¹MD/MBA/MPH Candidate at Yale University, New Haven, CT, USA
²Baylor College of Medicine, Houston, TX, USA
³Department of Dermatology and Skin Science, University of British Columbia, Vancouver, BC, Canada
⁴Pacific Dermaesthetics, Vancouver, BC, Canada

Conflict of interest:
Jason Rivers has been a consultant for Almirall; Harrison Nguyen has no conflicts to report.

Introduction

Actinic keratosis (AK), a common cutaneous lesion with the potential to transform into squamous cell carcinoma, has traditionally been treated with ablative and/or surgical procedures. Recently, a topical formulation combining 0.5% 5-fluorouracil with 10% salicylic acid (5-FU-SA) was introduced in Europe under the trade name Actikerall™ for the treatment of grade I/II AK. In a single randomized phase III trial, 5-FU-SA was shown to be superior to diclofenac 3% gel in hyaluronic acid, as measured by the histological clearance of one defined lesion (72% vs. 59.1%) and by complete clinical clearance (55.4% vs. 32.0%). 5-FU-SA should be applied once daily to a total area of up to 25 cm2, which may include the lesion(s) and a small area of surrounding skin (rim of healthy skin should not exceed 0.5 cm), for up to 12 weeks. The most commonly reported side effects are local inflammation and pruritus at the application site, and no serious adverse effects have been reported to date. Now commercially available in Canada, 5-FU-SA represents a patient-applied therapeutic option for the treatment of both overt and subclinical AK.

Background

• Actinic keratosis (AK) is a lesion considered to be on a continuum with squamous cell carcinoma (SCC).^1-6
• Invasive disease occurs in up to 10% of cases over time, which highlights the need for early recognition and adequate treatment of all AK, including subclinical lesions.⁷
• Although many AK never progress to SCC, their treatment has been recommended to preempt this eventuality.
• Treatment options can generally be stratified based on whether only discrete lesions are treated, or whether subclinical lesions are also targeted, which is referred to as field-directed therapy.
• Lesion-directed therapy has historically consisted of ablative and/or surgical procedures. However, several topical agents have emerged as attractive alternatives in the treatment of AK.
• Examples of topical agents available in Canada include 5-fluorouracil (5-FU), imiquimod (2.5%, 3.75%, and 5% formulations), diclofenac 3%, methylaminolevulinate/aminolevulinic acid (for photodynamic therapy), and ingenol mebutate.⁸
• In one controlled clinical trial, topical 5-FU applied to AK resulted in a 96% clearance after 4 weeks of twice daily application.9 However, high rates of severe localized tissue reactions with 5-FU have led to reduced patient compliance, and this, in part, may explain why the long-term clearance of AK in clinical practice is around 50%.⁸
• This problem has resulted in a search for therapeutic agents less likely to induce skin irritation.¹⁰
• In 2011, a topical formulation combining 0.5% 5-FU with 10% salicylic acid (5-FU-SA) was introduced to the European market under the trade name Actikerall™ for the topical treatment of grade 1/2 AK (slightly palpable and/or moderately thick hyperkeratotic lesions) in immunocompetent adult patients.^11,12 This preparation is not novel as the same agent has been used in Europe for more than 30 years in the treatment of plantar warts (Verrumal®).

In this brief review, we present some of the clinical data to support the use of 5-FU-SA in patient-directed* management of AK and we summarize the salient information that the provider should be aware of when prescribing this product.

*Health Canada has elected to classify Actikerall™ as neither lesion-directed nor field-directed. This was done to support the individual needs of patients; the locational distribution of a patient’s lesions will dictate whether a lesion-directed versus a field-directed approach is preferred.

Evidence from Clinical Trials

• The primary evidence used to support the efficacy of 5-FU-SA in the treatment of AK comes from a single randomized, multi-center, phase 3 trial.¹³ The study included 470 patients with histologically diagnosed AK on the face, forehead, or bald scalp. Subjects were randomly assigned to 5-FU-SA, diclofenac 3% gel in hyaluronic acid (diclofenac HA), or placebo (5-FU-SA vehicle). Treatment was continued until either complete resolution of the lesions was evident, or for a maximum of 12 weeks.
• Subjects were instructed to apply their assigned intervention directly to their lesions – once daily for the 5-FU-SA and vehicle groups and twice daily for the diclofenac group.
• The primary outcome – histological clearance of one defined lesion within 8 weeks of treatment cessation – was achieved in 72.0%, 59.1%, and 44.8% of patients treated with low-dose 5-FU-SA, diclofenac and placebo, respectively.
• In addition to the histological data, the rate of complete clinical clearance was also highest in the study group (55.4% vs. 32.0% and 15.1% for 5-FU-SA, diclofenac HA, and vehicle groups, respectively).¹³ Similar to the temporary lesion increase associated with other topical therapies, an ephemeral increase in mean lesion area was observed only in patients treated with 5-FU-SA at week 2.
• However, by the end of the treatment period, reduction in mean lesion area was more evident in the study medication group compared to the comparator and placebo groups.
• In a more recent non-interventional study, a reduction in number and size of AK after 0.5% 5-FU-SA therapy was observed even after a short period of use: target results were achieved in approximately half of patients within 6 weeks of treatment commencement.¹⁵
• Another study assessed the efficacy of low-dose 5-FU-SA versus cryosurgery in patients with grade II/III hyperkeratotic AK.¹⁶ In this open labelled, randomized trial, patients with histologically confirmed AK received either a 6-week course of once daily topical 5-FU-SA applied directly to lesions or up to two cryosurgical treatments spaced 3 weeks apart.
• 5-FU-SA achieved greater histological clearance as measured by mean lesion area and lower recurrence of lesions compared to cryosurgery at the 6-month follow-up.

Adverse Effects

• In the phase 3 mentioned above¹³, about 95% of patients in the study medication group reported treatment-emergent adverse effects (TEAEs), with local inflammation and pruritus at the application site being the most common.
• Approximately 60% of patients in the vehicle group also reported application site burning, suggesting the etiology of this sensation was likely related to dimethyl sulfoxide, which facilitates tissue absorption and is a known irritant present in the 5-FU-SA excipients.
• For patients who have difficulty tolerating the side effects, dosing can be reduced from daily applications to treatment three times a week.
• In spite of the relatively high rate of TEAEs, patients have reported a high level of satisfaction with the use of low-dose 5-FU-SA.¹⁵
• No serious adverse effects directly related to 5-FU-SA treatment, including usage as Verrumal® for warts, have been reported in either clinical studies or post-marketing surveillance.

Dosing and Administration

• Actikerall™ is a transparent, colorless to slightly orange-white solution that is packaged in 25 mL glass bottles, accompanied by a nylon brush that allows for easy application. It is recommended for application once daily to a total area of up to 25 cm2, which may include the lesion(s) and a small area of surrounding skin (rim of healthy skin should not exceed 0.5 cm), for up to 12 weeks (Table 1).

• If the lesions are located in areas with thin epidermis, the solution may be applied less frequently (e.g., 3 times per week).
• The solution should be allowed to dry on the skin but prior to re-application on subsequent days, the existing film should be peeled off, which can be facilitated by using warm water.
• A significant reduction in lesions is usually seen within 6 weeks of starting treatment, and patients most likely to benefit from the full 12-week course are those who have failed previous treatments with other modalities.¹⁵
• Patients should be advised that lesions may continue to regress for up to 8 weeks after cessation of therapy.
• 5-FU-SA is contraindicated for use during lactation or pregnancy. Other contraindications include renal insufficiency and concurrent usage of brivudine, sorivudine, or similar analogues. Of note, although these agents are structurally similar to acyclovir, which does not inhibit dihydropyrimidine dehydrogenase to any significant extent and is therefore safe to administer concurrently with 5-FU-SA.
• Additionally, instances of phenytoin toxicity related to the concurrent use of topical 5-FU-SA have been reported, so these patients should be tested at monthly intervals for plasma levels of phenytoin when this combination of therapies exists.¹¹
• 5-FU-SA should not be applied on bleeding lesions and has not been evaluated for the treatment of recurrent lesions.
• Patients should be educated on FU-SA’s flammability, propensity to desiccate quickly (the bottle needs to be closed tightly after use and it should be discarded if crystallization occurs), and ability to cause permanent stains on textiles and acrylics.

Conclusion

5-FU-SA represents a new addition to our treatment of AK, especially
for individuals who want to avoid the pain or potential consequences
associated with destructive therapy for isolated lesions. An emerging
role for 5-FU-SA may be in combination therapy with other agents
that have been unsuccessful in clearing hyperkeratotic lesions in the
treatment zone.

