Interleukin-10 reduces scar formation in both animal and human cutaneous wounds: results of two preclinical and phase II randomized control studies.

Cutaneous scarring affects up to 100 million people per annum. There is no effective scar reducing/preventing therapeutic developed to date. Interleukin (IL)-10 is an anti-inflammatory and antifibrotic cytokine. In the embryo it is important for scarless wound repair. We investigated the effect on wound healing and scarring of a double deletion of the IL-10 and IL-4 genes in a knockout (KO) mouse model, and also the effect of exogenous addition of recombinant human (rh) IL-10 into rat and human cutaneous incisions. Mouse study: Two incisions were made on the dorsal skin of 20 double IL-4/IL-10 KO mice and 20 wild-type (WT) controls. Rat study: Three concentrations of rhIL-10 were investigated. Four incisions were made on the dorsal skin of 30 rats. Each rat received two concentrations. Each incision receiving a concentration of rhIL-10 was matched with a control incision, which received either placebo or standard care. Human study: Eight concentrations of rhIL-10 were investigated. Four incisions were made on each arm of 175 healthy volunteers. Four incisions received four different concentrations, which were matched with four control incisions that received either standard care or placebo. KO mice healed with poor scar histology and increased inflammation. rhIL-10-treated rat incisions healed with decreased inflammation, better scar histology, and better macroscopic scar appearance. rhIL-10-treated human incisions at low concentrations healed with better macroscopic scar appearance and less red scars. IL-10 is an important cytokine in wound healing and its suppression of inflammation and scarring is demonstrated in mice and rats with a translational effect in humans.

Discovery and development of avotermin (recombinant human transforming growth factor beta 3): a new class of prophylactic therapeutic for the improvement of scarring.

Scarring in the skin following surgery or trauma may be associated with adverse aesthetic, functional, growth and psychological effects, such that both physicians and patients regard it as important to minimize the appearance of scars. The prophylactic improvement of cutaneous scar appearance represents a significant opportunity to improve the well-being of patients. Human recombinant transforming growth factor beta 3 (avotermin) is the first in a new class of therapeutic agents to address this medical need. Herein we describe scar-free healing in early embryonic development, including the identification of the cellular and molecular mechanisms underpinning the scarring process. This understanding has led to the discovery of novel therapeutics such as transforming growth factor beta 3, which can be administered to improve scar appearance in human subjects through pharmacological action. We discuss the pioneering development of transforming growth factor beta 3 in this new therapeutic area showing how it has been possible to translate preclinical concepts into clinical application, namely the improvement of scar appearance following surgery.

Prophylactic administration of avotermin for improvement of skin scarring: three double-blind, placebo-controlled, phase I/II studies.

BACKGROUND: Research into mechanisms of skin scarring identified transforming growth factor beta3 (TGFbeta3) as a potential antiscarring therapy. We assessed scar improvement with avotermin (recombinant, active, human TGFbeta3). METHODS: In three double-blind, placebo-controlled studies, intradermal avotermin (concentrations ranging from 0.25 to 500 ng/100 microL per linear cm wound margin) was administered to both margins of 1 cm, full-thickness skin incisions, before wounding and 24 h later, in healthy men and women. Treatments (avotermin and placebo or standard wound care) were randomly allocated to wound sites by a computer generated randomisation scheme, and within-participant controls compared avotermin versus placebo or standard wound care alone. Primary endpoints were visual assessment of scar formation at 6 months and 12 months after wounding in two studies, and from week 6 to month 7 after wounding in the third. Investigators, participants, and scar assessors were blinded to treatment. Efficacy analyses were intention to treat. These studies are registered with ClinicalTrials.gov, numbers NCT00847925, NCT00847795, and NCT00629811. RESULTS: In two studies, avotermin 50 ng/100 microL per linear cm significantly improved median score on a 100 mm visual analogue scale (VAS) by 5 mm (range -2 to 14; p=0.001) at month 6 and 8 mm (-29 to 18; p=0.0230) at month 12. In the third, avotermin significantly improved total scar scores at all concentrations versus placebo (mean improvement: from 14.84 mm [95 % CI 5.5-24.2] at 5 ng/100 microL per linear cm to 64.25 mm [49.4-79.1] at 500 ng/100 microL per linear cm). Nine [60%] scars treated with avotermin 50 ng/100 microL per linear cm showed 25% or less abnormal orientation of collagen fibres in the reticular dermis versus five [33%] placebo scars. After only 6 weeks from wounding, avotermin 500 ng/100 microL per linear cm improved VAS score by 16.12 mm (95% CI 10.61-21.63). Adverse events at wound sites were similar for avotermin and controls. Erythema and oedema were more frequent with avotermin than with placebo, but were transient and deemed to be consistent with normal wound healing. INTERPRETATION: Avotermin has potential to provide an accelerated and permanent improvement in scarring.

