Treatment of chronic ulcers

Abstract

The present invention provides a pharmaceutical formulation comprising LL-37 or a pharmaceutically-acceptable salt thereof and one or more pharmaceutically-acceptable diluent or carrier system, for use in a method of treatment of a chronic ulcer wound (such as a hard-to-heal venous leg ulcer or a diabetic foot ulcer), which method comprises: (a) topical application of the formulation to the ulcer; followed by (b) application of a dressing, and
wherein the application of the formulation provides for a dose of LL-37 at the wound site that is below about 80 μg of LL-37 applied per cm.sup.2 of wound area, and/or below about 26.7 μg of LL-37 applied per cm.sup.2 of wound area, per day of treatment.

Claims

1. A method of treating a chronic ulcer, wherein said method comprises: topically applying to said chronic ulcer a pharmaceutical formulation, wherein the pharmaceutical formulation is an aqueous solution comprising LL-37 or a pharmaceutically-acceptable salt thereof, wherein the pharmaceutical formulation comprises one thickening agent, wherein the dose of LL-37 applied per cm.sup.2 of wound area is between about 1 μg/cm.sup.2 and about 70 μg/cm.sup.2, and wherein the pharmaceutical formulation has a viscosity in the range of about 2 and about 10 Pa.Math.s.

2. The method as claimed in claim 1, wherein the dose of LL-37 applied per cm.sup.2 of wound area is between about 5 μg/cm.sup.2 and about 60 μg/cm.sup.2.

3. The method as claimed in claim 1, further comprising subsequently and separately applying a dressing to said chronic ulcer.

4. The method as claimed in claim 3, wherein the dressing is applied within no less than about 5 minutes and no more than about 30 minutes of the application of the pharmaceutical formulation.

5. The method as claimed in claim 2, further comprising subsequently and separately applying a dressing to said chronic ulcer.

6. The method as claimed in claim 5, wherein the dressing is applied within no less than about 5 minutes and no more than about 30 minutes of the application of the pharmaceutical formulation.

7. A method of treating a chronic ulcer, wherein said method comprises: topically applying to said chronic ulcer a pharmaceutical formulation, wherein the pharmaceutical formulation is an aqueous solution comprising LL-37 or a pharmaceutically-acceptable salt thereof, wherein the pharmaceutical formulation comprises one thickening agent; wherein the dose of LL-37 applied per cm.sup.2 of wound area is between about 0.3 μg/cm.sup.2 and about 23.3 μg/cm.sup.2 per day of treatment, and wherein the pharmaceutical formulation has a viscosity in the range of about 2 and about 10 Pa.Math.s.

8. The method as claimed in claim 7, wherein the dose of IL-37 applied is between about 1.6 μg/cm.sup.2 and about 20 μg/cm.sup.2 per day of treatment.

9. The method as claimed in claim 7, further comprising subsequently and separately applying a dressing to said chronic ulcer.

10. The method as claimed in claim 9, wherein the dressing is applied within no less than about 5 minutes and no more than about 30 minutes of the application of the pharmaceutical formulation.

11. The method as claimed in claim 8, further comprising subsequently and separately applying a dressing to said chronic ulcer.

12. The method as claimed in claim 11, wherein the dressing is applied within no less than about 5 minutes and no more than about 30 minutes of the application of the pharmaceutical formulation.

13. The method as claimed in claim 1, wherein the chronic ulcer is a venous leg ulcer.

14. The method as claimed in claim 1, wherein the chronic ulcer is a diabetic foot ulcer.

15. The method as claimed in claim 2, wherein the chronic ulcer is a venous leg ulcer.

16. The method as claimed in claim 2, wherein the chronic ulcer is a diabetic foot ulcer.

17. The method as claimed in claim 7, wherein the chronic ulcer is a venous leg ulcer.

18. The method as claimed in claim 7, wherein the chronic ulcer is a diabetic foot ulcer.

19. The method as claimed in claim 8, wherein the chronic ulcer is a venous leg ulcer.

20. The method as claimed in claim 8, wherein the chronic ulcer is a diabetic foot ulcer.

21. A method as claimed in claim 1, wherein the pharmaceutical formulation comprises one thickening agent being polyvinyl alcohol.

