Clinical efficacy of basic fibroblast growth factor (bFGF) for diabetic ulcer

ARTICLE

Auteur(s) : Hiroshi Uchi¹, Atsuyuki Igarashi², Kazunori Urabe¹, Tetsuya Koga³, Juichiro Nakayama⁴, Ryuzo Kawamori⁵, Kunihiko Tamaki⁶, Hideki Hirakata⁷, Takehiko Ohura⁸, Masutaka Furue¹

¹Department of Dermatology, Kyushu University Hospital, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan
²Department of Dermatology, Kanto Medical Center NTT EC, Tokyo, Japan
³Department of Dermatology, Fukuoka Red Cross Hospital, Fukuoka, Japan
⁴Department of Dermatology, Fukuoka University Hospital, Fukuoka, Japan
⁵Department of Medicine, Metabolism and Endocrinology, Juntendo University School of Medicine, Tokyo, Japan
⁶Department of Dermatology, The University of Tokyo Hospital, Tokyo, Japan
⁷Department of Nephrology, Fukuoka Red Cross Hospital, Fukuoka, Japan
⁸Wound Healing Research Center, Sapporo, Japan

accepté le 17 Mai 2009

Diabetic ulcer is one of the most intractable types of chronic ulcer and seriously affects the vital prognosis. Its main causes include diabetic peripheral neuropathy, impaired blood circulation, and increased susceptibility to infection. Foot sores from shoes and injury may serve as triggers [1-4]. In the healing stage, problems such as poor blood flow interfere with the inflammation and granulation processes, delaying healing. Diabetes aggravates tissue hypoxia by inhibiting microcirculation in wounded areas and decreasing capillary permeability as a result of reduced red cell deformability [1-4]. Increased susceptibility to infections is generally observed in diabetic patients, and infections in ulcerated areas aggravate tissue necrosis, which can lead to amputation and sepsis [5]. Good control of blood glucose levels results in the control of infections and improvement of blood circulation, and it is therefore the most important element in the general therapeutic policy for diabetic ulcers [1-4]. Locally, it is important to keep the affected areas clean and moist to promote wound closure and to promote granulation using skin ulcer treatment agents with granulation promoting effects [1-4].

Fibroblast growth factors (FGF) have two types of protein with different isoelectric points on the basic and acidic sides: basic FGF (bFGF) and acidic FGF [6, 7]. Abraham JA et al. succeeded in cloning the cDNA of human bFGF for the first time in 1986, clarifying the entire DNA sequence of the human bFGF gene [8]. Human bFGF is a single-stranded polypeptide without any sugar chain. Its molecular weight is about 17 kDa, and it has a high affinity for heparin [8-12]. bFGF has been reported to expedite the wound healing process in rats by promoting neovascularization, granulation and epithelisation [13-15].

This trial employed a topical formulation with a spray nozzle obtained by reconstituting the lyophilized bFGF cake with saline (Code No. KCB-1; Kaken Pharmaceutical Co., Ltd., Tokyo, Japan). In this formulation, bFGF was produced by genetic recombination that introduced the gene for human bFGF into E. coli. It was administered through a spray nozzle. The systemic absorption is negligible when it is sprayed over wound areas. A 0.01% bFGF preparation (brand name: Fiblast^® Spray) has been commonly used to treat bedsores and skin ulcers in Japan since it obtained marketing approval in April 2001 [16], but it has not yet obtained specific approval for diabetic ulcers.

In the present trial, we compared the efficacy of two doses of bFGF (0.001% and 0.01%) with a placebo in patients with diabetic ulcers.

Materials and methods

Study design and patients

Patients were enrolled in the trial only if the pulsation of the dorsalis pedis artery or posterior tibial artery was palpable or if the ankle-brachial index at rest was not less than 0.9 in patients with no palpable pulsation of either artery (excluding those with severe calcification due to maintenance hemodialysis or diabetes) in order to select patients with good peripheral circulation. The following patients were excluded: patients with a malignant tumor or its history, those with a history of hypersensitivity to bFGF, women with confirmed or suspected pregnancy, nursing women, women who desired to become pregnant during the trial, and patients receiving oral administration or injection of adrenocortical steroid (equivalent to > 20 mg/day of prednisolone).

All patients were required to sign an informed consent form before enrollment. The institutional review board (IRB) of participating medical institutions approved the conduct of the present trial.