References

1. Marks R, Rennie G, Selwood TS. Malignant transformation of solar keratoses to squamous cell carcinoma. Lancet. 1988 Apr 9;1(8589):795-7.
2. Cockerell CJ. Histopathology of incipient intraepidermal squamous cell carcinoma (“actinic keratosis”). J Am Acad Dermatol. 2000 Jan;42(1 Pt 2):11-7.
3. Ehrig T, Cockerell C, Piacquadio D, et al. Actinic keratoses and the incidence of occult squamous cell carcinoma: a clinical-histopathologic correlation. Dermatol Surg. 2006 Oct;32(10):1261-5.
4. Quaedvlieg PJ, Tirsi E, Thissen MR, et al. Actinic keratosis: how to differentiate the good from the bad ones? Eur J Dermatol. 2006 Jul-Aug;16(4):335-9.
5. Fuchs A, Marmur E. The kinetics of skin cancer: progression of actinic keratosis to squamous cell carcinoma. Dermatol Surg. 2007 Sep;33(9):1099-101.
6. Criscione VD, Weinstock MA, Naylor MF, et al. Actinic keratoses: Natural history and risk of malignant transformation in the Veterans Affairs Topical Tretinoin Chemoprevention Trial. Cancer. 2009 Jun 1;115(11):2523-30.
7. Stockfleth E, Ortonne JP, Alomar A. Actinic Keratosis and field cancerisation. Eur J Dermatol. 2011 Feb; 21(Suppl 1):3-12.
8. Samrao A, Cockerell CJ. Pharmacotherapeutic management of actinic keratosis: focus on newer topical agents. Am J Clin Dermatol. 2013 Aug;14(4):273-7.
9. Krawtchenko N, Roewert-Huber J, Ulrich M, et al. A randomised study of topical 5% imiquimod vs. topical 5-fluorouracil vs. cryosurgery in immunocompetent patients with actinic keratoses: a comparison of clinical and histological outcomes including 1-year follow-up. Br J Dermatol. 2007 Dec;157 Suppl 2:34-40.
10. Perl M, Goldenberg G. Field therapy in the treatment of actinic keratosis. Cutis. 2014 Apr;93(4):172-3.
11. The electronic Medicines Compendium (eMC). Actikerall 5mg/g and 100mg/g cutaneous solution. Summary of product characteristics. Almirall Limited, Uxbridge, Middlesex, UK. Last updated June 4, 2014.
12. Olsen EA, Abernethy ML, Kulp-Shorten C, et al. A double-blind, vehicle-controlled study evaluating masoprocol cream in the treatment of actinic keratoses on the head and neck. J Am Acad Dermatol. 1991 May;24(5 Pt 1):738-43.
13. Stockfleth E, Kerl H, Zwingers T, et al. Low-dose 5-fluorouracil in combination with salicylic acid as a new lesion-directed option to treat topically actinic keratoses: histological and clinical study results. Br J Dermatol. 2011 Nov;165(5):1101-8.
14. Frost C, Williams G, Green A. High incidence and regression rates of solar keratoses in a Queensland community. J Invest Dermatol. 2000 Aug;115(2):273-7.
15. Szeimies RM, Dirschka T, Prechtl A, et al. Efficacy of low-dose 5-fluorouracil/salicylic acid in actinic keratoses in relation to treatment duration. J Dtsch Dermatol Ges. 2015 May;13(5):430-8.
16. Simon JC, Dominicus R, Karl L, et al. A prospective randomized exploratory study comparing the efficacy of once-daily topical 0.5% 5-fluorouracil in combination with 10.0% salicylic acid (5-FU/SA) vs. cryosurgery for the treatment of hyperkeratotic actinic keratosis. J Eur Acad Dermatol Venereol. 2015 May;29(5):881-9.

¹MD/MBA/MPH Candidate at Yale University, New Haven, CT, USA;
²Baylor College of Medicine, Houston, TX, USA
³Department of Dermatology and Skin Science, University of British Columbia, Vancouver, BC, Canada
⁴Pacific Dermaesthetics, Vancouver, BC, Canada

Conflict of interest:
Jason Rivers has been a consultant for Almirall; Harrison Nguyen has no conflicts to report.

ABSTRACT
Actinic keratosis (AK), a common cutaneous lesion with the potential to transform into squamous cell carcinoma, has traditionally been treated with ablative and/or surgical procedures. Recently, a topical formulation combining 0.5% 5-fluorouracil with 10% salicylic acid (5-FU-SA) was introduced in Europe under the trade name Actikerall™ for the treatment of grade I/II AKs. In a single randomized phase III trial, 5-FU-SA was shown to be superior to diclofenac 3% gel in hyaluronic acid, as measured by the histological clearance of one defined lesion (72% vs. 59.1%) and by complete clinical clearance (55.4% vs. 32.0%). 5-FU-SA should be applied once daily to a total area of up to 25 cm², which may include the lesion(s) and a small area of surrounding skin (rim of healthy skin should not exceed 0.5 cm), for up to 12 weeks. The most common side effects are local inflammation and pruritus at the application site, and no serious adverse effects have been reported to date. Now commercially available in Canada, 5-FU-SA represents a patient applied therapeutic option for the treatment of both overt and subclinical AKs.

Key Words:
Actikerall, actinic keratosis, antineoplastic antimetabolite, drug combinations, immunosuppressive agents, keratolytic agents, salicylic acid, skin neoplasms, topical therapy

Introduction

Actinic keratosis (AK) is a lesion considered to be on a continuum with squamous cell carcinoma (SCC).^1-6 Invasive disease occurs in up to 10% of cases over time, which highlights the need for early recognition and adequate treatment of all AKs, including subclinical lesions.⁷ Although many AKs never progress to SCC, their treatment has been recommended to preempt this eventuality.

Treatment options can generally be stratified based on whether only discrete lesions are treated, or whether subclinical lesions are also targeted, which is referred to as eld-directed therapy. Lesion-directed therapy has historically consisted of ablative and/or surgical procedures. However, several topical agents have emerged as attractive alternatives in the treatment of AKs. Examples of topical agents available in Canada and/or the United States include 5- fluorouracil (5-FU), imiquimod (2.5%, 3.75%, and 5% formulations), diclofenac 3%, methylaminolevulinate/ aminolevulinic acid (for photodynamic therapy), and ingenol mebutate.⁸

In one controlled clinical trial, topical 5-FU applied to AKs resulted in 96% clearance after 4 weeks of twice daily application.⁹ However, high rates of severe localized tissue reactions with 5-FU have led to reduced patient compliance, and this, in part, may explain why the long-term clearance of AKs in clinical practice is around 50%.⁸ This problem has resulted in a search for therapeutic agents less likely to induce skin irritation.¹⁰ In 2011, a topical formulation combining 0.5% 5-FU with 10% salicylic acid (5-FU-SA) was introduced to the European market under the trade name Actikerall™ for the topical treatment of grade I/II AKs (slightly palpable and/or moderately thick hyperkeratotic lesions) in immunocompetent adult patients.^11,12 This preparation is not novel as the same agent has been used in Europe for more than 30 years in the treatment of plantar warts (Verrumal®).

Functionally, 5-FU is a pyrimidine analogue that inhibits nucleic acid synthesis and its efficacy is thought to be enhanced by the addition of salicylic acid, a keratolytic agent that facilitates penetration of 5-FU into AKs.¹³ In this brief review, we present some of the clinical data to support the use of 5-FU-SA in patient-directed* management of AKs and we summarize the salient information that the provider should be aware of when prescribing this product.

^{*Health Canada has elected to classify Actikerall™ as neither lesion-directed nor field-directed. This was done to support the individual needs of patients; the locational distribution of a patient’s lesions will dictate whether a lesion-directed versus a field-directed approach is preferred.}

Evidence from Clinical Trials

The primary evidence used to support the efficacy of 5-FU-SA in the treatment of AKs comes from a single randomized, multi- center, phase III trial.¹⁴ The study included 470 patients with histologically diagnosed AK on the face, forehead, or bald scalp. Subjects were randomly assigned to 5-FU-SA, diclofenac 3% gel in hyaluronic acid (diclofenac HA), or placebo (5-FU-SA vehicle) and treatment was continued until complete resolution of the lesions, or for a maximum of 12 weeks. Subjects were instructed to apply their assigned intervention directly to the lesions – once daily for the 5-FU-SA and vehicle groups, and twice daily for the diclofenac group. The primary outcome – histological clearance of one defined lesion within 8 weeks of treatment cessation – was achieved in 72.0%, 59.1%, and 44.8% of patients treated with low- dose 5-FU-SA, diclofenac and placebo, respectively. Of note, up to 25% of untreated AKs may regress spontaneously over a 1 year period,¹⁵ and therefore, this phenomenon does not fully explain the high rate of clearance noted in the aforementioned study’s placebo group.

