Doxycycline as a Modulator of Inflammation in Chronic Wounds
Doxycycline as a Modulator of Inflammation in Chronic Wounds
Doxycycline is a semisynthetic, chemically modified tetracycline compound that is rapidly absorbed and exerts biological effects independent of its antimicrobial activity. One such effect includes the inhibition of matrix metalloproteinases. Doxycycline has a long history as a collagenase inhibitor. This article will describe its mode of action and review its effectiveness in significantly reducing inflammation and elevated levels of proinflammatory cytokines within chronic wounds.
Doxycycline (DOX) is a semisynthetic, chemically modified tetracycline compound widely used to treat infections caused by both gram-negative and gram-positive microorganisms. This 4-ringed molecule was initially discovered in 1947. Since then, it has been well-established that DOX is rapidly absorbed with a prolonged half-life and exerts biological effects independent of its antimicrobial activity. One such effect includes the inhibition of matrix metalloproteinases (MMPs). Matrix metalloproteinases are a family of zinc-dependent enzymes with the ability to degrade all components of the extracellular matrix (ECM). Matrix metalloproteinases are produced by keratinocytes, endothelial cells, neutrophils, fibroblasts, macrophages, mast cells, and eosinophils. There are 4 distinct subsets of enzymes that exist within the MMP family: collagenases, gelatinases, and stromelysins, plus a few membrane-bound MMPs. Collagenases are the only enzymes in humans with the capacity to cleave the triple helix of type I, II, and III collagen, with gelatinases having the ability to cleave all other types. Gelatinases and collagenases also have the unique ability to activate against other material within the extracellular matrix when a disruption in their balance occurs. Inhibition of MMP activity by DOX, and other chemically modified tetracyclines (CMTs), occurs through the chelation of calcium and zinc ions. The chelation of calcium and zinc ions allows DOX and CMTs to inhibit matrix destruction mediated by MMP activity by 3 different processes: 1) the inhibition of already active MMPs; 2) the inhibition of pro-MMP activation; and 3) the down regulation of MMP expression. In addition, DOX and other CMTs indirectly prevent the degradation of connective tissue by protecting the host defense protein a-1 antitrypsin (α-1 PI), which inhibits leukocyte elastase. Excessive MMPs degrade and inactivate a-1 PI; the inhibition of MMPs thereby leads to higher tissue levels of α-1 PI, which reduce leukocyte elastase activity. Leukocyte elastases are responsible for the degradation and inactivation of tissue inhibitors of metalloproteinase (TIMPs). Tissue inhibitors of metalloproteinase are naturally occurring inhibitors of MMPs. Tissue inhibitors of metalloproteinase keep MMPs in balance including stromelysin (MMP-3), the only MMP that is relatively insensitive to direct inhibition by DOX. It is important to note there is no evidence that DOX or CMTs act as TIMPs inhibitors.
(Enlarge Image)
Free radicals are known to cause cell damage and function as inhibitory factors in the healing process. The production of reactive oxygen species (ROS) associated with chronic wounds can originate from several potential sources. During the healing process, various inflammatory cells — neutrophils; macrophages; fibroblasts, particularly senescent fibroblasts; and endothelial cells — are all prominent in chronic wounds, and are capable of producing ROS. However, in chronic wounds, activated neutrophils and macrophages produce extremely large amounts of superoxides, as well as its derivatives, via the phagocytic isoform of nicotinamide adenine dinucleotide phosphate oxidases. When polymorphonuclear neutrophils (PMNs) are recruited and activated at the wound site, they consume an increased amount of oxygen, which is converted into ROS in a process known as a "respiratory" or "oxidative" burst. This burst requires the consumption of large amounts of molecular oxygen, increasing oxygen consumption by at least 50%, and resulting in the generation of superoxide anions. Most of the superoxide anions formed are converted into hydrogen peroxide, which is converted into highly toxic hydroxyl radicals, creating a second source of ROS. These highly toxic hydroxyl free radicals enhance the synthesis and activation of even more pro-MMPs and the inactivation of a-1 PI. Doxycycline has been proven to scavenge these ROS, thereby protecting against their catabolic activities. This is partly due to its antioxidant effect on PMNs. Doxycycline prevents the oxidative conversion of pro-MMPs in the ECM into active MMPs. This process is not dependent upon the metal-ion binding properties of DOX and other CMTs.