Distortion-dependent unhooking of interstrand cross-links in mammalian cell extracts.

Interstrand cross-links (ICLs) are formed by many chemotherapeutic agents and may also arise endogenously. The mechanisms used to repair these lesions remain unclear in mammalian cells. Repair in Escherichia coli and Saccharomyces cerevisiae requires an initial unhooking step to release the tethered DNA strands. We used a panel of linear substrates containing different site-specific ICLs to characterize how structure affects ICL processing in mammalian cell extracts. We demonstrate that ICL-induced distortions affect NER-dependent and -independent processing events. The NER-dependent pathway produces dual incisions 5' to the site of the ICL as described previously [Bessho, T., et al. (1997) Mol. Cell. Biol. 17 (12), 6822-6830] but does not release the cross-link. Surprisingly, we also found that the interstrand cross-linked duplexes were unhooked in mammalian cell extracts in a manner independent of the NER pathway. Unhooking occurred identically in extracts prepared from human and rodent cells and is dependent on ATP hydrolysis and metal ions. The structure of the unhooked product was characterized and was found to contain the remnant of the cross-link. Both the NER-mediated dual 5' incisions and unhooking reactions were greatly stimulated by ICL-induced distortions, including increased local flexibility and disruption of base pairs surrounding the site of the ICL. These results suggest that in DNA not undergoing transcription or replication, distortions induced by the presence of an ICL could contribute significantly to initial cross-link recognition and processing.

End modification of a linear DNA duplex enhances NER-mediated excision of an internal Pt(II)-lesion.

The study of DNA repair has been facilitated by the development of extract-based in vitro assay systems and the use of synthetic DNA duplexes that contain site-specific lesions as repair substrates. Unfortunately, exposed DNA termini can be a liability when working in crude cell extracts because they are targets for DNA end-modifying enzymes and binding sites for proteins that recognize DNA termini. In particular, the double-strand break repair protein Ku is an abundant DNA end-binding protein that has been shown to interfere with nucleotide excision repair (NER) in vitro. To facilitate the investigation of NER in whole-cell extracts, we explored ways of modifying the exposed ends of synthetic repair substrates to prevent Ku binding and improve in vitro NER efficiency. Replacement of six contiguous phosphodiester linkages at the 3'-ends of the duplex repair substrate with nuclease-resistant nonionic methylphosphonate linkages resulted in a 280-fold decrease in binding affinity between Ku and the modified duplex. These results are consistent with the published crystal structure of a Ku/DNA complex [Walker et al. (2001) Nature 412, 607-614] and show that the 3'-terminal phosphodiester linkages of linear DNA duplexes are important determinants in DNA end-binding by Ku. Using HeLa whole-cell extracts and a 149-base pair DNA duplex repair substrate, we tested the effects of modification of exposed DNA termini on NER-mediated in vitro excision of a 1,3-GTG-Pt(II) intrastrand cross-link. Methylphosphonate modification at the 3'-ends of the repair substrate resulted in a 1.6-fold increase in excision. Derivatization of the 5'-ends of the duplex with biotin and subsequent conjugation with streptavidin to block Ku binding resulted in a 2.3-fold increase excision. By combining these modifications, we were able to effectively reduce Ku-derived interference of NER excision in vitro and observed a 4.4-fold increase in platinum lesion excision. These modifications are easy to incorporate into synthetic oligonucleotides and may find general utility whenever synthetic linear duplex DNAs are used as substrates to investigate DNA repair in whole-cell extracts.

Synthesis, cytotoxicity, DNA interaction and cell cycle studies of trans-diiodophosphine Pt(II) complexes.