In addition to the histological data, the rate of complete clinical clearance was also highest in the study group (55.4% vs. 32.0% and 15.1% for 5-FU-SA, diclofenac HA, and vehicle groups, respectively).¹⁴ Similar to the temporary lesion increase associated with other topical therapies, an ephemeral increase in mean lesion area was observed only in patients treated with 5-FU-SA at week 2. However, by the end of the treatment period, reduction in mean lesion area was more evident in the study medication group compared to the comparator and placebo groups (355.9 mm², 345.7 mm², and 341.4 mm², respectively). In a more recent non- interventional study, the reduction in number and size of AKs after 0.5% 5-FU-SA therapy was observed even after a short period of use: target results were achieved in approximately half of patients within 6 weeks of treatment commencement.¹⁶

Another study assessed the efficacy of low-dose 5-FU-SA versus cryosurgery in patients with grade II/III hyperkeratotic AKs.¹⁷ In this open labelled, randomized trial, patients with histologically con rmed AK received either a 6-week course of once daily topical 5-FU-SA applied directly to lesions or up to two cryosurgical treatments spaced 3 weeks apart. Although the sample size (33 per treatment arm) was not powered to draw statistically significant conclusions, 5-FU-SA achieved greater histological clearance as measured by mean lesion area and lower recurrence of lesions compared to cryosurgery at the 6-month follow-up.

Non-invasive assessment using reflectance confocal microscopy and high-definition optical coherence tomography has provided insight into the in vivo pharmacodynamic changes induced by 5-FU-SA. In one study, AKs were assessed 2 weeks after the last treatment with 5-FU-SA, and the measurement of stratum corneum and epidermis thickness showed significant reduction in both clinical and subclinical lesions.¹⁸ Moreover, histological characteristics of AK – including scaling, detached corneocytes, atypical honeycombing, round nucleated cells in the spinosum granulosum, round vessels, and inflammatory cells were all markedly reduced in lesions treated with 5-FU-SA.¹⁸

Unlike 5-FU alone, there have been no studies to assess 5-FU- SA’s ability to treat superficial basal cell carcinoma or Bowen’s disease.¹⁹

Adverse Effects

In the first trial mentioned above,¹⁴ 7.4% (35/470) of patients withdrew from the study prematurely: 14 patients from the 5-FU-SA group, 16 patients from the diclofenac group, and 5 patients from the vehicle group. About 95% of patients in the study medication group reported treatment-emergent adverse effects (TEAEs), with local inflammation and pruritus at the application site being the most common. Approximately 60% of patients in the vehicle group also reported application site burning, suggesting the etiology of this sensation was likely related to dimethyl sulfoxide, which facilitates tissue absorption and is a known irritant present in the 5-FU-SA excipients. For patients who have difficulty tolerating the side effects, dosing can be reduced from daily application to treatment 3 times a week. In spite of the relatively high rate of TEAEs, patients have reported a high level of satisfaction with the use of low-dose 5-FU-SA.¹⁶ No serious adverse effects directly related to 5-FU-SA treatment, including usage as Verrumal® for warts, have been reported in either clinical studies or post-marketing surveillance.

Dosing and Administration

Actikerall™ is a transparent, colorless to slightly orange-white solution packaged in 25 mL glass bottles, accompanied by a nylon brush that allows for easy application. 5-FU-SA is recommended for application once daily to a total area of up to 25 cm², which may include the lesion(s) and a small area of surrounding skin (rim of healthy skin should not exceed 0.5 cm), for up to 12 weeks (Table 1). However, if the patient has lesions in areas with thin epidermis, the solution may be applied less frequently (e.g., 3 times per week). To avoid excess application, the brush can be wiped on the neck of the bottle. The solution should be allowed to dry on the skin but prior to re-application on subsequent days, the existing film should be peeled off, which can be facilitated by using warm water.

As noted, significant reduction in the lesion count is usually
observed within 6 weeks of starting treatment, and patients most
likely to benefit from the full 12-week course are those who have
failed previous therapy with other modalities.¹⁶ Patients should be advised that lesions may continue to regress for up to 8 weeks after cessation of therapy.

The use of 5-FU-SA in areas other than the face and scalp has
only been reported in the setting of small case series. In one
publication,²¹ two patients with multiple AKs on the dorsal aspect of the hands achieved complete clearance after sequential treatment with diclofenac 3% gel and 5-FU-SA. A notable observation has been a lower therapeutic response of AKs located on the upper extremities compared to lesions on the face and scalp.¹⁶ This finding has also been observed with photodynamic therapy, ingenol mebutate and other topical agents used to treat AKs. Although the reason for this observation remains poorly understood, one hypothesis is that increased skin thickness in the upper extremities restricts drug absorption, thus limiting its therapeutic efficacy.²²

5-FU-SA is contraindicated for use during lactation or pregnancy.
Other contraindications include renal insufficiency and
concurrent usage of brivudine, sorivudine, or similar analogues.
The latter consideration is related to the ability of these agents to inhibit the enzyme dihydropyrimidine dehydrogenase, which plays a critical role in breakdown of fluorouracil.²³ Of note, although these agents are structurally similar to acyclovir, the latter drug does not inhibit dihydropyrimidine dehydrogenase to any significant extent and is therefore safe to administer concurrently with 5-FU-SA. Additionally, instances of phenytoin toxicity related to the concurrent use of topical 5-FU-SA have been reported, so these patients should be tested at monthly intervals for plasma levels of phenytoin when this combination of therapies exists.¹¹ 5-FU-SA should not be applied on bleeding lesions and has not been evaluated for the treatment of recurrent lesions. With regards to user experience and safety, the patient should be educated on the solution’s flammability, propensity to desiccate quickly (the bottle needs to be closed tightly after use and it should be discarded if crystallization occurs), and ability to cause permanent stains on textiles and acrylics

Cipher Pharmaceuticals, which owns the rights to Actikerall™ in Canada, has indicated that each bottle will be sold for a
wholesale price of $36.25.²⁴ Commercial availability commenced in February 2016.

Conclusion

5-FU-SA represents a new addition to our treatment of AK,
especially for individuals who want to avoid the pain or potential consequences associated with destructive therapy for isolated lesions. An emerging role for 5-FU-SA may be in combination therapy with other agents that have been unsuccessful in clearing hyperkeratotic lesions in the treatment zone.