Nitric oxide (NO) is similar to ROS in that it also requires a delicate balance within the microwound environment. This small, gaseous free radical is involved in extracellular and intracellular neurotransmission, cell-mediated cytotoxicity, and endothelium-dependent relaxation of vascular smooth muscle. Nitric oxide is also involved in inflammatory and autoimmune-mediated tissue destruction, and is produced by the nitric oxide synthase (NOS) family of enzymes. When NO and ROS are combined, they form peroxynitrite, a potent-free radical known to cause extensive tissue destruction. Because of this, excessive amounts of NO are extremely cytotoxic to the microwound environment. Wound infections and extended inflammation are also associated with an overproduction of NO in chronic wounds. This over expression has been directly linked to delayed healing in both chronic venous ulcers and diabetic foot ulcers. It is important to note that an under expression of NO has also been connected to delayed healing in diabetic foot ulcers. Decreased levels of NO in diabetic foot ulcers may be correctible with L-arginine, a semiessential amino acid, by using dietary supplements. Doxycycline reduces cytokine-induced NO production by inhibiting the expression of NOS through reducing inducible NOS levels.
Another potential indirect mechanism in which DOX and other CMTs inhibit the breakdown of the ECM is through the inhibition of the proinflammatory cytokines including tumor necrosis factor (TNF-a) and interleukin-1b (IL-1b). Chronic wound fluid has been found to contain up to a 100-fold increase in the levels of these cytokines. Proinflammatory cytokines synthesize MMPs and suppress TIMPs, resulting in an imbalance of the microwound environment with degradation of collagen, growth factors, and growth factor receptor sites, as well as other vital components of the ECM. Once growth factors are degraded, communication between the various cells participating in healing stops, and healing is delayed. As this delay continues, the proinflammatory cytokine cascade is amplified, and wound fluid becomes absent of DNA synthesis, ultimately resulting in senescent or mitotically incompetent cells.
Many researchers have reported a synergistic effect between DOX/CMTs and nonsteroidal anti-inflammatory drugs (NSAIDs) in different animal and human models of disease, even though they work through different pathways. For example, in the arthritic joint, oral tetracycline uptake is increased by as much as 150% without affecting blood levels of the drug. This phenomenon facilitates the uptake into therapeutic target areas. The combination of these 2 therapies (NSAIDs and tetracycline) has significantly decreased tissue destruction and inflammation when used in rheumatoid arthritis and osteoarthritis. Although NSAIDs do not directly affect MMP functions, researchers have found that they do potentiate the anticollagenolytic/proteolytic potential of tetracyclines by reducing edema, thereby facilitating the entry of tetracyclines into sites of inflammation. There are a number of studies demonstrating the benefit of DOX and/or CMTs in the treatment of chronic wounds. This synergistic effect may also be beneficial when combined with hyperbaric medicine in the treatment of a number of different wound types. For example, it may benefit patients with radiation proctitis and/or cystitis who are undergoing hyperbaric oxygen therapy for bleeding. These patients may also gain from the synergistic effect of combining DOX/CMT and NSAIDs to their treatment, as there is a significant inflammatory process involved in these radiation injuries, characterized by elevated production of MMPs in the tissue.
Pain is a frequent symptom associated with chronic wounds. Doxycycline and CMTs have a demonstrated ability to reduce pain associated with inflammation. Evidence supports the fact that the underlying cause of chronic wound pain is, in part, related to inflammation. A large prospective study of 758 patients found that the more chronic the duration of increased inflammation, the higher the reported wound pain (P = 0.022). As chronic wounds begin to heal, wound pain is reduced further, suggesting a link between prolonged inflammation and pain pathways. In 2009, a team of researchers demonstrated that ibuprofen can down-regulate proinflammatory cytokines when applied topically. This finding has led to the commercialization of ibuprofen-impregnated foam dressings for wound analgesia. The combination therapy of DOX/CMTs with NSAIDs, which both work on different pathways, may be of value in reducing chronic wound pain as well.