The dosage of bFGF of the present trial was set at a high dose (0.01%) and a low dose (0.001%), because the efficacy of bFGF showed a bell-shaped dose-response pattern with a peak at 0.01% in a dose-response trial conducted at 0.001%, 0.01%, 0.05%, and 0.1% in patients with various types of skin ulcers and also because a similar dose-response pattern was observed in non-clinical trials conducted using animal models [17]. The 0.01% and 0.001% bFGF preparations contained 500 μg (0.01%) and 50 μg (0.001%), respectively, of bFGF in 5 mL of a solvent (0.0005% benzalkonium chloride in saline). A placebo group was given the solvent mentioned above that was used as control. The trial drugs were indistinguishable from each other in appearance. Participants in the blinded trial included physicians, evaluators, patients, and monitors. The trial drugs were administered once a day, spraying 5 puffs, which is equivalent to 0 μg (placebo), 3 μg (0.001%), 30 μg (0.01%) of bFGF, 5 cm from the targeted ulcer for 8 weeks. Researchers instructed each patient in the method of drug application, and checked the compliance of medication at each visit.

All the patients continued the appropriate treatments to control blood glucose levels after the administration of the trial drug. The control levels were checked by measuring HbA1c at 8 weeks after the first administration of trial drug or at the point of discontinuing the administration. Targeted ulcers were irrigated with saline before administering the trial drugs and covered with the same type of silicone gauze at all participating institutions in order to protect new granulations after treatment. It was permitted to remove callus and crusts on a regular basis as a debridement, but extensive operative excision of the wound margin was prohibited. During the trial period, excessive loads on the targeted ulcers were appropriately avoided.

The number of patients in each of the three groups was balanced with respect to four factors (size and depth of ulcers, under dialysis or not, and control of blood glucose levels) stratified by a computer-generated randomization program. Patients were assigned to groups by telephone or fax at the KCB-1 Registration Center (ADJUST Co., Ltd., Hokkaido, Japan).

Efficacy evaluation

According to the operating procedure, three evaluators independently assessed the size and depth of ulcers and determined the grade of ulcer shrinkage (Grade 0: cure (complete epithelization); and Grade 1: marked reduction in the area or depth of ulcer (75% or greater reductions)), which were main outcome measures, by using the photographs of ulcer for each patient. The color and granulation formation of the ulcer were evaluated by the photographs for each patient.

The judgment was adopted when two of the three evaluators gave the same evaluation results. If the judgments of the three evaluators were perfectly different, the evaluation process was repeated until at least two evaluators gave the same results.

The area of ulcer (mm²) was calculated by multiplying the major axis (mm) with the minor axis (mm). The area and depth reduction rates (Ra and Rd) were defined as follows: Ra = (Area _{at baseline} – Area _{at time}) × 100/Area _{at baseline} and Rd = (Depth _{at baseline} – Depth _{at time}) × 100/Depth _{at baseline}.

The primary outcome was Grade 1 achievement in the area of ulcers during the 8-week observation period. The secondary outcome was cure (Grade 0 achievement) during the 8-week observation period.

Safety evaluation

Statistical analysis

The sample size was determined based on the Grade 1 achievement rate. In a 12-week preliminary trial conducted using 0.01% bFGF in patients with diabetic ulcers (data unpublished), it was observed that 5 of 8 patients achieved Grade 1 after 8 weeks’ administration. Assuming that the Grade 1 achievement rate in the placebo group and 0.01% bFGF group were 30% and 60%, respectively, the planned sample size of 50 patients on each group would provide a power of 80% to detect a percent difference of 30% at a significant level of 0.05. If, however, the percent difference in the Grade 1 achievement rate was 25%, the power would decrease to 64%. We set the minimum number of patients which could have been shown the statistical significant difference because this study was positioned as an exploratory study.

Fisher’s exact test or Kruskal-Wallis test was employed to compare baseline characteristics among the study groups (using The FREQ Procedures or The NPAR1WAY Procedures) Dose-response patterns for the Grade 1 achievement rate and the cure rate in the placebo, 0.001% bFGF and 0.01% bFGF groups were selected from “linear change” and “no difference between the bFGF groups”, and analyzed. Fisher’s exact test and Cochran-Armitage trend test were employed for these analyses and p values were adjusted for multiple comparisons (using The MULTITEST Procedures with Sidak adjustment). Non-evaluable patients were included in analyses as “Not achieved” and “Not cured”.

The evaluation of efficacy was analyzed based on the “per protocol” set, which included the patients who complied with the protocol in this trial.

