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

Results

As shown in Figure 1, the level of TNF in the conditioned medium of RAW cells that were not stimulated by lipopolysaccharide (LPS) was very low (1.7 ± 1.1ng/mL, mean ± standard error). Addition of 0.01-percent LPS to the RAW cells increased TNF concentration approximately 160 fold (276 ± 12ng/mL). Addition of doxycycline at 2mg/mL concentration (4mMolar) had no effect on TNF levels in the conditioned medium of the LPS-stimulated RAW cells (273 ± 11ng/mL). In contrast, addition of doxycycline at concentrations of 20, 200, and 2,000mg/mL to the LPS-stimulated RAW cells caused significant (p<0.05) decreases in concentrations of TNF in the conditioned medium that progressively decreased to levels in control RAW cells not exposed to LPS (15 ± 1ng/mL, 1.8 ± 1ng/mL, and 1.6 ± 1ng/mL, respectively). Based on this dose response curve, the concentration of doxycycline that inhibits 50 percent of the TNF release (IC50) is approximately 20mg/mL, or 40mM.

Inhibition of TNF release by doxycycline. Cultures of RAW murine macrophage cells were cultured in 24-well plates in medium without lipopolysaccharide (control) or with 0.001-percent LPS and doxycycline at concentrations of 0, 2, 20, 200, and 2,000B5g/mL. After eight hours, the concentration of TNF was measured with ELISA. Asterisks indicate significant reduction in TNF levels compared to LPS with no doxycycline.

As shown in Figure 2, the chronic wound fluid contained a high level of protease activity, 180 ± 20ng/mL. For comparison, we reported previously that acute wound fluid from mastectomy drains contained about 1ng/mL of protease activity, while the average level of protease activity from 40 chronic wounds was approximately 60ng/mL.[6] Addition of doxycycline at 50, 500, and 5,000mM significantly (p<0.05) reduced the protease activity by 44 percent, 75 percent, and 89 percent, respectively. Addition of tetracycline at 50, 500, and 5,000mM also reduced protease activity by 6 percent, 33 percent, and 61 percent, respectively, but tetracycline was not as effective as doxycycline. Addition of ilomastat at 20, 200, and 2,000mM concentrations reduced protease activity by 78 percent, 89 percent, and 92 percent, respectively. Thus, ilomastat was the most effective inhibitor of the chronic wound proteolytic activity, followed by doxycycline, then tetracycline, which is consistent with their reported IC50 values for collagenase.[27,28]

Inhibition of protease activity in chronic wound fluid by doxycycline. Protease activity was measured in a fluid from a chronic wound using azocoll substrate. Doxycycline inhibited protease activity in the wound fluid in a dose-dependent manner, achieving 89-percent inhibition at 5,000mM. Tetracycline also reduced protease activity, achieving 61-percent inhibition at 5,000mM. Ilomastat inhibited 92-percent activity at 2,000mM.

The small sample sizes in the two treatment arms in this initial pilot study limit generalization of the results. Nevertheless, as seen in Figure 3, all four of the chronic wounds treated with doxycycline healed (times to healing were 4, 7, 24, and 30 weeks). In contrast, only one of the three patients treated with vehicle healed during the initial 20 week treatment period (patient V3 at week 4, Figure 4). Patient V2's wound showed no trend for healing after 20 weeks of treatment with vehicle hydrogel. However, after 12 weeks of open label treatment with doxycycline, the ulcer had reduced in size by about 60%. Patient V1's wound remained essentially unchanged during the 20 week treatment period, and he did not choose to receive doxycycline treatment. Chi squared analysis of the healing outcome of the seven patients at 34 weeks indicated that topical doxycycline treatment significantly (p = 0.05) increased healing of the ulcers compared to treatment with vehicle hydrogel.

Effect of topical one-percent doxycycline on healing of chronic, diabetic, lower-extremity ulcers. Diabetic patients with chronic lower extremity ulcers were enrolled in a randomized controlled trial of daily, topical one-percent doxycycline in CMC hydrogel. All four of the chronic wounds treated with doxycycline healed (times to healing were 4, 7, 24, and 30 weeks).

Effect of topical CMC hydrogel vehicle on healing of chronic diabetic lower-extremity ulcers. Diabetic patients with chronic lower extremity ulcers were enrolled in a randomized controlled trial of daily, topical CMC hydrogel. Only patient V3 healed during the initial 20 week treatment period (at week 4). Patient V2 wound showed no trend for healing after 20 weeks of treatment with vehicle hydrogel, but after 12 weeks of open label treatment with doxycycline, the ulcer had reduced in size by about 60 percent. Patient V1's wound remained essentially unchanged during the 20-week treatment period, and the patient did not elect to receive doxycycline. Chi-squared analysis of the healing outcome of the seven patients at 34 weeks indicated that topical doxycycline treatment significantly (p=0.05) increased healing of the ulcers compared to treatment with vehicle hydrogel.

In this pilot study, another major objective was to determine safety of topical doxycycline treatment. No adverse events were noted that were attributable to either the doxycycline treatment or to the vehicle treatment.

Multiple confounding variables are known to influence healing of diabetic lower-extremity ulcers. These include the initial ulcer size, the duration of the ulcer, the age of the patient, co-morbidity conditions, such as renal or hepatic diseases, smoking history, and compliance with treatment protocol, especially offloading of plantar ulcers. It is difficult in this initial pilot study to adequately control for all these confounding variables. Nevertheless, as shown in Table 1 , there was no obvious segregation of confounding variables to one of the treatment arms. For example, the wound areas at the time of enrollment were not significantly different. In fact, the average area for doxycycline-treated wounds was about 2cm2 larger than the average area for vehicle-treated wounds (5.33 ± 4.59cm2 versus 3.47 ± 3.48cm2, respectively, mean ± standard deviation). Six of the seven patients were men, reflecting the demographics of the VA study population. The average age of the four patients treated with doxycycline was 57 years (range from 46-68 years), while the average age of the three patients treated with vehicle was 70 years (range from 64-78 years). There was no notable difference in smoking history or in the average number of years patients had diabetes (10 years in doxycycline-treated and 11 years in vehicle-treated) in the two treatment arms. The most common co-morbid condition was hypertension, followed by coronary artery disease (CAD), and congestive heart failure (CHF). Ulcer duration in the doxycycline-treated group averaged three months, with a range from 2 to 5 months. Patients V2 and V3 had ulcers for six and seven months before enrollment, while patient V1 had an ulcer for 24 months before enrollment.

Comments

3090D553-9492-4563-8681-AD288FA52ACE

processing....