References

1. Marks R, Rennie G, Selwood TS. Malignant transformation of solar keratoses to squamous cell carcinoma. Lancet. 1988 Apr 9;1(8589):795-7.
2. Cockerell CJ. Histopathology of incipient intraepidermal squamous cell carcinoma (“actinic keratosis”). J Am Acad Dermatol. 2000 Jan;42(1 Pt 2):11-7.
3. Ehrig T, Cockerell C, Piacquadio D, et al. Actinic keratoses and the incidence of occult squamous cell carcinoma: a clinical-histopathologic correlation. Dermatol Surg. 2006 Oct;32(10):1261-5.
4. Quaedvlieg PJ, Tirsi E, Thissen MR, et al. Actinic keratosis: how to differentiate the good from the bad ones? Eur J Dermatol. 2006 Jul-Aug;16(4):335-9.
5. Fuchs A, Marmur E. The kinetics of skin cancer: progression of actinic keratosis to squamous cell carcinoma. Dermatol Surg. 2007 Sep;33(9):1099-101.
6. Criscione VD, Weinstock MA, Naylor MF, et al. Actinic keratoses: Natural history and risk of malignant transformation in the Veterans Affairs Topical Tretinoin Chemoprevention Trial. Cancer. 2009 Jun 1;115(11):2523-30.
7. Stockfleth E, Ortonne JP, Alomar A. Actinic Keratosis and field cancerisation. Eur J Dermatol. 2011 Feb; 21(Suppl 1):3-12.
8. Samrao A, Cockerell CJ. Pharmacotherapeutic management of actinic keratosis: focus on newer topical agents. Am J Clin Dermatol. 2013 Aug;14(4):273-7.
9. Krawtchenko N, Roewert-Huber J, Ulrich M, et al. A randomised study of topical 5% imiquimod vs. topical 5-fluorouracil vs. cryosurgery in immunocompetent patients with actinic keratoses: a comparison of clinical and histological outcomes including 1-year follow-up. Br J Dermatol. 2007 Dec;157 Suppl 2: 34-40.
10. Perl M, Goldenberg G. Field therapy in the treatment of actinic keratosis. Cutis. 2014 Apr;93(4):172-3.
11. The electronic Medicines Compendium (eMC). Actikerall 5mg/g and 100mg/g cutaneous solution. Summary of product characteristics. Almirall Limited, Uxbridge, Middlesex, UK. Last updated June 4, 2014. Available at: https://www. medicines.org.uk/emc/medicine/24614. Accessed April 3, 2016.
12. Olsen EA, Abernethy ML, Kulp-Shorten C, et al. A double-blind, vehiclecontrolled study evaluating masoprocol cream in the treatment of actinic keratoses on the head and neck. J Am Acad Dermatol. 1991 May;24(5 Pt 1): 738-43.
13. Ceilley RI. Mechanisms of action of topical 5-fluorouracil: review and implications for the treatment of dermatological disorders. J Dermatolog Treat. 2012 Apr;23(2):83-9.
14. Stockfleth E, Kerl H, Zwingers T, et al. Low-dose 5-fluorouracil in combination with salicylic acid as a new lesion-directed option to treat topically actinic keratoses: histological and clinical study results. Br J Dermatol. 2011 Nov;165(5):1101-8.
15. Frost C, Williams G, Green A. High incidence and regression rates of solar keratoses in a queensland community. J Invest Dermatol. 2000 Aug;115(2):273-7.
16. Szeimies RM, Dirschka T, Prechtl A, et al. Efficacy of low-dose 5-fluorouracil/salicylic acid in actinic keratoses in relation to treatment duration. J Dtsch Dermatol Ges. 2015 May;13(5):430-8.
17. Simon JC, Dominicus R, Karl L, et al. A prospective randomized exploratory study comparing the efficacy of once-daily topical 0.5% 5-fluorouracil in combination with 10.0% salicylic acid (5-FU/SA) vs. cryosurgery for the treatment of hyperkeratotic actinic keratosis. J Eur Acad Dermatol Venereol. 2015 May;29(5):881-9.
18. Malvehy J, Alarcon I, Montoya J, et al. Treatment monitoring of 0.5% 5-fluorouracil and 10% salicylic acid in clinical and subclinical actinic keratoses with the combination of optical coherence tomography and reflectance confocal microscopy. J Eur Acad Dermatol Venereol. 2016 Feb;30(2):258-65.
19. Metterle L, Nelson C, Patel N. Intralesional 5-fluorouracil (FU) as a treatment for nonmelanoma skin cancer (NMSC): A review. J Am Acad Dermatol. 2016 Mar;74(3):552-7.
20. Actikerall™: fluorouracil and salicylic acid solution (0.5%/10%) topical antineoplastic agent [Product monograph]; August 7, 2015. Cipher Pharmaceuticals Inc., Mississauga, ON.
21. Dirschka T, Lear JT. Sequential treatment of multiple actinic keratoses with solaraze and actikerall. Case Rep Dermatol. 2014 May;6(2):164-8.
22. Lebwohl M, Swanson N, Anderson LL, et al. Ingenol mebutate gel for actinic keratosis. N Engl J Med. 2012 Mar 15;366(11):1010-9.
23. De Clercq E. Discovery and development of BVDU (brivudin) as a therapeutic for the treatment of herpes zoster. Biochem Pharmacol. 2004 Dec 15;68(12):2301-15.
24. Markowitz L. Personal communication. January 18, 2016.

Department of Dermatology, Baylor College of Medicine, Houston, TX, USA

Conflict of interest:
None Reported.

ABSTRACT
First described in the context of diabetes, advanced glycation end products (AGEs) are formed through a type of non-enzymatic reaction called glycation. Increased accumulation of AGEs in human tissue has now been associated with end stage renal disease, chronic obstructive pulmonary disease, and, recently, skin aging. Characteristic findings of aging skin, including decreased resistance to mechanical stress, impaired wound healing, and distorted dermal vasculature, can be in part attributable to glycation. Multiple factors mediate cutaneous senescence, and these factors are generally characterized as endogenous (e.g., telomere shortening) or exogenous (e.g., ultraviolet radiation exposure). Interestingly, AGEs exert their pathophysiological effects from both endogenous and exogenous routes. The former entails the consumption of sugar in the diet, which then covalently binds an electron from a donor molecule to form an AGE. The latter process mostly refers to the formation of AGEs through cooking. Recent studies have revealed that certain methods of food preparation (i.e., grilling, frying, and roasting) produce much higher levels of AGEs than water-based cooking methods such as boiling and steaming. Moreover, several dietary compounds have emerged as promising candidates for the inhibition of glycation-mediated aging. In this review, we summarize the evidence supporting the critical role of glycation in skin aging and highlight preliminary studies on dietary strategies that may be able to combat this process.

Key Words:
AGEs, advanced glycation end products, collagen, dietary sucrose, fibroblasts, nutrition, skin aging

Background: Glycation and Aging Skin

Societal obsession with the process of aging dates back to ancient history, and myths related to the conservation of youth—ranging from a bathing fountain that confers eternal youth to a philosopher’s stone that could be used to create an elixir of life—populate both past and contemporary folklore. However, it is only within recent years that aging has been investigated from an empirical approach, as it continues to garner increasing attention from the scientific community. While several hypotheses have been proposed to explain the pathophysiology responsible for senescence, no single theory accounts for the diverse phenomena observed. Rather, aging appears to be a multifactorial process that results from a complex interplay of several factors and mechanisms.

Nevertheless, stratification of factors and mechanisms contributing to senescence is critical for the development of initial strategies in combating the aging process. The skin is an excellent paradigm for studying aging, in large part due to its easy accessibility. Moreover, in addition to its vulnerability to internal aging processes because of its diverse role in cellular processes, such as metabolism and immunity, the skin is subject to a variety of external stressors as the chief barrier between the body and the environment.

Aging factors can generally be classified as exogenous or endogenous. As ultraviolet (UV) radiation exposure is so strongly associated with a host of age-related skin diseases, endogenous and exogenous factors can theoretically be studied somewhat independently in the skin by differentiating between UVprotected and UV-exposed sites.¹ Endogenously aged skin displays characteristic morphological features with resultant alterations in functionality. These include epidermal, dermal, and extracellular matrix atrophy leading to increased fragility, diminished collagen and elastin resulting in fine wrinkle formation, and marked vascular changes disrupting thermoregulation and nutrient supply. Endogenously aged skin also displays decreased mitotic activity, resulting in delayed wound healing, as well as decreased glandular function, resulting in disturbed re-epithelialization of
deep cutaneous wounds. Also seen is a reduction of melanocytes and Langerhans cells manifesting as hair graying and higher rates of infection, respectively.^2-10 Exogenously aged skin, in which environmental factors such as UV radiation act in concert with endogenous processes, shares many of the characteristics of endogenously aged skin. In addition, exogenously aged skin displays a thickened epidermis and aggregation of abnormal elastic fibers in the dermis (i.e., solar elastosis).¹

Among the many mechanisms thought to underlie aging, glycation has emerged in recent years as one of the most widely studied processes. Testament to the rapidly growing attention from the scientific community, a cursory literature search will yield thousands of articles related to glycation, the majority of them published in the last decade. Glycation refers to the nonenzymatic process of proteins, lipids, or nucleic acids covalently bonding to sugar molecules, usually glucose or fructose. The lack of enzyme mediation is the key differentiator between glycation and glycosylation. Glycosylation occurs at defined sites on the target molecule and is usually critical to the target molecule’s function. In contrast, glycation appears to occur at random molecular sites and generally results in the inhibition of the target molecule’s ability to function.The products of glycation are called advanced glycation end products (AGEs).

Increased accumulation of AGEs was first directly correlated to the development of diabetic complications. Since then, AGEs have been implicated in a host of other pathologies, including atherosclerosis, end stage renal disease, and chronic obstructive pulmonary disease.¹¹ (It should be noted that AGE levels have been shown to vary by race and gender, and until larger studies are done to create ethnic- and gender-specific reference values, increased accumulation of AGEs should be defined as levels that are elevated for all demographic groups.¹²) Not coincidentally, many of the pathologies associated with AGEs, including diabetic sequelae, are closely related to senescence.

This extends to aging skin, as methods of AGE detection, such as immunostaining, have demonstrated the prevalence of glycation in aged skin. Glycation results in characteristic structural, morphological, and functional changes in the skin, a process colloquially known as “sugar sag.” With glucose and fructose playing such a prominent role in the mechanism, it is not surprising that diet plays a critical role in glycation and thus aging skin.

Perhaps more surprising, studies have shown that consumption of AGEs is not only tied to the sugar content of food, but is also affected by the method of cooking. Furthermore, as the
connection between diet and aging is more clearly characterized, a host of dietary compounds have surfaced as potential therapeutic candidates in the inhibition of AGE-mediated changes. In this review, we explore glycation as it pertains to skin aging and highlight evidence that demonstrates the quintessential role of diet in modifying the degree to which AGE-related processes are able to alter the largest organ of the human body.

