Tattoo removal with ingenol mebutate

Introduction

Tattooing has increased in popularity in recent years among both women and men.^1,2 In 2002, a Harris poll showed a tattoo prevalence of 16%, and in 2006, a North American survey found that 24% of 18–50-year olds had tattoos.³ A survey of 16–64-year olds (n=8,656) in Australia found that 14.5% of respondents had a tattoo.¹ In the USA, ≈50,000 new tattoos are placed every year, and 24% of college students and 10% of adult males have been estimated to have tattoos.⁴ Unfortunately, up to 20%–50% of wearers regret obtaining their tattoos,^4,5 with one survey reporting that 28% of tattooed individuals regretted the decision within 1 month.⁶ However, the percentage of individuals with tattoos who undergo tattoo removal treatment is reported to be around 6%–8%.^5,7

Tattoo removal dates back to ancient Egyptian times, with ancient Greeks purporting to use inter alia, garlic mixed with cantharidin,⁴ an acantholytic vesicant (with a number of traditional dermatological uses) that can be obtained from beetles in the family Meloidae.⁸ Currently lasers, primarily Quality-switched lasers, are generally used for tattoo removal⁹ and can, for instance, achieve up to 95% clearance of black and blue tattoos.^10,11 Laser treatment is believed to result in both chemical cleavage of pigment and/or fragmentation the pigment particles,² with the fragments phagocytosed by the posttreatment inflammatory infiltrate. Subsequent local redistribution of fragments, and perhaps removal via the lymphatics, is believed to result in the tattoo becoming clinically inapparent.^10,11 Laser treatments are typically spaced by 1–2 months, with the overall treatment course often prolonged and costly, requiring from 4 to more than 10 treatment sessions.² Tattoo removal can also be incomplete, with tattoos containing a mixture of different colors presenting a particular challenge.^2,10,11 However, picosecond laser technology continues to evolve, with new wavelength lasers improving the removal of difficult-to-remove colors.^10,11

Ingenol mebutate is a topical therapeutic agent derived from the sap of Euphorbia peplus^12,13 and is licensed for use in field-directed treatment of actinic keratosis, with treatment involving a daily application of 0.05% ingenol mebutate for 2 or 3 days.^14,15 The treatment, by removing both actinic keratosis and surrounding mutated keratinocytes, seeks to reduce future risk of squamous cell carcinoma development.^16,17 The drug (originally called PEP005) activates protein kinase C,¹⁸ and the treatment is associated with a transient inflammatory response,¹⁹ with a generally favorable cosmetic outcome in human studies.^16,20,21 Studies in mice have shown that topical ingenol mebutate treatment results in 1) the loss of the epithelial layer within 1–2 days, with keratinocytes undergoing primary necrosis¹³ (or possibly pyroptosis²²), 2) dermal hemorrhage,^22,23 and 3) an acute inflammatory response¹⁷ involving a pronounced neutrophil infiltrate within 24 hours.^13,24 This is followed by eschar formation and reepithelialization, which begins within 48 hours after treatment initiation.^17,22 Here, we show that ingenol mebutate treatment of skin tattoos in SKH/hr hairless mice resulted in their complete removal. The dye-containing tattoo microspheres used herein were followed by histology and could clearly be seen to have relocated from the dermis into the posttreatment eschar.

Methods

Animal ethics statement

Mouse work was conducted in accordance with the “Australian code for the care and use of animals for scientific purposes” as defined by the National Health and Medical Research Council of Australia. The mouse work was approved by the QIMR Berghofer Medical Research Institute’s animal ethics committee (P891). Mice were euthanized using carbon dioxide.

SKH1/hr mice

Outbred SKH1/hr mice were obtained from Charles River Laboratories (Wilmington, NC, USA), and a breeding colony for outbred SKH1/hr mice was established at QIMR Berghofer Medical Research Institute.