The presence of hypergranulation tissue in chronic wounds is believed to result from an extended inflammatory response. Excessive amounts of transforming growth factor-β (TGF-β) accelerate the healing response by increasing collagen deposition. Few articles have been published on this subject, and the majority of those published relate to the care of equines. The predisposing factors for hypergranulation tissue formation in horses include hypoxia, infection, and trauma/pressure combined with a prolonged inflammatory reaction. The focus of this research has been on TGF-β, which is generally acknowledged to be a cytokine with the ability to retard or accelerate granulation tissue formation. A prolonged inflammatory phase with amplified proinflammatory cytokine leads to elevated protease activity and impaired growth factor functions, which may account for the presence of hypergranulation tissue in chronic wounds. It is interesting that TGF-β has also been identified as the key factor in the development of hypertrophic scars and keloids in humans, because both hypergranulation tissue and hypertrophic scars and keloids are associated with a prolonged inflammatory response that ends with an overproduction of connective tissue. it may therefore decrease or inhibit the development of hypergranulation tissue in chronic wounds, and may decrease or improve hypertrophic scarring and keloid formation.
Doxycycline and CMT have been found to inhibit osteoclast-mediated bone resorption by inhibiting osteoclastic action and inducing apoptosis of osteoclasts. Simultaneously, DOX and CMT enhance osteoblastic activity. This is not surprising due to the fact that collagen is the major component of bone and represents at least 90% of its organic matrix. The final process of bone remodeling and resorption is controlled by MMPs and TIMPs.136 Doxycycline and CMT are bone-seeking agents that accumulate in high concentrations in bone and act, similarly to bisphosphonates, as antiresorptive drugs. Long-term use of bisphosphonates is associated with enhanced bone resorption. When DOX/CMTs are combined with bisphosphonates, they increase the amount of bone by suppressing resorption, while DOX/CMTs significantly increase the number of active osteoblastic cells.
The negative impact of reperfusion injury in the healing cascade is partly due to the inflammatory response of damaged tissues. Leukocytes, particularly PMNs, carried to the area upon reperfusion release a host of inflammatory factors, such as interleukins and free radicals, in response to tissue injury. Changes in endothelial cells occurring during ischemia promote PMN binding to these cells, priming PMN synthesis of oxygen radicals and release of cytotoxic proteins which lead to vascular damage and tissue injury. Doxycycline inhibits PMN superoxide synthesis and degranulation, and suppresses PMN-mediated red blood cells, fibroblast, and endothelial cytotoxicity, thereby providing protection from ischemia-reperfusion induced injuries.
Doxycycline and CMTs as modulating agents of MMPs must be used with care. Simply inhibiting MMPs in chronic wounds is not an appropriate strategy as MMP activity is important for cytokines and chemokine production to attract cells into wound areas. To control MMPs with anti-inflammatory drugs, they must be used during the correct phase of healing. The use of these drugs in the acute wound healing phase has been associated with a delay in healing in select clinical scenarios.
Although no published protocols on the dosing of DOX or CMTs in chronic wounds could be found, the majority of the articles referenced cite a subantimicrobial dose. One such drug (Periostat), administered as a 20 mg dose of DOX twice daily, is the only drug therapy approved by the United States Food and Drug Administration (FDA) for adult periodontitis as a collagenase inhibitor. A second drug (Oracea) is FDA-approved for the treatment of rosacea through its anti-inflammatory/anticollagenolytic effect. The latter is a sustained-release, 40 mg DOX tablet, with 10 mg of the 40 mg coated to delay release. Using these drugs for chronic wounds may require a loading dose to ensure there are no bacteria sensitive to the drug before starting any subantimicrobial dose. Long-term use of subantimicrobial levels of DOX has not caused resistance to the drug.