Biochemical Processes in AGE Formation

First described over a century ago, glycation entails a series of simple and complex non-enzymatic reactions. In the key step, known as the Maillard reaction, electrophilic carbonyl groups of the sugar molecule react with free amino groups of proteins, lipids, or nucleic acids, leading to the formation of a Schiff base. This non-stable Schiff base contains a carbon-nitrogen double bond, with the nitrogen atom connected to an aryl or alkyl group. The Schiff base rapidly undergoes re-arrangement to form a more stable ketoamine, termed the Amadori product. At this juncture, the Amadori product can: (1) undergo the reverse reaction; (2) react irreversibly with lysine or arginine functional groups to produce stable AGEs in the form of protein adducts or protein cross-links; or (3) undergo further breakdown reactions, such as oxidation, dehydration, and polymerization, to give rise to numerous other AGEs.¹³ AGE formation is accelerated by an increased rate of protein turnover, hyperglycemia, temperatures above 120° C (248° F), and the presence of oxygen, reactive oxygen species, or active transition metals.¹⁴

AGEs comprise a highly heterogenous group of molecules. The first, and perhaps most well-known, physiological AGE to be described was glycated hemoglobin (hemoglobin A1C), now widely used to measure glycemic control in diabetes. However, the most prevalent AGE in the human body, including the skin, is carboxymethyl-lysine (CML), which is formed by oxidative degeneration of Amadori products or by direct addition of glyoxal to lysine. In the skin, CML is found in the normal epidermis, aged and diabetic dermis, and photoaging-actinic elastosis.^15-17 Other AGEs detected in skin include pentosidine, glyoxal, methylglyoxal, glucosepane, fructoselysine, carboxyethyl-lysine, glyoxal-lysine dimer, and methylglyoxal-lysine dimer.¹⁸

AGEs and the Skin

AGEs accumulate in various tissues as a function, as well as
a marker, of chronological age.¹⁹ Proteins with slow turnover rates, such as collagen, are especially susceptible to modification by glycation. Collagen in the skin, in fact, has a half-life of approximately 15 years and thus can undergo up to a 50% increase in glycation over an individual’s lifetime.²⁰

Collagen is critical not only to the mechanical framework of the skin but also to several cellular processes, and is impaired by glycation in multiple ways. First, intermolecular cross-linking modifies collagen’s biomechanical properties, resulting in increased stiffness and vulnerability to mechanical stimuli.²¹ Second, the formation of AGEs on collagen side chains alters the protein’s charge and interferes with its active sites, thereby distorting the protein’s ability to interact properly with surrounding cells and matrix proteins.²² Third, the ability to convert L-arginine to nitric oxide, a critical cofactor in the crosslinking of collagen fibers, is impaired.²³ Finally, glycated collagen is highly resistant to degradation by matrix metalloproteinases (MMPs). This further retards the process of collagen turnover and replacement with functional proteins.²⁴

Other cutaneous extracellular matrix proteins are functionally affected by glycation, including elastin and fibronectin. This further compounds dermal dysfunction,^18,25 as glycation crosslinked collagen, elastin, and fibronectin cannot be repaired like their normal counterparts.

Interestingly, CML-modified elastin is mostly found in sites of solar elastosis and is nearly absent in sun-protected skin. This suggests that UV-radiation can mediate AGE formation in some capacity or, at the least, render cells more sensitive to external stimuli.²⁶ It is hypothesized that UV-radiation accomplishes this through the formation of superoxide anion radicals, hydrogen peroxide, and hydroxyl radicals. This induces oxidative stress and accelerates the production of AGEs.²⁷ AGEs themselves are very reactive molecules and can act as electron donors in the formation of free radicals. Occurring in conjunction with the decline of the enzymatic system that eliminates free radicals during the aging process, these properties lead to a “vicious cycle” of AGE formation in the setting of UV exposure.

Formed both intracellularly and extracellularly, AGEs can also have an effect on intracellular molecular function. In the skin, the intermediate filaments of fibroblasts (vimentin) and keratinocytes (cytokeratin 10) have been shown to be susceptible to glycation modification.²⁸ Analogous to the diverse role of collagen in the skin, intermediate filaments are essential to both the maintenance of cytoskeletal stability and the coordination of numerous cellular functions. Fibroblasts with glycated vimentin demonstrate a reduced contractile capacity, and these modified fibroblasts are found to accumulate in skin biopsies of aged donors.²⁸

In fact, general cellular function may be compromised in the presence of high concentrations of AGEs. In vitro, human dermal fibroblasts display higher rates of premature senescence and apoptosis, which likely explains the decreased collagen and extracellular matrix protein synthesis observed in both cell culture and aged skin biopsies.^29,30 Similarly, keratinocytes exposed to AGEs express increased levels of pro-inflammatory mediators, suffer from decreased mobility, and also undergo premature senescence in the presence of AGEs.³¹

In addition to intermediate filaments, proteasomal machinery and DNA can undergo glycation. Proteasomal machinery, which functions to remove altered intracellular proteins, decline
functionally in vitro when treated with glyoxal.³² Similar in vitro findings were observed when human epidermal keratinocytes and fibroblasts were treated with glyoxal, leading to accumulation of CML in histones, cleavage of DNA, and, ultimately, arrest of cellular growth.³³

Beyond the modification of host molecular physicochemistry, AGEs also exert detrimental effects through the binding to specialized cellular surface receptors, called the Receptor for
AGEs (RAGE). RAGE is a multiligand protein that, when activated, can trigger several cellular signaling pathways, including the mitogen-activated protein kinases (MAPKs), extracellular signalregulated kinases (ERK), phosphatidyl-inositol-3-kinase (PI3K), and nuclear factor kappa-beta (NFκ-β) pathways.³⁴ These pathways are known to mediate various pathogenic mechanisms through the alteration of cell cycle regulators, gene expression, inflammation, and extracellular protein synthesis.³⁴ Not surprisingly, RAGE is found to be highly expressed in the skin and is present at even higher levels in both UV-exposed anatomical sites and aged skin.³⁵

Combating AGE with Diet

Nearly 70 years ago, Urbach and Lentz reported that the level of sugar both in the blood and in the skin is decreased with a diet low in sugar.³⁶ Although its significance was not appreciated at the time, this finding demonstrated a quintessential connection between diet and skin health. We now understand that food is a source of both monosaccharides that, in high amounts, catalyze the production of AGEs in the body, and preformed AGEs.³⁷

Preformed AGEs are absorbed by the gut with approximately 30% efficiency. They can then enter the circulation, where they may induce protein cross-linking, inflammation, and intracellular oxidative stress. The end result is the amplification of a similar “vicious cycle,” which may be as detrimental as the consumption of excess dietary sugar ³⁸ Interestingly, preformed AGEs largely result from exogenous synthesis mediated by the food cooking process. Grilling, frying, deep fat frying, and roasting methods are all known to produce higher levels of AGEs in food. In contrast, methods of preparation that are water-based, such as boiling and steaming, produce a logarithmically lower amount of AGEs.³⁹

A diet low in AGEs correlated with a reduction in inflammatory biomarkers (i.e., tumor necrosis factor-alpha, interleukin-6, and C-reactive protein) in diabetic human patients, as well as an improvement in wound healing and other diabetes-associated sequelae in mice.^40,41 Other authors have cited the relatively youthful appearance that is often associated with the elderly Asian population as evidence of the long-term impact of employing water-based cooking practices, which are characteristic of Asian cooking.³⁷

Tight glycemic control over a 4-month period can result in a reduction of glycated collagen formation by 25%.^37,38 Consumption of a low-sugar diet prepared through waterbased cooking methods would limit both the consumption of preformed exogenous AGES and endogenous production through physiological glycation. Avoiding foods that result in higher levels of AGEs, such as donuts, barbecued meats, and dark-colored soft drinks, can be an effective strategy for slowing “sugar sag.”³⁹

A diet low in AGEs correlated with a reduction in inflammatory biomarkers (i.e., tumor necrosis factor-alpha, interleukin-6, and C-reactive protein) in diabetic human patients, as well as an improvement in wound healing and other diabetes-associated sequelae in mice.^40,41 Other authors have cited the relatively youthful appearance that is often associated with the elderly Asian population as evidence of the long-term impact of employing water-based cooking practices, which are characteristic of Asian cooking.³⁷

Tight glycemic control over a 4-month period can result in a reduction of glycated collagen formation by 25%.^37,38 Consumption of a low-sugar diet prepared through waterbased cooking methods would limit both the consumption of preformed exogenous AGES and endogenous production through physiological glycation. Avoiding foods that result in higher levels of AGEs, such as donuts, barbecued meats, and dark-colored soft drinks, can be an effective strategy for slowing “sugar sag.”³⁹

Of interest, several culinary herbs and spices are believed to be capable of inhibiting the endogenous production of AGEs (specifically fructose-induced glycation). These include
cinnamon, cloves, oregano, and allspice.⁴² Other dietary compounds that have been linked to inhibition of AGE formation based on in vitro data and preliminary animal models include ginger, garlic, α-lipoic acid, carnitine, taurine, carnosine, flavonoids (e.g., green tea catechins), benfotiamine, α-tocopherol,niacinamide, pyridoxal, sodium selenite, selenium yeast, riboflavin, zinc, and manganese.^42-44 The cosmeceutical industry has taken notice of this data, and several have recently released topical products containing carnosine and α-lipoic acid, with claims related to anti-AGE formation.³⁸ However, data is lacking as to whether topical administration of these compounds is as effective as dietary delivery in slowing the aging process.

