Treatment of Chronic Ulcers in Diabetic Patients with a Topical Metalloproteinase Inhibitor, Doxycycline

Gloria A. Chin, MD, MS; Tera G. Thigpin; Karen J. Perrin, BSN, ARNP; Lyle L. Moldawer, PhD; Gregory S. Schultz, PhD


Wounds. 2003;15(10) 

In This Article

Abstract and Introduction

Chronic diabetic ulcers are characterized by elevated levels of pro-inflammatory cytokines and proteases, which are thought to impair wound healing by degrading essential molecules. There is a need for an inexpensive, safe, and simple-to-use agent that promotes healing by selectively reducing levels of pro-inflammatory cytokines and proteases. Doxycycline, a member of the tetracycline family of antibiotics that inhibits metalloproteinases, was evaluated in vitro for inhibition of tumor necrosis factor-alpha (TNF) converting enzyme (TACE) and for inhibition of protease activities in human chronic wound fluid. In addition, topical doxycycline treatment was evaluated in a pilot, randomized, controlled trial of chronic, diabetic, lower-extremity foot ulcers. Incubation of lipopolysaccharide (LPS)-stimulated murine macrophages with doxycycline at 200mg/mL totally prevented the release of TNF. Doxycycline also reduced proteolytic degradation of azocoll, a substrate for matrix metalloproteinases (MMPs), by a chronic wound fluid in a dose-dependent manner, achieving 89-percent reduction at 2.3mg/mL. All four chronic ulcers of diabetic patients treated daily with topical one-percent doxycycline healed in the 20-week treatment period, whereas only one of three patients treated with vehicle healed (p=0.05). No adverse events were attributed to doxycycline treatment. These results demonstrate that doxycycline effectively inhibits TACE and MMPs and suggest that topical one-percent doxycycline treatment improves healing of chronic, diabetic, foot ulcers, presumably by reducing levels of TNF and MMPs.

It was estimated in 1998 that there were over two million, and perhaps up to five million, chronic wounds annually in the US alone.[1] Diabetic foot ulcers account for five percent of these ulcers and 50 to 80 percent of all lower-extremity amputations. Optimal conventional treatments for chronic wounds are based on the concepts of wound bed preparation, which include eliminating necrotic tissue and fibrinous exudate, controlling infection, establishing moisture balance, and optimizing the epidermal margin.[2] However, despite applying the principles of wound bed preparation, some chronic wounds fail to heal in a timely fashion and are candidates for advanced interventions, including topical growth factors,[3,4] bioengineered skin substitutes,[5] or surgical intervention for closure. Although advanced technologies have been proven to be effective in controlled clinical studies, they are expensive and are not universally available to all patients. Thus, there is a need for an inexpensive, readily available, simple therapy that can be added to the concept of wound bed preparation.

Multiple studies have established that the molecular and cellular environments of chronic skin wounds differ dramatically from the acute wound environment. In general, chronic wounds typically contain increased levels of pro-inflammatory cytokines and increased levels of proteases that are able to degrade essential mitogenic factors.[6,7,8,9] These observations led to the hypothesis that correcting these molecular abnormalities would promote healing of chronic wounds.[10] Studies from Trengove and colleagues[6] support this hypothesis by demonstrating that elevated levels of pro-inflammatory cytokines and proteases decreased in chronic venous stasis ulcers as healing progressed. These results suggest that an inexpensive, simple-to-administer, topical agent that reduced inflammation and protease activities might be an effective adjunctive therapy for wound bed preparation. Doxycycline, an antibiotic of the tetracycline family of drugs, is an inhibitor of matrix metalloproteinases (MMPs),[11] and several animal studies reported that treatment with doxycycline or other tetracycline analogues improved healing parameters. For example, systemic treatment of rats with doxycycline increased tensile strength of rat intestinal anastomoses on Day 3 after surgery,[12] and systemic tetracycline reduced the incidence of ulceration in alkali-injured rabbit eyes.[13] Topical application of a chemically modified tetracycline, which lacks antibiotic activity but retains MMP inhibitor activity, increased hydroxyproline levels and decreased collagenase levels in skin wounds of streptozotocin-induced diabetic rats.[14] Thus, members of the tetracycline family of molecules, such as doxycycline, appear to reduce MMP activities in various animal wound models and improve parameters of healing.

Another major difference in the molecular environments between acute, healing wounds and chronic wounds is elevated levels of pro-inflammatory TH1 cytokines, including TNF, and interleukin-1 (IL-1). There are several important relationships between proteases and pro-inflammatory cytokines that appear to influence wound healing. For example, TNF induced synthesis of IL-1 in human dermal fibroblasts,[15] and both TNF and IL-1 stimulated synthesis of collagenase (MMP-1) by dermal fibroblasts.[16] Furthermore, TNF inhibited synthesis of collagen mRNA and protein in cultured human fibroblasts.[17] Thus, chronically elevated levels of TNF in wounds would be expected to impair healing by reducing synthesis of collagen and increasing synthesis of MMPs.

Previous experiments showed that TNF is synthesized as a 26kDa transmembrane precursor protein that is processed to a 17kDa soluble protein and secreted from cells after proteolytic cleavage by a metalloproteinase enzyme, named TNF converting enzyme (TACE).[18,19,20] The proteolytic activity of TACE was inhibited in vitro by various synthetic MMP inhibitors.[18,21,22,23] In addition, systemic treatment of mice with these inhibitors, including doxycycline, prevented endotoxin-induced lethality by preventing secretion of TNF into serum.[18,21,22,23,24]

To better understand the possible effects of doxycycline on the environment of chronic wounds, we investigated the in-vitro effects of doxycycline on inhibiting the release of TNF from cultured macrophages and on inhibiting protease activities in a sample of human chronic wound fluid. In addition, we performed a pilot, randomized, controlled trial to evaluate the safety and efficacy of topical one-percent doxycycline treatment on healing of chronic ulcers in diabetic patients.