Abstract and Introduction
Abstract
Doxycycline is a semisynthetic, chemically modified tetracycline compound that is rapidly absorbed and exerts biological effects independent of its antimicrobial activity. One such effect includes the inhibition of matrix metalloproteinases. Doxycycline has a long history as a collagenase inhibitor. This article will describe its mode of action and review its effectiveness in significantly reducing inflammation and elevated levels of proinflammatory cytokines within chronic wounds.
Introduction
Doxycycline (DOX) is a semisynthetic, chemically modified tetracycline compound widely used to treat infections caused by both gram-negative and gram-positive microorganisms. This 4-ringed molecule was initially discovered in 1947. Since then, it has been well-established that DOX is rapidly absorbed with a prolonged half-life and exerts biological effects independent of its antimicrobial activity. One such effect includes the inhibition of matrix metalloproteinases (MMPs). Matrix metalloproteinases are a family of zinc-dependent enzymes with the ability to degrade all components of the extracellular matrix (ECM). Matrix metalloproteinases are produced by keratinocytes, endothelial cells, neutrophils, fibroblasts, macrophages, mast cells, and eosinophils. There are 4 distinct subsets of enzymes that exist within the MMP family: collagenases, gelatinases, and stromelysins, plus a few membrane-bound MMPs. Collagenases are the only enzymes in humans with the capacity to cleave the triple helix of type I, II, and III collagen, with gelatinases having the ability to cleave all other types. Gelatinases and collagenases also have the unique ability to activate against other material within the extracellular matrix when a disruption in their balance occurs. Inhibition of MMP activity by DOX, and other chemically modified tetracyclines (CMTs), occurs through the chelation of calcium and zinc ions. The chelation of calcium and zinc ions allows DOX and CMTs to inhibit matrix destruction mediated by MMP activity by 3 different processes: 1) the inhibition of already active MMPs; 2) the inhibition of pro-MMP activation; and 3) the down regulation of MMP expression. In addition, DOX and other CMTs indirectly prevent the degradation of connective tissue by protecting the host defense protein a-1 antitrypsin (α-1 PI), which inhibits leukocyte elastase. Excessive MMPs degrade and inactivate a-1 PI; the inhibition of MMPs thereby leads to higher tissue levels of α-1 PI, which reduce leukocyte elastase activity. Leukocyte elastases are responsible for the degradation and inactivation of tissue inhibitors of metalloproteinase (TIMPs). Tissue inhibitors of metalloproteinase are naturally occurring inhibitors of MMPs. Tissue inhibitors of metalloproteinase keep MMPs in balance including stromelysin (MMP-3), the only MMP that is relatively insensitive to direct inhibition by DOX. It is important to note there is no evidence that DOX or CMTs act as TIMPs inhibitors.
(Enlarge Image)
Effect on Reactive Oxygen Species
Free radicals are known to cause cell damage and function as inhibitory factors in the healing process. The production of reactive oxygen species (ROS) associated with chronic wounds can originate from several potential sources. During the healing process, various inflammatory cells — neutrophils; macrophages; fibroblasts, particularly senescent fibroblasts; and endothelial cells — are all prominent in chronic wounds, and are capable of producing ROS. However, in chronic wounds, activated neutrophils and macrophages produce extremely large amounts of superoxides, as well as its derivatives, via the phagocytic isoform of nicotinamide adenine dinucleotide phosphate oxidases. When polymorphonuclear neutrophils (PMNs) are recruited and activated at the wound site, they consume an increased amount of oxygen, which is converted into ROS in a process known as a "respiratory" or "oxidative" burst. This burst requires the consumption of large amounts of molecular oxygen, increasing oxygen consumption by at least 50%, and resulting in the generation of superoxide anions. Most of the superoxide anions formed are converted into hydrogen peroxide, which is converted into highly toxic hydroxyl radicals, creating a second source of ROS. These highly toxic hydroxyl free radicals enhance the synthesis and activation of even more pro-MMPs and the inactivation of a-1 PI. Doxycycline has been proven to scavenge these ROS, thereby protecting against their catabolic activities. This is partly due to its antioxidant effect on PMNs. Doxycycline prevents the oxidative conversion of pro-MMPs in the ECM into active MMPs. This process is not dependent upon the metal-ion binding properties of DOX and other CMTs.