Results

The Grade 1 achievement rate (75% or greater reductions) in the area of ulcers determined (primary outcome) was 57.5% (27/47 patients) in the placebo group, 72.3% (34/47) in the 0.001% bFGF group, and 82.2% (37/45) in the 0.01% bFGF group. A significant difference was noted between the 0.01% bFGF group and the placebo group (p = 0.025; figure 2). Dose-dependent, linear increases were noted (p = 0.009; figure 2). The cure rate (secondary outcome) was 46.8% (22/47) in the placebo group, 57.4% (27/47) in the 0.001% bFGF group, and 66.7% (30/45) in the 0.01% bFGF group. Although no significant differences between groups were observed (figure 3), a dose-response relationship similar to that observed with the Grade 1 achievement rate in the area of ulcers was noted. The Grade 1 achievement rate (75% or greater reductions) in the depth of ulcers determined was 55.3% (26/47 patients) in the placebo group, 61.7% (29/47) in the 0.001% bFGF group, and 71.1% (32/45) in the 0.01% bFGF group (figure 4). And also, no significant differences were observed in the Grade 1 achievement rate in the depth of ulcers in each group, but a linear dose-response relationship similar to the Grade 1 achievement rate in the area of ulcers was observed. The ulcer area reduction rates determined at the stage of final observation are shown by Box-and-Whisker plots (figure 5). Though no statistically significant difference was observed, the 0.01% bFGF group had a greater rate of ulcer area reduction at the final observation compared to the placebo group. The color and appearance of granulation suggested the formation of better quality granulation in the 0.01% bFGF group compared to the placebo group (data not shown).

Adverse events were observed in 3 patients in the placebo group (5.9%; 3 events; wound infection in 1 and pain at the administration site in 2), 1 patient in the 0.001% bFGF group (2.1%; 2 events; increase in ALP and LDH), and 3 patients in the 0.01% bFGF group (6.1%; 3 events; wound infection in 1, increase in the exudate in 1, and pain upon spraying in 1). None of these adverse events was serious or severe. The incidences were not associated with the study groups. The treatment was terminated by only 2 placebo-treated patients due to pain at the administration site.
Table 1 Characteristics of the patients

Discussion

A high Grade 1 achievement rate and cure rate of 59.3% and 46.8%, respectively, were obtained in the placebo group, because standardized care was fully provided to treat diabetic ulcers and the present trial targeted only small ulcers measuring 900 mm² or less. However, the Grade 1 achievement rate showed dose-dependent, linear increases, showing significant differences between the placebo and 0.01% bFGF groups. Consequently, the healing process for diabetic ulcers, which consists of inflammation, granulation, and remodeling, is similar to that for other types of skin ulcers, and the recommended clinical dose of bFGF should not be different for diabetic ulcers and other types of skin ulcer.

The percent difference in the Grade 1 achievement rate between the placebo group and 0.01% bFGF group was 24.7% in this 8-week treatment trial, and failed to reach the target of 30%. Coupled with the percent difference of 19.9% in the cure rate between the placebo group and 0.01% bFGF group, however, this difference of 24.7% achieved in the 8-week treatment period was thought to indicate a clinically significant difference between the two groups. In the present trial, Grade 1 was not achieved in 20 of the 47 patients in the placebo group, 13 of the 47 patients in 0.001% bFGF group, and 8 of the 45 in 0.01% bFGF group. This finding suggests that the conservative treatment period that can be recommended with bFGF is no longer than 8 weeks. It will be necessary to further study the reasons for the insufficient responses to bFGF in some patients, including the relationship with the pathological features of diabetes.

The results of stratified analyses conducted with respect to the presence or absence of dialysis and control of blood glucose levels showed that neither factor affected the efficacy of bFGF, with the drug being significantly more ffective in the 0.01% bFGF group than in the placebo group.

Cell growth factor drugs applicable to treat diabetic ulcers at present include bFGF preparations similar to KCB-1 in China, PDGF preparations (gel) in the US, Europe, Canada, and South Korea, and EGF preparations (solution) in South Korea. The EGF preparations are applicable as orphan drugs. Four clinical trials were conducted to evaluate the efficacy of the PDGF preparations for diabetic ulcers, and a higher cure rate (primary outcome) was obtained in the PDGF group (50%) than in the placebo group (35%) after treatment for 20 weeks [22]. Compared to these findings, our findings indicate that the efficacy of bFGF is comparable to that of PDGF although these findings and ours cannot be appropriately compared because of the differences in the formulations used, patient selection criteria, and study designs such as the treatment period.