Tattoo application and ingenol mebutate treatment

Mice were tattooed on their backs with one ≈1 cm × ≈1 cm cross using human-grade blue tattoo ink composed of a fluorescent dye encapsulated within polymethylmethacrylate microspheres (FireFly BMX1000, Marine Blue; Protat Tattoo Supplies, Adelaide, SA, Australia). The tattoos were applied using a Harvard Apparatus tattooing machine (Harvard Apparatus Ltd., Holliston, MA, USA). Two weeks later, the mice were randomly allocated into two groups, one received ingenol mebutate (0.1% in gel) and the other gel alone (placebo; both supplied by Peplin Ltd., Brisbane, QLD, Australia).¹⁷ Treatments were applied topically (50 µL) to the tattoo, daily for 2 days. The gels were spread to ensure that the gel covered the entire tattoo. The gel contains isopropyl alcohol, hydroxyethylcellulose, citric acid monohydrate, sodium citrate, benzyl alcohol, and purified water.

Histology

Skin samples at the tattoo site were cut out with a scalpel and fixed in 10% formalin and processed for paraffin embedding at the indicated times after ingenol mebutate treatment. Paraffin sections were stained with hematoxylin and eosin and were viewed with a Nikon Eclipse E800 fluorescent microscope and images taken with a Nikon DXM 1200F digital camera attachment (Nikon, Sydney, NSW, Australia). Slides were illuminated with normal bright light and UV light (DAPI setting).

Results

Tattoo removal with 0.1% ingenol mebutate

Tattooed crosses were placed on the backs of hairless SKH1/hr mice, and after 2 weeks, the tattooed areas (Figure 1A and B, Day 0) were treated once per day for 2 days with placebo gel or 0.1% ingenol mebutate gel. Two days after ingenol mebutate treatment initiation, an eschar began to form at the treatment site, which was fully formed by Day 8 (Figure 1A). Around Day 20, the eschar dropped off and no tattoo remained visible in the skin, with the site continuing to heal over the following weeks (Figure 1A). Placebo-treated tattoos showed no significant change over this period (Figure 1A). A cohort of five mice is shown to illustrate the consistency of the results obtained, with tattoos shown before treatment (Day 0) and 220 days after treatment (Figure 1B). Placebo treatment again had no significant effect (Figure 1B). Ingenol mebutate gel at 0.05% (a concentration used in humans¹⁶) was also partially effective at tattoo removal in SKH/hr mice (data not shown).

Figure 1 Tattoo removal with ingenol mebutate. Notes: (A) Photographs of two tattoos (≈1 cm × ≈1 cm blue crosses), one treated with placebo and the other with ingenol mebutate, and both followed over time (till Day 220). A fully formed eschar can be seen on Day 8 over the ingenol mebutate-treated tattoo site. (B) Pictures of five tattoos on five mice treated with placebo gel and five tattoos on another five mice treated with ingenol mebutate. Pictures were taken before treatment (Day 0) and 220 days after treatment.

The topical ingenol mebutate treatment used herein for tattoo removal showed similar changes in the skin of SKH1/hr mice to those reported previously when ingenol mebutate was used for field-directed treatment of skin lesions.^17,25 Inflammation (erythema) was evident within a day, and eschar formation began on Day 2, with the skin recovering around Day 20 (Figure 1A). Ingenol mebutate treatment (as reported previously in these mice¹⁷) resulted in some skin contraction (suggesting dermal disruption), with slightly darker pink irregular markings remaining within the treatment areas (Figure 1A and B, Day 220). Such changes are not seen in humans after ingenol mebutate treatment.^16,20,21

Histological examination of ingenol mebutate-treated tattoos

For the tattoos, a microsphere-based product was used, which contained a fluorescent dye that fluoresces under UV light. The light-blue colored tattoo microspheres could thus be clearly seen in hematoxylin and eosin sections illuminated with both white and UV light. The microspheres were clearly visible in the dermis prior to treatment (Figure 2A, dermis). After ingenol mebutate treatment, hemorrhage, mononuclear cell infiltrates (shown previously to be neutrophils^13,24), and loss of epidermis were apparent within 1–2 days, as described previously in these mice.¹⁷ Importantly, by Day 4, the microspheres could be clearly seen located within the purulent exudate forming an eschar over the dermis (Figure 2B, eschar). A resolving mononuclear cell infiltrate could also be seen in the dermis (Figure 2B, N). By Day 7, the epithelium had fully reformed (slightly thickened²⁶) and the eschar had darkened and crusted, with tattoo microspheres clearly located within the eschar (Figure 2C, eschar).