22. A method as claimed in claim 2, wherein the pharmaceutical formulation comprises one thickening agent being polyvinyl alcohol.

23. A method as claimed in claim 7, wherein the pharmaceutical formulation comprises one thickening agent being polyvinyl alcohol.

24. A method as claimed in claim 8, wherein the pharmaceutical formulation comprises one thickening agent being polyvinyl alcohol.

Description

EXAMPLE

(1) Double-Blind, Randomised, Placebo-Controlled, Safety and Pilot-Dose Response Study of LL-37 in HTH VLUs

(2) A double-blind, randomised, placebo-controlled, Phase I/II safety and pilot-dose response study was conducted on human subjects. The first subject was enrolled in August 2012 and the last subject completed in April 2013.

(3) The study was designed, conducted and reported in accordance with the International Conference on Harmonisation (ICH) Harmonised Tripartite Guidelines for Good Clinical Practice (GCP), with applicable local regulations and ethical principles laid down in the Declaration of Helsinki and was conducted across three centres in Sweden.

(4) Selection Criteria

(5) Study subjects comprised either male subjects of at least 18 years of age, or post-menopausal or clinically sterile women, with VLUs or combined venous-arterial ulcers with a predominant venous component.

(6) Exclusion criteria included if subjects had known predominant aetiology other than VLU within the target ulcer, and malignant disease (excluding basal cell carcinoma), unless in remission for 5 years or more.

(7) After obtaining necessary regulatory (Swedish Medicinal Products Agency (MPA) and ethical (regional Ethics Committee) approvals, and written informed consent, a total of 56 subjects attended a screening visit followed by a 3-week open-label run-in period.

(8) Target wounds with an area greater than 30 cm.sup.2 and less than 2 cm.sup.2 were excluded initially. However, in order to increase the inclusion rate and decrease the total study time, criteria were amended after randomisation of 21 subjects, to exclude areas greater than 40 cm.sup.2 and less than 1 cm.sup.2. If an eligible subject had more than one wound satisfying the entry criteria, the investigator identified the wound with the most appropriate size, shape and localisation as target wound. Only one wound per subject was treated in the study.

(9) During the 3-week run-in period, subjects received, every third day (±1) and not more than two times per week, six applications (in total) of a placebo preparation (see below) in combination with standard compression bandaging (as defined by the Swedish MPA), comprising elastic bandaging and appropriate dressing to control wound exudation.

(10) If the ulcerative area decrease was greater than between 7% and 13% (depending on size) per week relative to the start of the run-in period, the ulcer was not defined as HTH for the purposes of this study. In such cases, subjects were excluded from the study and the outcome was recorded as screening failure.

(11) After a run-in period, 34 eligible subjects underwent baseline assessments before randomisation to receive active treatment (one of three treatment strengths) or placebo.

(12) Formulations and Treatment

(13) Following the above run-in period, a 4-week treatment period was initiated, including 8 visits, an end-of-treatment visit 3 days (±1 day) after the last treatment, and a follow-up visit after an additional 4 weeks (±1 to 7 days).

(14) Subjects were randomised into four treatment groups to receive LL-37 (one of three alternative strengths), or placebo.

(15) Wound bed preparation prior to treatment included debridement of non-viable tissue, treatment of clinical infection and selection of the most appropriate dressing (see below). The wound was cleaned using tap water, gently dried with a gauze pad and, if necessary, curettage was applied prior to application of the trial product or placebo.