Since glycation is accelerated in the presence of reactive oxygen species, antioxidants should theoretically be effective in limiting the production of new AGEs. They may also impact AGE-induced tissue damage. One intriguing study looked at the effects of the antioxidant resveratrol. Popularly known for its abundance in red wine, resveratrol is a natural phenol produced by several plants in response to injury and is found in the skin of grapes, blueberries, raspberries, and mulberries. In one study, resveratrol inhibited AGE-induced proliferation and collagen synthesis activity in vascular smooth muscle cells belonging to strokeprone rats.⁴⁵ Another study found that it decreased the frequency of DNA breaks in methylglyoxal treated mouse oocytes. Although resveratrol does not appear to reverse the glycation process itself, these studies suggest that it can reduce AGE-induced tissue damage.⁴⁶ While these findings are promising, to our knowledge these laboratory results have not yet been demonstrated in human studies.

In one of the few human studies successfully conducted on antiAGE therapeutics, L-carnitine supplementation for 6 months in hemodialysis patients significantly decreased levels of AGEs in the skin.⁴⁷ L-carnitine, which is naturally abundant in meat, poultry, fish, and dairy products, is an antioxidant. Furthermore, it may function synergistically to neutralize oxidative stress when given with α-lipoic acid.⁴⁸

It warrants mentioning that dietary caloric restriction, the most effective strategy for slowing the general aging process known to date, may function to some degree by preventing accumulation of AGEs in the human body. Caloric restriction is capable of decreasing the levels of AGEs detected in rat and mice skin collagen and has resulted in an increased lifespan in mice models.^49,50

Conclusion: Obstacles and Future Directions

There is clearly an abundance of in vitro data and a handful of in vivo animal findings that support various options for dietary therapy directed against “sugar sag.” However, studies in humans are limited by logistical, ethical, and inherent study design issues. In a stimulating commentary as part of a review article on controversies in aging and nutrition, Draelos writes about the frustrating obstacles that she encountered when she attempted to study the impact of vitamin C supplementation on skin health.³⁸ Examples of problems she faced included: identifying a facility that offered affordable measurements of vitamin C levels not only in the serum but also in the skin; designing an ethical study that would include a control arm requiring subjects to adhere to a diet poor in vitamin C without any supplementation; and ensuring participant compliance to the diet and supplementation protocol while also minimizing confounding factors.Most of these challenges also exist in the human studies needed to identify and/or to verify evidence-based dietary strategies in combating glycation-mediated skin aging.

Nevertheless, the role of diet in skin aging is undeniable. As our understanding of how accumulation of AGEs affects a rapidly growing number of pathologies, it is inevitable that our research methods will evolve to better address the challenges that currently seem so discouraging. For instance, a research group reported in early 2014 that they were able to successfully create a model of reconstructed skin modified by glycated collagen to identify biological modifications of both epidermal and dermal markers.⁵¹ Perhaps the creation of an in vitro model that comprehensively and accurately represents aged human skin will serve as the next stepping stone in translating therapeutic findings from bench to bedside.

In the meantime, awareness of the critical impact of AGEformation in both diabetics and non-diabetics must be extended to all patients, regardless of their current health status. That task begins with clinicians. Dietary counseling should be incorporated into our regular interactions with patients, alongside essential discussions about UV-protection and avoidance of tobacco. After all, these are the three most important known exogenous aging factors. Their common grouping is reflective of their interconnected nature and their action in concert to disturb homeostasis.