Platinum complexes, bearing aliphatic amines and phosphine ligands in trans configuration with iodide as leaving groups, are synthesized and characterized. The crystal structure of trans-PtI2(isopropylamine)(PPh3) is reported. The complex bearing isopropylamine is demonstrated to be the best candidate as its cytotoxic activity is comparable to or better than cisplatin. A remarkably higher interaction of the complexes with DNA is reported as compared to the parent chlorido series. Cell cycle studies of the complexes in six human cell lines are performed and also compared with the previous series.

Avotermin for scar improvement following scar revision surgery: a randomized, double-blind, within-patient, placebo-controlled, phase II clinical trial.

BACKGROUND: Skin scarring is associated with psychosocial distress and has a negative effect on quality of life. The transforming growth factor (TGF)-ß family of cytokines plays a key role in scarring. TGF-ß3 improves scar appearance in a range of mammalian species. This study was performed to assess the efficacy of intradermal avotermin (TGF-ß3) for the improvement of scar appearance following scar revision surgery. METHODS: Sixty patients (35 men and 25 women; age, 19 to 78 years; 53 Caucasians; scar length, 5 to 21 cm) received intradermal avotermin (200 ng/100 µl/linear cm wound margin) and placebo to outer wound segments immediately after, and again 24 hours after, complete (group 1) or staged (group 2) scar revision surgery. A within-patient design was chosen to control for interindividual factors that affect scarring. The primary efficacy variable was a total scar score derived from a visual analogue scale, scored by a lay panel from standardized photographs from months 1 through 7 following treatment. RESULTS: : Primary endpoint data from the combined surgical groups showed that avotermin significantly improved scar appearance compared with placebo (total scar score difference, 21.93 mm; p = 0.04). Profilometry showed a greater reduction in scar surface area from baseline with avotermin treatment compared with placebo, significant in group 2 at months 7 and 12 (difference, 41.99 mm and 25.85 mm, respectively; p = 0.03 for both comparisons). Histologic analysis from group 2 showed that, compared with placebo treatment, collagen organization in avotermin-treated scars more closely resembled normal skin in 14 of 19 cases. Avotermin was well tolerated. CONCLUSION: Avotermin administration following scar revision surgery is well tolerated and significantly improves scar appearance compared with placebo. CLINICAL QUESTION/LEVEL OF EVIDENCE: Therapeutic, I.(Figure is included in full-text article.).

Therapies with emerging evidence of efficacy: avotermin for the improvement of scarring.

Many patients are dissatisfied with scars on both visible and non-visible body sites and would value any opportunity to improve or minimise scarring following surgery. Approximately 44 million procedures in the US and 42 million procedures in the EU per annum could benefit from scar reduction therapy. A wide range of non-invasive and invasive techniques have been used in an attempt to improve scarring although robust, prospective clinical trials to support the efficacy of these therapies are lacking. Differences in wound healing and scar outcome between early fetal and adult wounds led to interest in the role of the TGFbeta family of cytokines in scar formation and the identification of TGFbeta3 (avotermin) as a potential therapeutic agent for the improvement of scar appearance. Extensive pre-clinical and human Phase I and II clinical trial programmes have confirmed the scar improving efficacy of avotermin which produces macroscopic and histological improvements in scar architecture, with improved restitution of the epidermis and an organisation of dermal extracellular matrix that more closely resembles normal skin. Avotermin is safe and well tolerated and is currently in Phase III of clinical development, with the first study, in patients undergoing scar revision surgery, fully recruited.

Scar-improving efficacy of avotermin administered into the wound margins of skin incisions as evaluated by a randomized, double-blind, placebo-controlled, phase II clinical trial.

BACKGROUND: The authors report on a prospective, randomized, placebo-controlled phase II trial to investigate avotermin (transforming growth factor beta-3) for reducing scarring resulting from acute incised skin wounds. METHODS: Seventy-one healthy male subjects (18 to 45 years) received avotermin at 50 or 200 ng/100 µl/linear centimeter of wound margin. Subjects received three standardized 1-cm incisional wounds on the inner aspect of each upper arm. Wounds were randomized to receive (into each margin): no injection (standard wound care only), one intradermal injection of avotermin or placebo (immediately before surgery), or two injections of avotermin or placebo (immediately before surgery and 24 hours later). The primary efficacy variable was a 10-cm visual analog scale score, which assessed how closely scars resembled normal skin, administered at month 12 by an independent external scar assessment panel (a panel of lay public individuals). RESULTS: Avotermin at 200 ng/100 µl/linear centimeter, administered once or twice, achieved significant improvements in scar appearance compared with controls (p<0.02 for all comparisons). The 50-ng dose, administered twice, achieved significant improvements in scar appearance versus placebo (p=0.043). Treatment was well tolerated. CONCLUSION: These results confirm that avotermin is the first of a new class of regenerative medicines that reduce scarring when administered once or twice to the approximated margins of acute skin incisions.