(16) 0.7 mL of a sterile concentrate of LL-37 in water at concentrations of 2.5, 8 or 16 mg of LL-37 per mL was provided in sealed glass vials (APL, Umeå, Sweden), which were stored prior to the trial at between 2 and 8° C. Immediately before application to the wound, this was diluted with 5 mL of a viscous diluent solution comprising 13.1% (w/w) of polyvinyl alcohol in acetate buffered saline (APL AB) to give final concentrations of either 0.5, 1.6 or 3.2 mg/mL, respectively. Placebo comprised exactly the same solution, but without LL-37 present. The diluted viscous liquid solution of active treatment or placebo was applied topically at an amount of 25 μL/cm.sup.2 with a 1 mL graded syringe at the centre of the cleansed and dried ulcer, and distributed evenly over the entire ulcer area using the rounded head of the syringe plunger that had been used in mixing the study drug.

(17) After a 5-minute wait (to prevent LL-37 or placebo from being absorbed by the dressing material), the above procedure was followed by application of a dressing and a compression bandage.

(18) For moderate to heavy exudating wounds, highly absorbent dressings made of polyurethane foam, such as Mepilex®, Allevyn® and Tegaderm®, were used. Alternatively, the absorbent hydrofibre dressing Aquacel® was used. For slight to moderate exudative wounds, non-adherent dressings were used, with or without an adhesive edge. The dressings used were Solvaline® N or the combined polyurethane/hydrofibre dressing Versiva®. For dry wounds, Aquacel® hydrofibre dressing was used.

(19) In combination with these other dressings, gauze pads were used to offload sensitive parts during compression. A 100% viscose, ductile, soft and absorbent cushion was used, including Cellona® or Soffban®. Comprilan® was the most commonly used compression bandage used in the study. Two bandages (8 and 10 cm wide) were tied with approximately 50% stretch from the base of the toes in a spiral to just beneath the knee. Alternatively, ZipZoc® medical stocking plus Coplus® or a similar self-adhesive bandage or Profore Lite® in combination with some of the above mentioned wound dressings were allowed. A long-stretch bandage of Dauer-type could also be used. Because of the high resting pressure with this type of bandage, it was temporarily removed during the night or when the subject was in a resting position for a long period.

(20) Additional treatment included specific topical creams for the treatment of skin problems. Zinc paste or Cavilon® were used on per-ulcer skin to protect the skin near the wound edge from maceration. Hydrocortisone cream or ointment (topical steroid group I or hydrocortisone-imidazole cream) was used for peri-ulcer eczema. If the skin was dry and scaly, Canoderm or Propyderm cream was allowed.

(21) The above procedure was repeated every third day (±1) over a 4 week treatment period, not more than two times per week, representing a total of eight applications.

(22) Subsequent to treatment, subjects were followed up for a period of 4 weeks (±1 to 7 days).

(23) The study assessed the rate of wound healing, pain at wound site both at the time of dressing and over the previous 24 hours using Visual Analogue Scale (VAS). Any incidence of complete healing was recorded even though statistically significant results were not expected based on the short duration of the trial and the small sample size. Wound size measurements were performed repeatedly over the study period to indicate the extent of wound healing. At every other visit, wound area was measured using the Visitrak™ planimetry device, pain at the wound site and wound characteristics (odour, slough, granulation and necrotic tissue) were recorded. The wound condition was to be described using photographs of the wound.

(24) Adverse events (AEs) and local tolerability were assessed at all visits. Subjects were advised to report any AEs and were given an emergency card with a phone number to call in case of any significant AEs. Blood was collected for laboratory safety assessment and investigation of development of antibodies against LL-37. Changes in laboratory values and vital signs were assessed, and analysis of auto-antibody formation against LL-37 was performed.

(25) Safety endpoints included: incidence of severe local reactions in wound and adjacent skin as exemplified by clinical signs of inflammation (oedema, redness, odour and raised temperature) or skin irritation (scaling, redness, papules, vesicles, pustules); any local reaction was recorded on a graded scale (0 to 3); incidence of greater than 30% increase in wound area relative to baseline (randomisation visit); change in laboratory values and vital signs from baseline; overall incidence of AEs.

