Chemically modified tetracyclines: The novel host modulating agents.

Periodontal pathogens and destructive host responses are involved in the initiation and progression of periodontitis. The emergence of host response modulation as a treatment concept has resulted from our improved understanding of the pathogenesis of periodontal disease. A variety of drugs have been evaluated as host modulation agents (HMA), including Non Steroidal Anti Inflammatory Drugs (NSAIDS), bisphosphonates, tetracyclines, enamel matrix proteins and bone morphogenetic proteins. Chemically modified tetracyclines (CMTs) are one such group of drugs which have been viewed as potential host modulating agents by their anticollagenolytic property. The CMTs are designed to be more potent inhibitors of pro inflammatory mediators and can increase the levels of anti inflammatory mediators.

INTRODUCTION

Chronic periodontitis is a complex immune inflammatory disease instigated by anaerobic gram negative bacteria.[1] Theories about the pathogenesis of periodontitis have evolved from a purely plaque-associated disease to a more recent hypothesis which has considerable emphasis on host response. The disease process is the result of a complex interplay between bacterial challenges and the response of the host.[2] Periodontal pathogens produce harmful by-products and enzymes (e.g. hyaluronidases, collagenases, proteases) that break down extracellular matrices, such as collagen, as well as host cell membranes in order to produce nutrients for their growth and subsequent tissue invasion. Many of the microbial surface protein and lipopolysaccharide (LPS) molecules are responsible for eliciting a host immune response, resulting in local tissue inflammation. Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans and other periodontal pathogens possess multiple virulence factors such as cytoplasmic membranes, peptidoglycans, outer membrane proteins, lipopolysaccharide, capsules and cell-surface fimbriae.

Once immune and inflammatory processes are initiated, various inflammatory mediators such as matrix metalloproteinases (MMPs), cytokines and prostaglandins are released from leukocytes, fibroblasts or other tissue-derived cells. Proteases can degrade the collagen structure of periodontal tissues and thus create inroads for further leukocyte infiltration. Although the production of collagenase from infiltrating neutrophils and resident periodontal tissue cells is part of the natural host response to infection, in periodontal disease and other chronic inflammatory diseases, there is an imbalance between the level of activated tissue-destroying MMPs and their endogenous inhibitors.[3]

Treatment of periodontal disease has, traditionally, been focused on the reduction of bacterial load in the periodontal pocket by mechanical debridement and also by the use of topical and systemic antibiotics as an adjunct. Periodontal therapy is currently aimed at reducing the bacterial overload and modulating the host response to these microbial factors.[4]

Tetracyclines appear to fit this profile by having both antibacterial and non-antibacterial properties. The perceived advantages of this group of antibiotics were their effectiveness against anaerobic gram-negative periodontopathogens in the subgingival plaque, increased concentration in gingival crevicular fluid at levels 2-10 times greater than that of serum after a single 250 mg dose, the substantivity property which enabled them to bind to the biological tissues and get released over a period of time, resulting in prolonged efficacy and anti-collagenase property. Peak concentrations of 5-12 μg/ml were reached in the gingival crevicular fluid (GCF) at 3.5-7 hours.[4]

The tetracyclines have been used locally and systemically as antimicrobial agents and more recently systemically as host-modulating agents (HMAs). Sub-antimicrobial dose doxycycline (SDD) remains, at present, the only systemic host response modulator specifically indicated as an adjunctive treatment for periodontitis. SDD is currently the only FDA-approved, systemically administered HMT indicated specifically in the treatment of periodontitis. SDD is a 20 mg dose of doxycycline (Periostat), taken twice daily for 3 months up to a maximum of 9 months. SDD as well as the other members of tetracycline family has the ability to down regulate MMPs by a variety of synergistic mechanisms, including reductions in cytokine levels and stimulates osteoblastic activity. But, tetracyclines have major disadvantages like gastrointestinal disturbances and development of antibiotic-resistant microorganisms which led to development of CMTs.[5] Currently, three groups of tetracyclines are available. Tetracycline natural products, tetracycline semisynthetic compounds and chemically modified tetracyclines (CMTs).