References

1. Zouboulis CC, Makrantonaki E. Clinical aspects and molecular diagnostics of skin aging. Clin Dermatol. 2011 Jan-Feb;29(1):3-14.
2. Elias PM, Ghadially R. The aged epidermal permeability barrier: basis for functional abnormalities. Clin Geriatr Med. 2002 Feb;18(1):103-20, vii.
3. Ye J, Garg A, Calhoun C, et al. Alterations in cytokine regulation in aged epidermis: implications for permeability barrier homeostasis and inflammation. I. IL-1 gene family. Exp Dermatol. 2002 Jun;11(3):209-16.
4. Tsutsumi M, Denda M. Paradoxical effects of beta-estradiol on epidermal permeability barrier homeostasis. Br J Dermatol. 2007 Oct;157(4):776-9.
5. Fimmel S, Kurfurst R, Bonte F, et al. Responsiveness to androgens and effectiveness of antisense oligonucleotides against the androgen receptor on human epidermal keratinocytes is dependent on the age of the donor and the location of cell origin. Horm Metab Res. 2007 Feb;39(2):157-65.
6. Makrantonaki E, Vogel K, Fimmel S, et al. Interplay of IGF-I and 17betaestradiol at age-specific levels in human sebocytes and fibroblasts in vitro. Exp Gerontol. 2008 Oct;43(10):939-46.
7. Ashcroft GS, Horan MA, Ferguson MW. The effects of ageing on wound healing: immunolocalisation of growth factors and their receptors in a murine incisional model. J Anat. 1997 Apr;190 ( Pt 3):351-65.
8. Bhushan M, Cumberbatch M, Dearman RJ, et al. Tumour necrosis factoralpha-induced migration of human Langerhans cells: the influence of ageing. Br J Dermatol. 2002 Jan;146(1):32-40.
9. Zouboulis CC, Boschnakow A. Chronological ageing and photoageing of the human sebaceous gland. Clin Exp Dermatol. 2001 Oct;26(7):600-7.
10. Chung JH, Yano K, Lee MK, et al. Differential effects of photoaging vs intrinsic aging on the vascularization of human skin. Arch Dermatol. 2002 Nov;138(11):1437-42.
11. Van Puyvelde K, Mets T, Njemini R, et al. Effect of advanced glycation end product intake on inflammation and aging: a systematic review. Nutr Rev. 2014 Oct;72(10):638-50.
12. Mook-Kanamori MJ, Selim MM, Takiddin AH, et al. Ethnic and gender differences in advanced glycation end products measured by skin autofluorescence. Dermatoendocrinol. 2013 Apr 1;5(2):325-30.
13. Ahmed N. Advanced glycation endproducts–role in pathology of diabetic complications. Diabetes Res Clin Pract. 2005 Jan;67(1):3-21.
14. Thorpe SR, Baynes JW. Maillard reaction products in tissue proteins: new products and new perspectives. Amino Acids. 2003 Dec;25(3-4):275-81.
15. Kawabata K, Yoshikawa H, Saruwatari K, et al. The presence of N(epsilon)-(carboxymethyl) lysine in the human epidermis. Biochim Biophys Acta. 2011 Oct;1814(10):1246-52.
16. Dyer DG, Dunn JA, Thorpe SR, et al. Accumulation of Maillard reaction products in skin collagen in diabetes and aging. J Clin Invest. 1993 Jun;91(6):2463-9.
17. Jeanmaire C, Danoux L, Pauly G. Glycation during human dermal intrinsic and actinic ageing: an in vivo and in vitro model study. Br J Dermatol. 2001 Jul;145(1):10-8.
18. Gkogkolou P, Bohm M. Advanced glycation end products: Key players in skin aging? Dermatoendocrinol. 2012 Jul 1;4(3):259-70
19. Hipkiss AR. Accumulation of altered proteins and ageing: causes and effects. Exp Gerontol. 2006 May;41(5):464-73.
20. Dunn JA, McCance DR, Thorpe SR, et al. Age-dependent accumulation of N epsilon-(carboxymethyl)lysine and N epsilon-(carboxymethyl) hydroxylysine in human skin collagen. Biochemistry. 1991 Feb 5;30(5): 1205-10.
21. Avery NC, Bailey AJ. The effects of the Maillard reaction on the physical properties and cell interactions of collagen. Pathol Biol (Paris). 2006 Sep;54(7):387-95.
22. Haitoglou CS, Tsilibary EC, Brownlee M, et al. Altered cellular interactions between endothelial cells and nonenzymatically glucosylated laminin/type IV collagen. J Biol Chem. 1992 Jun 25;267(18):12404-7.
23. Goldin A, Beckman JA, Schmidt AM, et al. Advanced glycation end products: sparking the development of diabetic vascular injury. Circulation. 2006 Aug 8;114(6):597-605.
24. DeGroot J, Verzijl N, Wenting-Van Wijk MJ, et al. Age-related decrease in susceptibility of human articular cartilage to matrix metalloproteinasemediated degradation: the role of advanced glycation end products. Arthritis Rheum. 2001 Nov;44(11):2562-71.
25. Nowotny K, Grune T. Degradation of oxidized and glycoxidized collagen: role of collagen cross-linking. Arch Biochem Biophys. 2014 Jan 15;542:56-64.
26. Yoshinaga E, Kawada A, Ono K, et al. N(varepsilon)-(carboxymethyl)lysine modification of elastin alters its biological properties: implications for the accumulation of abnormal elastic fibers in actinic elastosis. J Invest Dermatol. 2012 Feb;132(2):315-23.
27. Masaki H, Okano Y, Sakurai H. Generation of active oxygen species from advanced glycation end-products (AGEs) during ultraviolet light A (UVA) irradiation and a possible mechanism for cell damaging. Biochim Biophys Acta. 1999 Jun 28;1428(1):45-56.
28. Kueper T, Grune T, Prahl S, et al. Vimentin is the specific target in skin glycation. Structural prerequisites, functional consequences, and role in skin aging. J Biol Chem. 2007 Aug 10;282(32):23427-36.
29. Alikhani Z, Alikhani M, Boyd CM, et al. Advanced glycation end products enhance expression of pro-apoptotic genes and stimulate fibroblast apoptosis through cytoplasmic and mitochondrial pathways. J Biol Chem. 2005 Apr 1;280(13):12087-95.
30. Zhu P, Ren M, Yang C, et al. Involvement of RAGE, MAPK and NF-kappaB pathways in AGEs-induced MMP-9 activation in HaCaT keratinocytes. Exp Dermatol. 2012 Feb;21(2):123-9.
31. Zhu P, Yang C, Chen LH, et al. Impairment of human keratinocyte mobility and proliferation by advanced glycation end products-modified BSA. Arch Dermatol Res. 2011 Jul;303(5):339-50.
32. Bulteau AL, Verbeke P, Petropoulos I, et al. Proteasome inhibition in glyoxal-treated fibroblasts and resistance of glycated glucose-6-phosphate dehydrogenase to 20 S proteasome degradation in vitro. J Biol Chem. 2001 Dec 7;276(49):45662-8.
33. Roberts MJ, Wondrak GT, Laurean DC, et al. DNA damage by carbonyl stress in human skin cells. Mutat Res. 2003 Jan 28;522(1-2):45-56.
34. Bierhaus A, Humpert PM, Morcos M, et al. Understanding RAGE, the receptor for advanced glycation end products. J Mol Med (Berl). 2005 Nov;83(11):876-86.
35. Lohwasser C, Neureiter D, Weigle B, et al. The receptor for advanced glycation end products is highly expressed in the skin and upregulated by advanced glycation end products and tumor necrosis factor-alpha. J Invest Dermatol. 2006 Feb;126(2):291-9.
36. Urbach E, Lentz JW. Carbohydrate metabolism and the skin. Arch Derm Syphilol. 1945 Nov-Dec;52:301-16.
37. Danby FW. Nutrition and aging skin: sugar and glycation. Clin Dermatol. 2010 Jul-Aug;28(4):409-11.
38. Draelos ZD. Aging skin: the role of diet: facts and controversies. Clin Dermatol. 2013 Nov-Dec;31(6):701-6.
39. O’Brien J, Morrissey PA. Nutritional and toxicological aspects of the Maillard browning reaction in foods. Crit Rev Food Sci Nutr. 1989 28(3):211-48.
40. Vlassara H, Striker GE. AGE restriction in diabetes mellitus: a paradigm shift. Nat Rev Endocrinol. 2011 Sep;7(9):526-39.
41. Peppa M, Brem H, Ehrlich P, et al. Adverse effects of dietary glycotoxins on wound healing in genetically diabetic mice. Diabetes. 2003 Nov;52(11): 2805-13.
42. Dearlove RP, Greenspan P, Hartle DK, et al. Inhibition of protein glycation by extracts of culinary herbs and spices. J Med Food. 2008 Jun;11(2):275-81.
43. Thirunavukkarasu V, Nandhini AT, Anuradha CV. Fructose diet-induced skin collagen abnormalities are prevented by lipoic acid. Exp Diabesity Res. 2004 Oct-Dec;5(4):237-44.
44. Tarwadi KV, Agte VV. Effect of micronutrients on methylglyoxal-mediated in vitro glycation of albumin. Biol Trace Elem Res. 2011 Nov;143(2):717-25.
45. Mizutani K, Ikeda K, Yamori Y. Resveratrol inhibits AGEs-induced proliferation and collagen synthesis activity in vascular smooth muscle cells from stroke-prone spontaneously hypertensive rats. Biochem Biophys Res Commun. 2000 Jul 21;274(1):61-7.
46. Liu Y, He XQ, Huang X, et al. Resveratrol protects mouse oocytes from methylglyoxal-induced oxidative damage. PLoS One. 2013 8(10):e77960.
47. Fukami K, Yamagishi S, Sakai K, et al. Potential inhibitory effects of L-carnitine supplementation on tissue advanced glycation end products in patients with hemodialysis. Rejuvenation Res. 2013 Dec;16(6):460-6.
48. Hagen TM, Liu J, Lykkesfeldt J, et al. Feeding acetyl-L-carnitine and lipoic acid to old rats significantly improves metabolic function while decreasing oxidative stress. Proc Natl Acad Sci USA. 2002 Feb 19;99(4):1870-5.
49. Cefalu WT, Bell-Farrow AD, Wang ZQ, et al. Caloric restriction decreases age-dependent accumulation of the glycoxidation products, N epsilon- (carboxymethyl)lysine and pentosidine, in rat skin collagen. J Gerontol A Biol Sci Med Sci. 1995 Nov;50(6):B337-41.
50. Sell DR, Kleinman NR, Monnier VM. Longitudinal determination of skin collagen glycation and glycoxidation rates predicts early death in C57BL/6NNIA mice. FASEB J. 2000 Jan;14(1):145-56.
51. Pageon H, Zucchi H, Rousset F, et al. Skin aging by glycation: lessons from the reconstructed skin model. Clin Chem Lab Med. 2014 Jan 1;52(1):169-74.

¹Department of Dermatology, Baylor College of Medicine, Houston, TX, USA
²Department of Dermatology, University of Texas Medical School at Houston, Houston, TX, USA
³Center for Clinical Studies, Houston, TX, USA

Conflict of interest:
None reported

ABSTRACT
Herpes labialis is a frequently occurring viral infection of the lips and oral mucosa. Recurring lesions are induced by viral reactivation and replication, but the symptoms leading to morbidity, such as pain and inflammation, are immune-mediated. The introduction of 5% acyclovir/1% hydrocortisone in a topical cream (Xerese™) represents a therapeutic strategy directed at both of these pathogenic processes. Applied at the onset of prodromal symptoms, this combination treatment has a good safety profile and is more effective in reducing healing time than antiviral or anti-inflammatory agents alone. Although it was US FDA-approved for herpes labialis in 2009, Xerese™ has only recently been approved for use in Canada in October 2013. Herein, we review the basic science and clinical studies that support the efficacy of this topical combination acyclovir-hydrocortisone product in treating herpes labialis and examine its safety profile, as well as touch upon other therapies that have been shown to be effective in treating this common viral condition.

Key Words:
cold sores, herpes labialis, Xerese, viral infection, Canada, drug approval

Introduction

Herpes labialis (colloquially known as “cold sores”) is a common viral infection characterized by vesicular lesions of the lips and oral mucosa. It is estimated to affect 1 in 5 Canadians annually and is associated with a negative stigma that can lead to depression, fear of rejection, and isolation for infected individuals during an outbreak.¹ Herpes labialis is mostly caused by the herpes simplex virus-1 (HSV-1), which enters the nerve during primary infection and remains latent in the ganglionic neuron for the rest of the individual’s life. Periodically, the virus travels back down the nerve to the skin and replicates, producing a clinical episode of reactivated HSV-1 infection. Intralesional viral replication is halted by the host immune response approximately 7 days after primary infection and 3 days after recurrent infection;² however, inflammation secondary to immune defense is also the cause of redness, swelling, and tenderness that is characteristic of herpes labialis lesions. As a result, although viral clearance happens rapidly following reactivation, the lesion often takes 7-10 days to heal completely.