(26) Efficacy endpoints included: wound healing rate within the study period; a number of ulcers attaining a greater than 30% area reduction from baseline at randomization; and changes in local pain using VAS score (0 to 10) changes in wound characteristics: (scores for slough, exudation, granulation tissue, necrosis) from baseline (scale 0 to 3).

(27) Analyses

(28) Statistical analyses were carried out using SAS® software (version 9.2 or higher; SAS Institute, Cary, N.C., US).

(29) The wound area was modelled over time for each subject to estimate the healing rate over the treatment period and the relationship between healing rate and dose was investigated.

(30) Estimates of healing rate constants (i.e. the change in wound area or mathematically transformed wound area/day) were derived with 95% confidence intervals (Cis) for each of the four treatment groups. An exponential decay model was fitted to the data with two parameters representing the initial wound area and the rate of healing. The model was of the form:
Y=α×e.sup.−βt
where α denotes the initial wound area (i.e. at randomisation visit), β denotes the reduction in size expressed as healing rate per day, Y denotes the wound area and t denotes the time in days from the randomisation visit.

(31) Additional analyses incorporating all run-in data was conducted to assess how the application of study treatments affected the healing process. This was assessed using a “broken-stick” approach, approximating pre- and post-randomisation data by a linear response over time after suitable data transformation (e.g. log, square root).

(32) If appropriate, pain scores were analysed using a repeated measures analysis to assess the overall difference between treatments over time. The model included terms for treatment, and time and the interaction between the two.

(33) The incidence of any AEs was estimated using the percentage of subjects in each treatment group who experienced at least one AE, with associated 95% CIs. Cl was calculated using the method detailed in Altman et al, Statistics with Confidence, BMJ Publication Group (2000), which was appropriate for small samples, or low incidences. The relationship between incidence of AEs and dose was investigated if appropriate. The overall incidence of grade 3 (severe) local reactions was presented for the LL-37 treated subjects with 95% CIs. Laboratory data were to be shown in plots of post-treatment values versus baseline, with reference ranges.

(34) Results

(35) A total of 31 subjects received the full course of eight treatment applications. Of the remaining three subjects, two (in 0.5 mg/mL treatment group) received seven applications and one subject (in placebo arm) received three applications.

(36) The exponential decay model fitted to the wound area provided a good fit to the data.

(37) The healing rate constant for the 0.5 mg/mL dose of LL-37 (0.039) differed significantly from that for placebo (0.007, p=0.003) representing a faster reduction in wound area (i.e. improved healing). A non-linear mixed effect model was used and the significance of the difference between the parameters was calculated by taking the estimate of the difference and dividing by its standard error, and comparing the result against a t-distribution.

(38) The estimated healing rate constant for the 1.6 mg/mL dose of LL-37, 0.019, also represented a faster reduction in wound area compared to placebo, although was not statistically significant at the 5% level (p=0.09). There was no statistically significant difference between the highest dose of LL-37 (healing rate constant 0.004) group and the placebo group. The low and intermediate dose groups were statistically indistinguishable. The estimated healing rate constants, with a measure of mean reduction in wound area, for each treatment are shown in Table 1 below.

(39) TABLE-US-00002 TABLE 1 Estimates of wound healing rates Estimated Estimated difference Estimated healing relative initial rate constant.sup.1 to control area (cm.sup.2) (cm.sup.2/day) (cm.sup.2/day) Mean Mean Mean p- Treatment (95% C.I.) (95% C.I.) (95% C.I.) value Placebo 9.222 (6.133 0.007 (−0.001 (n = 9) to 12.312) to 0.014) LL-37 5.036 (1.742 0.039 (0.020 0.032 (0.012 0.003 0.5 mg/mL to 8.329) to 0.058) to 0.053) (n = 8) LL-37 7.498 (4.583 0.019 (0.007 0.012 (−0.002 0.088 1.6 mg/mL to 10.414) to 0.031) to 0.026) (n = 9) LL-37 5.872 (2.573 0.004 (−0.007 −0.003 (−0.016 0.656 3.2 mg/mL to 9.171) to 0.015) to 0.010) n = 8) .sup.1Where the lower 95% confidence limit is negative this represents a positive healing rate constant over time since the model fitted assumes a negative constant (i.e. decrease in wound area).