A novel approach for the treatment of periodontal disease is the use of host-modulating therapy (HMTs) along with conventional mechanical therapy. One of the most promising groups of potential HMTs is the CMTs. These nonantibiotic tetracyclines analogs are nothing but the tetracycline molecules which have been modified to eliminate the antimicrobial property, but retain the host modulatory, anticollagenolytic property.[6] CMTs are one such group of drugs, which has been viewed as potential HMAs. Golub et al.[7] in 1987 recognized that the antimicrobial and anti-collagenase properties of tetracyclines resided in different parts of four ringed structures. They altered the structure of tetracyclines which had led to the development of CMTs. Since that time several CMTs have been developed. Among them CMT-1, CMT-3 and CMT-8 have been tested for periodontal applications.

Structure of CMT

Golub et al. discovered that the carbon 4 position side chain was responsible for the antimicrobial activity of tetracyclines [Figure 1]. CMTs were produced by removing the dimethylamino group from the carbon-4 position of the A ring of the four ringed (A, B, C, D) structure. The resulting compound, 4-dedimethylamino tetracycline (CMT-1) did not have antimicrobial property but the anti-collagenase activity was retained both in vitro and in vivo. Further modifications in the central structure of tetracyclines by addition or deletion of functional groups resulted in the formation of other CMTs. Currently, about 10 CMTs have been developed.

Figure 1

Structure of tetracyclines

They are [Figures 2–5]:

Figure 2

Structure of tetracycline, doxycycline and CMT -1

Figure 5

Structure of CMT-6, CMT-7 and CMT-8

Structure of CMT-2 and CMT-3

CMT-1 (4-dedimethylaminotetracycline)

CMT-2 (tetracyclinonitrite)

CMT-3 (6-deoxy-6-demethyl- 4-dedimethylamino tetracycline)

CMT-4 (7-chloro-4-de-dimethylamino tetracycline)

CMT-5 (tetracycline pyrrazole)

CMT-6 (4-dedimethylamino. 4-hydroxytetracycline)

CMT-7 (12-deoxy-4-de-dimethyamino tetracycline) and

CMT-8 (4-dedimethylaminodoxycycline) have been developed.

The Ca+2 and Zn+2-binding sites at the carbonyl oxygen and hydroxyl group of c-11 and c-12 positions are responsible for anti-collagenase action of CMTs. CMT-5 is a pyrazole analog of tetracycline, formed by replacement of carbonyl oxygen at c-11 and hydroxyl group at C-12 by nitrogen atoms [Figure 4]. It does not have metal-binding site and therefore it is inactive against MMPs.[8] So, the only CMT found to have lost its anti-collagenase property was CMT-5.[9] Currently, CMT-3 is the only CMT being tested in human clinical trials of cancer patients.[10]

Figure 4

Structure of CMT-4 and CMT-5

The advantages of CMTs over conventional tetracyclines are their rapid absorption, a longer serum half life than tetracycline, long-term systemic administration does not result in gastrointestinal toxicity, no development of antibiotic-resistant microflora and can be used for prolonged periods.[11]

Mechanism of action

CMTs are used as HMT agents in the management of periodontitis by inhibition of MMPs, inhibition of proinflammatory cytokines, inducible nitric oxide synthase (iNOS) and inhibition of bone resorption, enhancement of the attachment of fibroblasts and connective tissues to the tooth surface [Figure 6].[12]

Figure 6

Schematic illustration of the pathogenesis of periodontitis, including targets for host modulation, depicting the action of CMTs on MMPs

Inhibition of MMPs

Among the above actions, the anti-MMP effect of CMTs has been widely discussed in the management of periodontitis.[11] The anti-MMP actions of CMTs include direct inhibition of the active MMPs by the virtue of Ca2+ and Zn2+-binding sites, inhibition of reactive oxygen species-mediated activation of pro-MMPs, proteolysis of pro-MMPs into enzymatically inactive fragments, protection of α-1 proteinase inhibitor from MMPs, reduction in the activity of serine proteinases. Polymorphonuclear leucocytes (PMNs) provide the major source of collagenases that mediate the connective tissue breakdown during inflammatory periodontal disease, while the fibroblasts contribute the collagenase required for connective tissue remodeling in normal gingiva. The anti-collagenase activity of CMTs is specific against the collagenase produced from neutrophils but not the fibroblasts. This non-antimicrobial action of CMTs is important as it would help in the reduction of pathologic concentrations of collagenases without affecting the normal collagen turnover required to maintain the tissue integrity. The CMT-3 is specifically active against MMP-2, MMP-9 and MMP-14 isozymes due to its pleiotropic action toward MMPs. It exerts an inhibitory effect on MMPs in micromolar concentrations by decreasing trypsinogen-2 and inducible nitric oxide (iNOS) production.[13] A comparative evaluation of six different CMTs in inhibition of MMPs showed that the CMT-8 was most effective inhibitor of periodontal breakdown. CMT-8,-1, -3, -4, -7 and doxycycline inhibited tumor necrosis factor-α (TNF-α), interleukin-1 (IL-1), interleukin- 6 (IL-6) and MMPs in descending order.[12]

