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

Disclosures

Wounds. 2003;15(10) 

In This Article

Methods

Wound fluid was collected from a patient with a nonhealing pressure ulcer of greater than six months duration using a previously described procedure.[6] Briefly, the ulcer was covered with an occlusive dressing for one hour, and the fluid that spontaneously collected was aspirated, centrifuged at 10,000 x g for 15 minutes, and the supernatant solution was stored in multiple aliquots at -80°C.

The effect of doxycycline on TACE activity was assessed by measuring the release of soluble TNF from a murine macrophage cell line (RAW) following stimulation by lipopolysaccharide.[21] Briefly, RAW cells were plated at 5,000 cells per well in a 24-well microtiter plate in RPMI 1640 medium supplemented with 10-percent fetal calf serum, 2mM glutamine, and 50mg/mL gentamicin, and cultured at 37°C in five-percent carbon dioxide. Doxycycline (Sigma Chemical Co., St. Louis, Missouri) was added to give final concentrations of 0, 2, 20, 200, and 2,000mg/mL in the culture medium. RAW cells were then stimulated with 1mg/mL of Escherichia coli (serotype 0127:B8) lipopolysaccharide, and cell culture medium was harvested eight hours later. Levels of TNF protein in the samples of RAW cells culture medium were measured using an enzyme-linked immunosorbent assay (ELISA) (R&D Systems, Minneapolis, Minnesota).

The effect of doxycycline, tetracycline (Sigma Chemical Co., St. Louis, Missouri), and ilomastat (Elastin Products Company, Owensville, Missouri) on protease activity in chronic wound fluid was measured using minor modifications of a previously described assay that utilizes azocoll (Sigma Chemical Co., St. Louis, Missouri) as the substrate.[25] Azocoll is insoluble bovine hide that is covalently coupled with dye molecules. Proteolysis of the insoluble azocoll substrate generates soluble colored peptide fragments. Since the hide is composed predominately of intact collagen molecules, proteolytic hydrolysis of the azocoll substrate preferentially measures the combined activities of MMPs, including collagenases (MMP-1, MMP-8, and MMP-13) and gelatinases (MMP-2 and MMP-9). The rate of hydrolysis of azocoll by non-MMPs, such as neutrophil elastase, is relatively slow.

Azocoll was washed and suspended in 50mM Tris-HCl, 1mM CaCl2, pH 7.8, at a final concentration of 5mg/mL. Samples (50mL) of wound fluid, buffer, or inhibitors (50mL) were mixed with 900mL of azocoll suspension in a 1.5mL conical microcentrifuge tube. Ilomastat, a potent synthetic inhibitor of MMPs, was included as a positive control for MMP inhibition.[26] Reaction tubes were placed in a mixer that inverted the tubes 30 times per minute. After six hours incubation at 37°C, the reactions were centrifuged at 10,000 x g for five minutes, and the absorbance of the supernatant solution was measured at 520nm with a spectrophotometer. Levels of protease activity in samples were calculated by interpolation from a standard curve generated by crude collagenase from Clostridium histoliticum (Sigma Chemical Co., St. Louis, Missouri) and were expressed as mg of collagenase equivalents per mL of wound fluid. Protease levels in samples were calculated as the average of three replicate assays and were compared for statistical significance by ANOVA with Tukey's post-hoc test.

Enrollment criteria. Seven diabetic patients with full-thickness, lower-extremity ulcers were enrolled in a randomized, double-blinded, placebo-controlled study comparing the effect on healing of once-daily, topical one-percent doxycycline gel treatment with vehicle treatment and standardized good wound care. Following a thorough history and physical exam at the screening visit, patients who met the following criteria were enrolled: ulcer duration of four weeks to two years; ulcer dimensions between 1.7 to 12cm2; bacterial counts from punch biopsy of 8D1x106/gm of tissue; transcutaneous oxygen saturation (TcpO2) of 8E30mmHg; ankle brachial index (ABI) 8E0.8; and blood analyses within normal limits, including complete blood count (CBC), metabolic panel, and glycosylated hemoglobin A 1C (HbA1C) of up to six percent. Patients were randomly assigned to one of the treatment arms by the pharmacy at the time of enrollment.

Study drugs. Patients were provided with an opaque 60mL syringe equipped with a screw plunger that contained either the doxycycline hydrogel (1% doxycycline [w/v] neutralized with sodium hydroxide in a 2.7% [w/v] sodium carboxymethylcellulose [CMC] hydrogel with 1.7% [v/v] simethicone and 0.56% [v/v] polysorbate 20 antifoaming agents) or the vehicle hydrogel (2.7% CMC hydrogel with antifoaming agents). The contents of the syringes were unknown to the patient and the physician. The color of the vehicle hydrogel was matched to the light brown color of the doxycycline hydrogel with food dyes. The doxycycline hydrogel was stable for four weeks at 4°C, although patients usually received new study drugs every two weeks.

Wound treatment and assessment. Until the patients were enrolled, their wounds were dressed with moist-to-dry saline gauze dressings that were changed twice daily. Patients were trained to place the hydrogel on their wounds to a thickness of about 2mm (approximately the thickness of a dime), cover their wounds with dry gauze pads, and secure their dressings with soft outer wraps. Patients were fitted with an offloading shoe and were treated with the hydrogels until the ulcer healed, or for up to 20 weeks. If the ulcer was not healed after 20 weeks of treatment, the patient could elect to receive doxycycline treatment for an additional 12 weeks of open-label use. Wounds were clinically evaluated by the physician every two weeks, photographed, and the wound edge was traced on an acetate sheet. Wound areas were calculated by multiplying the longest perpendicular length and width dimensions of the wound, which were made at each biweekly clinic visit. The effect of doxycycline treatment on healing was compared to vehicle treatment using Chi-squared analysis of healed or not healed wounds at 20 weeks. At each clinic visit, patients were also assessed for any adverse events of treatment.

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