapy during the 6–8th week. Similar observations were made by various researchers in their respective studies on CsA-induced GO in animal models[1027] and in humans.[8911262829]

The mechanism by which Azi may decrease DIGO is unknown. The interaction between Azi and CsA is controversial. Some researchers reported the upregulating effect of Azi on the serum levels of CsA.[26] In one of the earlier studies, Azi alone showed no effect on gingiva or gingival fibroblasts, but the effects were appreciated only when it was used to counter the effects of CsA. In the same study, the authors suggested that Azi appears to restore part of the phagocytosis mechanism of collagen degradation.[10] Some authors have suggested that Azi may improve CsA-induced GO by blocking CsA-induced cell proliferation and collagen synthesis, and by activating MMP-2 in gingival fibroblasts with CsA-induced GO.[30] In a recent review, authors have explained the triple action of Azi, namely, antibiotic, immunomodulating, and anti-inflammatory effect, which makes Azi a useful drug, especially for periodontium.[31]

In the previous studies, administration of Azi resulted in significant regression or inhibition of CsA-induced GO in renal[892628] and cardiac transplant[29] patients, but effect on CsA + Nf induced GO was not evaluated profundity in dental literature. The present study demonstrates that short-term (7 days) course of Azi can effectively inhibit the GO induced by CsA + Nf combination, along with the simultaneous use of the drugs. However, the effect of plaque control and duration of the stability of the results was not evaluated in the present study.

CONCLUSION

Azi proved to be efficacious, safe, cost-effective for managing DIGO in the patients on combined drug therapy of CsA + Nf. In addition, we suggest that if tried in combination with drug withdrawal or drug replacement along with the meticulous oral hygiene and professional dental cleaning, Azi should be considered as a first choice drug in the management of DIGO cases. In addition, the efficacy of Azi in DIGO may be evaluated with the surgical management of severe and resistant cases of DIGO in future.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.