Skin tension or skin compression? Small circular wounds are likely to shrink, not gape.

The final appearance of a scar may be influenced by tension or mechanical factors [Borges AF. Scar prognosis of wounds. Br J Plast Surg 1960;13:47-54; Arem AJ, Madden JW. Effects of stress on healing wounds. J Surg Res 1976;20:93-102; Burgess LP, Morin GV, Rand M, et al. Wound healing. Relationship of wound closing tension to scar width in rats. Arch Otolaryngol Head Neck Surg 1990;116:798-802; Meyer M, McGrouther DA. A study relating wound tension to scar morphology in the pre-sternal scar using Langer's technique. Br J Plast Surg 1991;44:291-4] Karl Langer suggested that information could be gained about the tension inherent in skin, in all directions, by observing the wound edge retraction that occurred after making circular skin incisions [Langer K. On the anatomy and physiology of the skin II. Skin tension. Br J Plast Surg 1978;31:93-106]. Circular wounds may be used to demonstrate the orientation of the dominant axis of 'tension' in the skin but is this always a tensile stress as opposed to a compressive stress? This is the second article in a series documenting the mechanical properties of circular punch biopsy wounds. The aim of this study was to make detailed observations of the dimensional distortions of circular wounds on the face and neck, from which deductions could be made with regard to mechanical stress. One hundred and seventy-five benign head and neck lesions were excised from 72 volunteers using circular dermal punch biopsies. The distortions of the resulting wounds were observed to be elliptical in most cases. Measurements were taken of the maximum and minimum diameters of the wound and expressed as ratios of the size of the punch biopsy used for excision. The change in area from the area of the punch biopsy to that of the wound was also calculated. The maximum diameter of the wound was smaller than the diameter of the punch biopsy in 40.6% of cases, the minimum diameter of the wound was smaller in 97.7% of cases and the area of the wound was smaller than that of the punch biopsy in 90.3%. These dimensional changes varied between sites (P=0.0005, P=0.0001 and P<0.0001, respectively). We conclude that the reported rhomboidal or lattice structure [Ridge MD, Wright V. The directional effects of skin. A bioengineering study of skin with particular reference to Langer's Lines. J Invest Dermatol 1966;46:341-6] of skin has individual components which are under tensional force due to elastic retraction. Wounds smaller than the rhomboidal unit will reduce in area, due to the intact tensional forces in the individual dermal components, giving an appearance of the skin overall being under compression. Larger wounds, disrupting more of the lattice structure, will gape.

Scar redness in humans: how long does it persist after incisional and excisional wounding?

BACKGROUND: The natural history of scar redness in humans has never been formally described, and the point at which normal scar redness fades is unknown. METHODS: As part of a randomized, placebo-controlled, clinical trial investigating the effects of various doses of transforming growth factor-beta3 on scar quality, the authors observed the process of scar redness and maturation in non-drug-treated incisional and excisional wounds made on the upper inner arms of 103 volunteers. Scar photographic images were assessed by a review panel to ascertain the month during which redness faded for a particular scar. Scar histology documented the level of inflammation and angiogenesis. RESULTS: Scar redness faded at an average of 7 months. Scar redness for incisions faded significantly faster than excisions (p = 0.0001, Kruskal-Wallis test), and significant differences were also seen between anteriorly and posteriorly placed scars for incisions (p = 0.0008) and excisions (p = 0.0035), respectively. Month 12 histologic examination revealed the absence of any ongoing inflammatory processes in all scars. CONCLUSIONS: Scar redness fades on average at 7 months. This is influenced by the wound type and position. The authors advocate the use of the term "rubor perseverans" to describe the physiologic redness of a normal scar as it matures beyond the first month, a process that does not involve inflammation.

Maturation of the human scar: an observational study.