All adverse events observed in the present trial have already been reported. None was serious and all showed a recovery with or without an appropriate remedial action. There was no difference among the study groups in the incidence of adverse events, indicating that bFGF is safe for use in treating diabetic ulcers.

In the present trial, it was suggested that bFGF promotes granulation and epithelization and possibly shortens the time needed for healing in patients with diabetic ulcers, as well as in those with other types of skin ulcers. bFGF was therefore thought to provide benefits to patients with diabetic ulcers.

Acknowledgements

Financial support: This study was supported by Kaken Pharmaceutical Co., Ltd., which proposed the study protocol and was responsible for data collection and pre-specified statistical analysis. Preparation of the manuscript was consigned to the authors and the views expressed in this article do not necessarily reflect those of Kaken Pharmaceutical Co., Ltd. Conflict of interest: none.

Participated medical institutions and investigators

The following medical institutions and investigators participated in this trial:

Asahikawa Medical College Hospital; Azuma N, Asahikawa Red Cross Hospital; Abe K, Sapporo City General Hospital; Yoshida T, Hokkaido University Hospital; Akiyama M, Yamamoto Y, Nishi Sapporo National Hospital; Kato M, Manda Memorial Hospital; Nakayama H, Hokkaido Junkanki Hospital; Sakata J, Obihiro Kosei general Hospital; Kuwahara H, Nikko Memorial Hospital; Sakamoto D, Hakodate Central General Hospital; Kimura C, Akita University Hospital; Wako M, Tohoku University Hospital; Aiba S, Okuguchi Clinic of internal medicine; Okuguchi F, JR Sendai Hospital; Ichiki M, The Institute for Adult Diseases, Asahi Life Foundation; Ohnishi Y, Tokyo Teishin Hospital; Etoh T, Tokyo Metropolitan Police Hospital; Iozumi K, Tokyo Saiseikai Central Hospital; Atsumi Y, Tokyo Medical And Dental University Hospital Faculty of Medicine; Uchimura I, The University of Tokyo Hospital; Kikuchi K, The Fraternity Memorial Hospital; Kaneko T, Kanto Medical Center NTT EC, Idetsuki T, Japanese Red Cross Medical Center; Imakado S, JR Tokyo General Hospital; Kurose N, Kanto Central Hospital; Hino H, International Medical Center of Japan; Tamaki T, Tokyo Kosei-Nenkin Hospital; Nankou H, Diabetes Center Tokyo Women’s Medical University; Iwamoto Y, Kosei Hospital; Ogata F, Social Insurance Central General Hospital; Torii H, Labor Welfare Corporation Kanto Rosai Hospital; Adachi M, Toshiba Hospital; Hattori N, National Hospital Organization Nagoya Medical Center; Nitta Y, Japanese Red Cross Nagoya First Hospital; Hayashi Y, Anjo Kosei Hospital Kamata S, National Hospital Organization Kyoto Medical Center; Kohno S, Osaka City University Hospital; Ishii M, Osaka Kosei Nenkin Hospital; Okada N, Higashiosaka City General Hospital; Saruban H, Hoshigaoka Kosei Nenkin Hospital; Kato H, Yao Municipal Hospital; Takagi K, Ishikiriseiki Hospital; Kanetou K, Kobe University Hospital; Tahara S, Okayama University Hospital; Oono T, Hiroshima Red Cross and Atomic-bomb Survivors Hospital; Mashino T, Hiroshima City Asa Hospital; Niimi N, Matsue Red Cross Hospital; Kaji S, Tokushima University Hospital; Yamano M, Ehime university Hospital; Hashimoto K, Kitakyusyu Municipal Medical Center; Ishii H, Fukuoka City Hospital; Hashimoto T, Kyushu University Hospital; Moroi Y, Iwase M, Inoguchi T, Fukuoka University Hospital; Tamura K, Fukuoka Red Cross Hospital; Tsutsu N, Haradoi Hospital; Ikematsu H, Social Insurance Inatsuki Hospital; Tsujita J, Saga Medical School Hospital; Narisawa Y, Nagasaki University Hospital of Medicine and Dentistry; Sato S, Kumamoto University Hospital; Ishihara T, Jinnouchi Clinic Diabetes center; Jinnouchi H, Jinikai Hospital; Abe R, Urasoe General Hospital; Yoshida T, Tokuyama Clinic; Tokuyama K

References

2 Singer AJ, Clark RA. Cutaneous wound healing. N Engl J Med 1999; 341: 738-46.

3 Loots MA, Lamme EN, Zeegelaar J, Mekkes JR, Bos JD, Middelkoop E. Differences in cellular infiltrate and extracellular matrix of chronic diabetic and venous ulcers versus acute wounds. J Invest Dermatol 1998; 111: 850-7.