Figure 2 Histology of tattoo sites before and after ingenol mebutate treatment. Notes: Histology of tattoo sites stained with H&E and viewed under UV and normal light. The tattoo microspheres contain fluorescent dye, and can thus be clearly seen as bright, light-blue spheres. (A) Cross-section of skin showing tattoo microspheres in the dermis, 2 weeks after tattoo application. The dark purple staining layer above the dermis is the epidermis. (B) Day 4 postinitiation of ingenol mebutate treatment showing loss of epidermis and the presence of tattoo microspheres in the forming eschar. A resolving mononuclear cell infiltrate (N) is also apparent in the dermis. (C) Day 7 postinitiation of ingenol mebutate treatment showing a reestablished epidermis and tattoo microspheres predominantly located in the mature and crusting eschar. Abbreviations: H&E, hematoxylin and eosin; UV, ultraviolet.

Discussion

Here, we show that 0.1% ingenol mebutate gel was able to remove efficiently and consistently 2-week-old tattoos from SKH/hr hairless mice. The mechanism of tattoo removal by ingenol mebutate appears to be quite distinct from that seen after laser treatment. The tattoo dye-containing microspheres remained intact and were exuded out of the dermis into an eschar, which then dropped off as the skin healed. This mechanism of action would suggest that tattoo removal facilitated by ingenol mebutate is likely to be independent of the tattoo ink color.

Ingenol mebutate treatment induces a complex series of changes in the skin, including epithelial and dermal disruption, hemorrhage, inflammation, and neutrophil infiltration, followed by eschar formation and ultimately some cicatrix formation (Figures 1 and 2).¹⁷ Imiquimod, which like ingenol mebutate is used for treating actinic keratoses, was also reported similarly to be able to remove tattoos in a guinea pig model, with evident epidermal and dermal necrosis, hemorrhage, inflammation, neutrophil infiltration, and eschar formation.²⁷ Dermabrasion (to remove the epidermis) has also been used for tattoo removal, with tattoo pigment mobilized and extruded from the inflamed skin into a dressing covering the treatment site.^28,29 Increased tattoo pigment phagocytosis has also been proposed as a mechanism for imiquimod and dermabrasion.^29,30 However, ingenol mebutate activates PKC,¹⁸ which is reported to suppress phagocytic activity of neutrophils.^31,32 One might therefore speculate that the ingenol mebutate treatment-induced inflammatory response mobilized the tattoo microspheres via edema formation and degradation of the extracellular matrix,³³ with the microspheres (≈30 µm diameter) likely becoming encapsidated by connective tissue.³⁴ Purulent exudation, driven by tissue pressure (≈10 mmHg), may then carry the microspheres out of the skin into an eschar. A weakness of the current study is that no time series was undertaken; the efficacy of ingenol mebutate for removing older tattoos (potentially with more mature encapsidation³⁴) remains to be established.

Imiquimod has been reported to facilitate tattoo removal in conjunction with Quality-switched laser treatment in a guinea pig model.³⁵ Human studies have shown inconsistent results, with a study in 3 patients suggesting that imiquimod facilitated tattoo removal in conjunction with laser treatment,³⁰ whereas another study in 19 patients reported that imiquimod was ineffective in this setting.³⁶ Whether ingenol mebutate could synergize with laser treatment to facilitate tattoo removal in humans without cicatrix formation clearly remains to be tested, given that there are substantial differences between mouse and human skin.³⁷

Acknowledgments

S-J Cozzi was funded by an Industry Fellowship from the National Health and Medical Research Council (NHMRC), Australia. A Suhrbier is a Principal Research Fellow of the NHMRC. The research was conducted at QIMR Berghofer with consumables and research staff funded by Peplin Ltd. We thank the histopathology and animal house staff of QIMRB for excellent support.

Disclosure

A Suhrbier was a paid consultant for Peplin Ltd. and LEO Pharma. S-J Cozzi was an employee of LEO Pharma. SM Ogbourne was an employee of Peplin Ltd. A Suhrbier and S-J Cozzi are named inventors on a patent (WO/2012/176015) held by LEO Pharma, but receive no royalties or other remuneration. The authors report no other conflicts of interest in this work.

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