Anti-inflammatory actions

Inhibition of inducible nitric oxide synthase

CMT-3 inhibited the inducible nitricoxide synthase activity, thereby reducing nitric oxide which is one of the activators of MMPs. The peroxynitrite radical formed by the reaction of NO is highly cytotoxic, inhibits collagen and proteoglycan synthesis and upregulates the MMP expression. Inhibition of iNOS production causes reduction in the peroxynitrite levels, thus preventing denatuartion of proteins. CMT-3 and CMT-8 have shown maximum inhibitory effect on the iNOS and CMT-1 and-2 had an intermediary effect while CMT-5 was ineffective.[14]

Biologic role of CMT in inflammation and wound healing

CMTs inhibit release of IL-1β, IL-6, IL-8, TNF-α and prostaglandin-E2 (PGE2) from LPS-stimulated host immune cells by suppressing phosphorylation of the nuclear factor κ-B cell signaling pathway [Figure 7]. The CMT-3 inhibits cyclooxygenase-2 (COX-2)-mediated PGE-2 production. CMTs are designed to be more potent inhibitors of proinflammatory mediators and can increase levels of anti-inflammatory mediators such as IL-10. CMTs increase integrin expression on endothelial cells in inflammation, counteract the effects of transforming growth factor-β (TGF-β)-induced expression of MMPs, enhance phagocytosis by increased expression of FcγRIII, and stimulate fibroblasts to produce protease inhibitors like tissue inhibitors of matrix metalloproteases (TIMPs).[15]

Figure 7

Biologic role of CMTs in wound healing

Inhibition of bone resorption

CMTs such as CMT-3 and CMT-8 have been shown to inhibit osteoclastic bone resorption and promote bone formation, enhance wound healing and inhibit proteinases produced by periodontal pathogens. CMT-1, CMT-3, CMT-6, -7 and -8 were effective inhibitors of osteoblastic collagenase in culture. CMT-8 was the most potent among these. CMTs inhibit bone resorption by various mechanisms which include reduction in number of osteoclasts by inhibiting their development and inducing apoptosis, by altering the ruffled border and increasing the size of clear zone, by decreasing the production of osteoclastic enzymes like TRAP and Cathepsin-L which degrade organic components of bone, inhibits osteoclastogenesis, elevates intracellular calcium levels which makes the osteoclasts to detach from bone resorbing site, inhibits osteoclasts collagenase production and also decreases acid production, thereby inhibiting bone resorption, thus preventing the progression of periodontal disease.[16]

CMTs promote matrix and collagen deposition and inhibit bone resorption through anti-MMP and pro-TIMP actions and reduced activity of inflammatory cytokines (e.g. IL-1, IL-6, TNF-α) and PGE2. These pleiotropic mechanisms of CMT provide significant therapeutic potential for treating periodontitis and various other chronic inflammatory conditions.

Other medical uses:[17]

As antifungal agents

Inhibition of intimal thickening after arterial injury

Inhibition of orthodontic tooth displacement

Against advanced cancers

In diabetes mellitus

For rheumatoid arthritis

For acne and acute respiratory distress syndrome

Tumor metastasis.

CONCLUSION

CMTs are still in their infancy with regard to use in humans as they have not been approved due to concerns like excessive suppression of MMPs which may hinder the normal physiologic turnover of collagen. The CMT-3 has been shown to be the most promising agent among all CMTs. CMTs will likely emerge as drugs that have beneficial effects in a variety of disease status because of their host-modulating capabilities.