4 Dinh TL, Veves A. A review of the mechanisms implicated in the pathogenesis of the diabetic foot. Int J Low Extrem Wounds 2005; 4: 154-9.

5 Wieman TJ, Griffiths GD, Polk Jr HC, et al. Management of diabetic midfoot ulcers. Ann Surg 1992; 215: 627-32.

6 Bohlen P, Baird A, Esch F, Ling N, Gospodarowicz D. Isolation and partial molecular characterization of pituitary fibroblast growth factor. Proc Natl Acad Sci USA 1984; 81: 5364-8.

7 Thomas K, Rios-Candelore M, Fitzpatrick S. Purification and characterization of acidic fibroblast growth factor from bovine brain. Proc Natl Acad Sci USA 1984; 81: 357-61.

8 Abraham JA, Whang JL, Tumolo A, et al. Human basic fibroblast growth factor: nucleotide sequence and genomic organization. EMBO J 1986; 5: 2523-8.

9 Baird A, Bohlen P. Fibroblast growth factors. In: Sporn MB, Roberts AB, eds. Peptide Growth Factors and Their Receptors I. Heidelberg: Springer-Verlag, 1991: 369-418.

10 Ueno N, Baird A, Esch F, Ling N, Guillemin R. Isolation of an amino terminal extended form of basic fibroblast growth factor. Biochem Biophys Res Commun 1986; 138: 580-8.

11 Story MT, Esch F, Shimasaki S, Sasse J, Jacobs SC, Lawson RK. Amino-terminal sequence of a large form of basic fibroblast growth factor isolated from human benign prostatic hyperplastic tissue. Biochem Biophys Res Commun 1987; 142: 702-9.

12 Baird A, Esch F, Mormede P, et al. Molecular characterization of fibroblast growth factor: distribution and biological activities in various tissues. Recent Prog Horm Res 1986; 42: 143-205.

13 Tsuboi R, Rifkin DB. Recombinant basic fibroblast growth factor stimulates wound healing in healing-impaired db/db mice. J Exp Med 1990; 172: 245-51.

14 Greenhalgh DG, Sprugel KH, Murray MJ, Ross R. PDGF and FGF stimulate wound healing in the genetically diabetic mouse. Am J Pathol 1990; 136: 1235-46.

15 Hayward P, Hokanson J, Heggers J, et al. Fibroblast growth factor reserves the bacterial retardation of wound contraction. Am J Surg 1992; 163: 288-93.

16 Ishibashi Y, Soeda S, Ohura T, et al. Clinical effects of KCB-1, a solution of recombinant human basic fibroblast growth factor, on skin ulcers – A phase III study comparing with sugar and povidone iodine ointment. J Clin Ther Med (in Japanese) 1996; 12: 2159-87

17 Okumura M, Okuda T, Nakamura T, Yajima M. Acceleration of wound healing in diabetic mice by basic fibroblast growth factor. Biol Pharm Bull 1995; 19: 530-5.

18 Nevins M, Giannobile WV, McGuire MK, et al. Platelet-derived growth factor stimulates bone fill and rate of attachment level gain: results of a large multicenter randomized controlled trial. J Periodonto 2005; 76: 2205-15.

19 Pierce GF, Tarpley JE, Allman RM, et al. Tissue repair processes in healing chronic pressure ulcers treated with recombinant platelet-derived growth factor BB. Am J Pathol 1994; 145: 1399-410.

20 Mustoe TA, Cutler NR, Allman RM, et al. A phase II study to evaluate recombinant platelet-derived growth factor-BB in the treatment of stage 3 and 4 pressure ulcers. Arch Surg 1994; 129: 213-9.

21 Hong JP, Kim YW, Jung HD, Jung KI. The effect of various concentrations of human recombinant epidermal growth factor on split-thickness skin wounds. Int Wound J 2006; 3: 123-30.

22 Wieman TJ, Smiell JM, Su Y. Efficacy and safety of a topical gel formulation of recombinant human platelet-derived growth factor-BB (becaplermin) in patients with chronic neuropathic diabetic ulcers: A phase III randomized placebo-controlled double-blind study. Diabetes Care 1998; 21: 822-7.