BACKGROUND: The natural history of scar maturation in humans has never been formally described from either a clinical or a histologic standpoint. METHODS: The maturation of incisional scars was observed in 58 healthy male volunteers who each had 2 x 1-cm incisional wounds created on the inner aspect of both upper arms. The resulting scars were photographed digitally at monthly intervals for 12 months and excised for histologic analysis at specific time points. All histologic specimens were stained using Masson's trichrome and reviewed together with the corresponding digital clinical scar images to produce macroscopic and microscopic descriptions of the maturation process. RESULTS: Three distinct groups, each displaying a different rate of longitudinal progression of scar maturation, were identified from within the study group. The majority of volunteers belonged to a "representative" subset but distinct "poor" and "excellent" subsets were also identified. The poor subset invariably contained volunteers younger than 30 years of age, whereas the majority of the excellent subset comprised subjects older than 55 years of age. CONCLUSIONS: Scar maturation occurs as a series of defined macroscopic and microscopic stages over the course of 1 year. The rate of scar maturation varied within the study group, with older subjects (>55 years) displaying accelerated maturation, whereas a prolonged high turnover state and a retarded rate of maturation were observed in younger subjects (<30 years).

The dynamic rotation of Langer's lines on facial expression.

Karl Langer investigated directional variations in the mechanical and physical properties of skin [Gibson T. Editorial. Karl Langer (1819-1887) and his lines. Br J Plast Surg 1978;31:1-2]. He produced a series of diagrams depicting lines of cleavage in the skin [Langer K. On the anatomy and physiology of the skin I. The cleavability of the cutis. Br J Plast Surg 1978;31:3-8] and showed that the orientation of these lines coincided with the dominant axis of mechanical tension in the skin [Langer K. On the anatomy and physiology of the skin II. Skin tension. Br J Plast Surg 1978;31:93-106]. Previously these lines have been considered as a static feature. We set out to determine whether Langer's lines have a dynamic element and to define any rotation of the orientation of Langer's lines on the face with facial movement. One hundred and seventy-five naevi were excised from the face and neck of 72 volunteers using circular dermal punch biopsies. Prior to surgery a vertical line was marked on the skin through the centre of each naevus. After excision distortions of the resulting wounds were observed. The orientation of the long axis of each wound, in relation to the previously marked vertical line, was measured with a goniometer with the volunteer at rest and holding their face in five standardised facial expressions: mouth open, smiling, eyes tightly shut, frowning and eyebrows raised. The aim was to measure the orientation of the long axis of the wound with the face at rest and subsequent rotation of the wound with facial movement. After excision elliptical distortion was seen in 171 of the 175 wounds at rest. Twenty-nine wounds maintained the same orientation of distortion in all of the facial expressions. In the remaining wounds the long axis of the wound rotated by up to 90 degrees . The amount of rotation varied between sites (p>0.0001). We conclude that Langer's lines are not a static feature but are dynamic with rotation of up to 90 degrees . It is possible that this rotation in the axis of mechanical tension will affect the appearance of the resulting scar.

Visual analogue scale scoring and ranking: a suitable and sensitive method for assessing scar quality?

BACKGROUND: The field of scar assessment lacks a standard methodology. Previous methods have focused on a wide range of scar types, resulting in poorer sensitivity and diminishing their discriminatory effectiveness. METHODS: As part of a clinical trial investigating the scar-improving efficacy of transforming growth factor-beta3, the authors investigated the use of a visual analogue scale and scar ranking as scar assessment tools. Scar photographic images were assessed using a newly developed computerized scar assessment system by an external lay panel. RESULTS: A total of 4296 scar images were collected for visual analogue scale assessment and 2148 scar pairs were collected for scar ranking. Intrarater consistency was 100 percent for the ranking data, with differences very close to zero for the visual analogue scale consistency data. Reducing the number of assessors in the external panel significantly improved intraclass correlation coefficients. From month 1 to month 12, the correlation coefficients for the difference in visual analogue scale score showed that the assessors reliably noted the changes in the maturing scars. Combining logistic regression with an area under the curve of 0.72 in a receiver operating characteristic curve analysis, the visual analogue scale score was shown to be a highly statistically significant predictor of a good scar. CONCLUSIONS: The authors have shown the visual analogue scale scar scoring and scar ranking methods to be consistent, reliable, valid, and feasible. These methods for scar assessment are highly sensitive and capable of reliably measuring differences in scar quality, making them valuable techniques, reaching an unmet clinical need, and enabling investigation of changes in scar quality (e.g., with time or after therapeutic intervention).