Nitric oxide (NO) is similar to ROS in that it also requires a delicate balance within the microwound environment. This small, gaseous free radical is involved in extracellular and intracellular neurotransmission, cell-mediated cytotoxicity, and endothelium-dependent relaxation of vascular smooth muscle. Nitric oxide is also involved in inflammatory and autoimmune-mediated tissue destruction, and is produced by the nitric oxide synthase (NOS) family of enzymes. When NO and ROS are combined, they form peroxynitrite, a potent-free radical known to cause extensive tissue destruction. Because of this, excessive amounts of NO are extremely cytotoxic to the microwound environment. Wound infections and extended inflammation are also associated with an overproduction of NO in chronic wounds. This over expression has been directly linked to delayed healing in both chronic venous ulcers and diabetic foot ulcers. It is important to note that an under expression of NO has also been connected to delayed healing in diabetic foot ulcers. Decreased levels of NO in diabetic foot ulcers may be correctible with L-arginine, a semiessential amino acid, by using dietary supplements. Doxycycline reduces cytokine-induced NO production by inhibiting the expression of NOS through reducing inducible NOS levels.
Another potential indirect mechanism in which DOX and other CMTs inhibit the breakdown of the ECM is through the inhibition of the proinflammatory cytokines including tumor necrosis factor (TNF-a) and interleukin-1b (IL-1b). Chronic wound fluid has been found to contain up to a 100-fold increase in the levels of these cytokines. Proinflammatory cytokines synthesize MMPs and suppress TIMPs, resulting in an imbalance of the microwound environment with degradation of collagen, growth factors, and growth factor receptor sites, as well as other vital components of the ECM. Once growth factors are degraded, communication between the various cells participating in healing stops, and healing is delayed. As this delay continues, the proinflammatory cytokine cascade is amplified, and wound fluid becomes absent of DNA synthesis, ultimately resulting in senescent or mitotically incompetent cells.
Synergistic Effect Between Doxycycline/Chemically Modified Tetracyclines and Nonsteroidal Anti-inflammatory Drugs
Many researchers have reported a synergistic effect between DOX/CMTs and nonsteroidal anti-inflammatory drugs (NSAIDs) in different animal and human models of disease, even though they work through different pathways. For example, in the arthritic joint, oral tetracycline uptake is increased by as much as 150% without affecting blood levels of the drug. This phenomenon facilitates the uptake into therapeutic target areas. The combination of these 2 therapies (NSAIDs and tetracycline) has significantly decreased tissue destruction and inflammation when used in rheumatoid arthritis and osteoarthritis. Although NSAIDs do not directly affect MMP functions, researchers have found that they do potentiate the anticollagenolytic/proteolytic potential of tetracyclines by reducing edema, thereby facilitating the entry of tetracyclines into sites of inflammation. There are a number of studies demonstrating the benefit of DOX and/or CMTs in the treatment of chronic wounds. This synergistic effect may also be beneficial when combined with hyperbaric medicine in the treatment of a number of different wound types. For example, it may benefit patients with radiation proctitis and/or cystitis who are undergoing hyperbaric oxygen therapy for bleeding. These patients may also gain from the synergistic effect of combining DOX/CMT and NSAIDs to their treatment, as there is a significant inflammatory process involved in these radiation injuries, characterized by elevated production of MMPs in the tissue.
Pain is a frequent symptom associated with chronic wounds. Doxycycline and CMTs have a demonstrated ability to reduce pain associated with inflammation. Evidence supports the fact that the underlying cause of chronic wound pain is, in part, related to inflammation. A large prospective study of 758 patients found that the more chronic the duration of increased inflammation, the higher the reported wound pain (P = 0.022). As chronic wounds begin to heal, wound pain is reduced further, suggesting a link between prolonged inflammation and pain pathways. In 2009, a team of researchers demonstrated that ibuprofen can down-regulate proinflammatory cytokines when applied topically. This finding has led to the commercialization of ibuprofen-impregnated foam dressings for wound analgesia. The combination therapy of DOX/CMTs with NSAIDs, which both work on different pathways, may be of value in reducing chronic wound pain as well.