(40) Overall, the highest reduction in wound area was noted for 1.6 mg/mL dose of LL-37 across all visits. The mean reduction in wound area for 1.6 mg/mL dose of LL-37 was 5.29 cm.sup.2 from randomization to the end of the study (see Table 2 below).

(41) TABLE-US-00003 TABLE 2 Wound Area (cm.sup.2) at Each Visit LL-37 LL-37 LL-37 Placebo 0.5 mg/mL 1.6 mg/mL 3.2 mg/mL Mean ± S.D for the visits (N = 9) (N = 8) (N = 9) (N = 8) Visit 1 (Screening) 9.72 ± 8.475 4.59 ± 2.024 7.84 ± 4.789 8.41 ± 6.450 Visit 3 10.25 ± 9.875  4.39 ± 1.995 8.14 ± 4.997 7.83 ± 6.163 Visit 6 (Randomisation) 9.54 ± 8.246 5.11 ± 1.732 7.69 ± 4.072 8.50 ± 6.919 Visit 8 8.60 ± 8.171 3.89 ± 1.329 5.83 ± 2.156 7.95 ± 6.270 Visit 10 6.63 ± 5.569 3.18 ± 1.549 5.34 ± 1.943 7.03 ± 5.063 Visit 12 6.50 ± 7.777 2.26 ± 1.061 4.59 ± 2.266 5.93 ± 5.317 Visit 14 6.16 ± 6.850 1.93 ± 1.262 3.86 ± 3.029 5.89 ± 4.784 Visit 15 (End of Treatment) 6.52 ± 7.683 1.98 ± 1.276 3.34 ± 3.166 5.85 ± 4.648 Visit 16 (End of Study) 5.76 ± 7.574 1.33 ± 1.209 2.40 ± 3.269 6.19 ± 4.770 SD: Standard deviation; end of study refers to the follow-up visit performed approximately 4 weeks after the last treatment. SD: Standard deviation; end of study refers to the follow-up visit performed approximately 4 weeks after the last treatment.

(42) The healing rate constant for the exponential decay model exhibited a 6-fold difference for the lowest dose (0.5 mg/mL) group, and a 3-fold difference for the intermediate dose (1.6 mg/mL) group versus the placebo group (p=0.003 for 0.5 mg/mL and p=0.088 for 1.6 mg/mL). These formulations thus gave rise to a faster reduction in wound area, and thus an improved healing response. However, there was no difference between the estimated healing rates in the highest LL-37 dose (3.2 mg/mL) group and the placebo group.

(43) In order to incorporate the data from the run-in and to assess if the addition of treatment affected the rate of healing, a broken stick analysis was conducted and was consistent with the previous analyses. As shown in FIG. 1, both the lower two doses of LL-37 had greater reduction in wound area after randomisation than during the run-in, indicating statistically significant improved healing compared to pre-treatment values (p<0.001 and p=0.011 for 0.5 mg/mL and 1.6 mg/mL respectively). No significant difference between the post-randomisation rate and the run-in was seen for either the highest dose or placebo. The rate of change during the run-in was very close to zero, as would be expected since responders were excluded from the study.

CONCLUSIONS

(44) A marked and significant improvement in wound healing was observed in response to administration of the two lower doses of LL-37 (0.5 and 1.6 mg/mL). The results from this study suggest that doses of 0.5 (which equates to 13 μg of LL-37 per cm.sup.2 of wound area), and 1.6 mg/mL (which equates to 40 μg of LL-37 per cm.sup.2 of wound area) every third day results in significant stimulation of wound healing. Alternatively, a less frequent application of a higher dose may be appropriate.