The presence of hypergranulation tissue in chronic wounds is believed to result from an extended inflammatory response. Excessive amounts of transforming growth factor-β (TGF-β) accelerate the healing response by increasing collagen deposition. Few articles have been published on this subject, and the majority of those published relate to the care of equines. The predisposing factors for hypergranulation tissue formation in horses include hypoxia, infection, and trauma/pressure combined with a prolonged inflammatory reaction. The focus of this research has been on TGF-β, which is generally acknowledged to be a cytokine with the ability to retard or accelerate granulation tissue formation. A prolonged inflammatory phase with amplified proinflammatory cytokine leads to elevated protease activity and impaired growth factor functions, which may account for the presence of hypergranulation tissue in chronic wounds. It is interesting that TGF-β has also been identified as the key factor in the development of hypertrophic scars and keloids in humans, because both hypergranulation tissue and hypertrophic scars and keloids are associated with a prolonged inflammatory response that ends with an overproduction of connective tissue. it may therefore decrease or inhibit the development of hypergranulation tissue in chronic wounds, and may decrease or improve hypertrophic scarring and keloid formation.
Doxycycline and Chemically-modified Tetracyclines as Antiresorptive Drugs
Doxycycline and CMT have been found to inhibit osteoclast-mediated bone resorption by inhibiting osteoclastic action and inducing apoptosis of osteoclasts. Simultaneously, DOX and CMT enhance osteoblastic activity. This is not surprising due to the fact that collagen is the major component of bone and represents at least 90% of its organic matrix. The final process of bone remodeling and resorption is controlled by MMPs and TIMPs.136 Doxycycline and CMT are bone-seeking agents that accumulate in high concentrations in bone and act, similarly to bisphosphonates, as antiresorptive drugs. Long-term use of bisphosphonates is associated with enhanced bone resorption. When DOX/CMTs are combined with bisphosphonates, they increase the amount of bone by suppressing resorption, while DOX/CMTs significantly increase the number of active osteoblastic cells.
The negative impact of reperfusion injury in the healing cascade is partly due to the inflammatory response of damaged tissues. Leukocytes, particularly PMNs, carried to the area upon reperfusion release a host of inflammatory factors, such as interleukins and free radicals, in response to tissue injury. Changes in endothelial cells occurring during ischemia promote PMN binding to these cells, priming PMN synthesis of oxygen radicals and release of cytotoxic proteins which lead to vascular damage and tissue injury. Doxycycline inhibits PMN superoxide synthesis and degranulation, and suppresses PMN-mediated red blood cells, fibroblast, and endothelial cytotoxicity, thereby providing protection from ischemia-reperfusion induced injuries.
Doxycycline and CMTs as modulating agents of MMPs must be used with care. Simply inhibiting MMPs in chronic wounds is not an appropriate strategy as MMP activity is important for cytokines and chemokine production to attract cells into wound areas. To control MMPs with anti-inflammatory drugs, they must be used during the correct phase of healing. The use of these drugs in the acute wound healing phase has been associated with a delay in healing in select clinical scenarios.
Although no published protocols on the dosing of DOX or CMTs in chronic wounds could be found, the majority of the articles referenced cite a subantimicrobial dose. One such drug (Periostat), administered as a 20 mg dose of DOX twice daily, is the only drug therapy approved by the United States Food and Drug Administration (FDA) for adult periodontitis as a collagenase inhibitor. A second drug (Oracea) is FDA-approved for the treatment of rosacea through its anti-inflammatory/anticollagenolytic effect. The latter is a sustained-release, 40 mg DOX tablet, with 10 mg of the 40 mg coated to delay release. Using these drugs for chronic wounds may require a loading dose to ensure there are no bacteria sensitive to the drug before starting any subantimicrobial dose. Long-term use of subantimicrobial levels of DOX has not caused resistance to the drug.