Since the pathogenesis of herpes labialis is both viral- and immune-mediated, it is not surprising that administration of exclusively antiviral drugs has limited effects on the clinical parameters of the disease.³ Therefore, medications demonstrating dual mechanisms via inhibition of viral replication and modulation of the inflammatory response to facilitate healing, indicate a more successful therapeutic approach.⁴ Such an agent was introduced by Valeant Pharmaceuticals, consisting of 5% antiviral acyclovir plus 1% anti-inflammatory hydrocortisone (ACHC) in a topical cream formulation (Xerese™). Although it has been FDA-approved in the US for the treatment of recurrent herpes labials since 2009, authorization for Xerese™ in Canada was not officially granted until October 2013. In light of this recent Canadian approval, we review the data supporting the efficacy of this topical combination therapy and discuss the details regarding its clinical use, specifically incorporating our experience in prescribing ACHC for the past half-decade.

Drug Information

ACHC is intended for the early treatment of recurrent herpes simplex labialis (HSL) in adults and adolescents (12 years of age and older). It is designed for cutaneous use only and is applied to the lips and skin around the mouth. Usage should be avoided on the eyes, inside the mouth or nose, and on the genitals. The patient should be instructed to apply a thin layer across the affected area, including the outer margins of the cold sore. Treatment should be initiated at the first sign or symptom (prodromal stage), applying 5 times per day for a period of 5 consecutive days. If there is no noticeable improvement and/or the cold sore fails to heal within 2 weeks, patients are encouraged to seek further medical attention. At present, the efficacy of ACHC has not been established in the immunocompromised population.⁵

From Bench to Bedside: Duration and Efficacy

In order to obtain maximum clinical benefit from a topical antiviral medication, therapy should be initiated within 72 hours of onset of symptoms.⁶ Patients with recurrent herpes labialis experience a rapid onset of disease and a short viral shedding period, both of which make it difficult to measure responses to therapy.

In an early basic science study conducted in 2003, researchers used mice that had undergone adaptive transfer of immunity and infected the skin on the mice’s ear pinna with HSV-1.⁷ After the mice developed a zosteriform infection, treatment groups received topical ACHC, 5% acyclovir (Zovirax®), 1% hydrocortisone, or no treatment at all. Medication was applied 3 times daily for 4 days. The treatment groups were analyzed based on ear thickness increase and zoster score. The zoster scores were adapted from a scale previously described in another study, and the scores used were: 0 for unchanged ear, 1 for isolated zosteriform lesions, and 2-4 for describing the ulceration of confluent zosteriform lesions from mild to severe.⁸ ACHC outperformed both 5% acyclovir and 1% hydrocortisone creams, with an average increase in ear thickness of only 0.15 ± 0.03 mm compared to 0.48 ± 0.08 mm and 0.23 ± 0.03 mm, respectively. The average increase in ear thickness for ACHC was only 34% of that experienced by the mice in the control group, compared to 110% observed with acyclovir and 52% with hydrocortisone. The average zoster score for the ACHC group at day 9 was also the lowest of the four groups at 2.0 ± 0.2 (58% of control), compared to 2.4 ± 0.3 (70% of control) for acyclovir and 2.8 ± 0.2 (80% of control) for hydrocortisone.⁷

In a 2012 Phase 3 study, Strand et al instructed their human subjects to apply ACHC 5 times daily for 5 days at the onset of prodromal symptoms, preferably before the appearance of actual papules or vesicles. Of the 131 test subjects, 78 (59.5%) had nonulcerative recurrences, and 53 (40.5%) had ulcerative recurrences. At the follow-up visit, all 131 of the test subjects had returned to the stage of normal skin, 3 weeks after the last dose, with no signs or symptoms of herpes labialis recurrence. In the 40% of subjects who experienced ulcerative herpes lesions despite applying the ACHC cream, the mean maximum lesion area was 39 mm², which was a 48% decrease from the mean lesion area size of 75 mm² typically reported in immunocompetent adults.^9,10

A similar study published in 2011, also using a dosing regimen of applying cream 5 times daily for 5 days, studied a much larger patient population in a randomized, double-blind, placebocontrolled trial.¹¹ The 2,437 volunteers were randomized to receive either ACHC, acyclovir in the ACHC vehicle, or placebo in the form of the ACHC vehicle. Of the 1,443 subjects who experienced a recurrence of herpes labialis during the trial and initiated treatment, 42% used ACHC, 42% acyclovir, and 16% placebo. The authors reported that 58% of the patients on ACHC developed an ulcerative lesion, vs. 65% in the acyclovir group and 74% in the placebo group. In patients who experienced an ulcerative lesion, the healing times were reduced in those who received ACHC or acyclovir, compared with placebo. The patients using ACHC also had a smaller cumulative lesion area (~50% less) than the placebo group (Tables 1 and 2).

Finally, in a simulated 2002 trial, researchers tested the efficacy of ACHC in patients whose latent HSV-1 infection was intentionally reactivated using ultraviolet (UV) light.¹² Of the 380 subjects, 120 patients developed classical cold sores 2 days after UV light exposure, which was followed by initiation of treatment with either ACHC or placebo. Treatment with ACHC reduced lesion size, healing time, and lesion tenderness when compared with placebo. Healing time (defined as the time to restoration of normal skin) was reduced from 10.1 days in the placebo group to 9.0 days in the ACHC group (Table 2).

Adverse Effects

The combination cream of 5% acyclovir and 1% hydrocortisone has been shown to induce only minimal side effects when used to treat herpes labialis infections. In Strand et al’s 2012 Phase 3, open-label, multicenter study, 131 of 134 subjects were categorized with recurrence at the post-treatment visit.¹⁰ Of these 131 subjects, only 5 reported any adverse events. The events were classified as mild to moderate in intensity and consisted of secondary herpes labialis recurrences (n=2), infectious rhinitis (n=1), application site inflammation (n=1), and bronchial asthma (n=1). Additional studies have corroborated the safety of ACHC, observing only minor side effects.^11,12 The most common adverse reactions reported in clinical trials were drying or flaking of the skin, burning or tingling at the application sight, erythema, and pigmentation changes; these infrequent adverse effects occurred in less than 1% of patients studied.⁵

Other Therapies for Herpes Labialis

Prior to the authorization of Xerese™ by Health Canada, the mainstays of treatment for recurrent HSL included over-thecounter docosanol cream, and prescription-only members of the acyclovir family (oral and topical). If ACHC is contraindicated for use in a particular patient due to adverse effects, docosanol or acyclovir may provide therapeutic benefit.

Docosanol 10% cream (Abreva®) is an approved treatment for recurrent herpes labialis, with efficacy demonstrated in two identical double-blind, placebo-controlled studies conducted at 21 sites.¹³ Therapy was initiated at the onset of prodromal symptoms or the erythema stage in subjects who were otherwise healthy adults with documented histories of herpes labialis. Treatment was administered 5 times daily until healing occurred, with twice-daily visits to the investigative clinic for the first 7 days. For the 370 patients who were treated with docosanol, the median time to heal was 4.1 days, which was 18 hours shorter than the healing time for the 367 patients treated with placebo (Table 2). The patients treated with docosanol also reported earlier cessation of pain and exhibited complete healing, as well as experienced reduced lesion progression to the ulcer or soft crust stage.

A well-established mainstay in the treatment of recurrent herpes labialis is valacyclovir. This prodrug of acyclovir has proven to be a safe and effective therapy for long-term HSV suppression.¹⁴ It has been studied in children, pregnancy, and immunocompromised patients. The most common adverse events associated with oral valacyclovir are headache, rhinitis, infection, nausea, and pharyngitis, with all of these occurring infrequently. Despite many years of use by clinicians, HSV resistance remains low at approximately 0.1-0.4% in the UK and the US.¹⁴

A new form of acyclovir was recently approved by the US FDA in April 2013. This medication consists of acyclovir in the form of a mucoadhesive buccal tablet (ABT) (Sitavig®), which is applied to the upper gum region within the first hour of prodromal symptoms. A Phase 3 double-blind trial found that acyclovir, utilizing the proprietary Lauriad® technology, decreased the median duration time and development of primary vesicular lesions when compared to placebo (Tables 1 and 2).¹⁵

Lastly, a non-pharmacologic treatment for recurrent HSL involves low-level light therapy. A paper published in 2013 demonstrated that 1072 nm light-emitting diode therapy applied 3 times a day for 2 days was able to shorten healing time in patients with HSL to a median of 129 hours vs. 177 hours for the control group (Table 2).¹⁶

Clinical Observations

When taken daily (along with topical sunscreens), oral acyclovir, famciclovir, or valacyclovir are better at preventing herpes labialis than topical therapies are at treating outbreaks; however, it is the authors’ experience over the past 5 years that when used appropriately, ACHC is the superior topical therapy. Because the signs and symptoms of herpes labialis are attributable to both viral and inflammatory mechanisms, prescription topicals exerting only antiviral or anti-inflammatory activities have limited efficacy. Most over-the-counter therapies fail to target underlying pathogenic mechanisms (i.e., viral and inflammatory) and, thus, have little to no efficacy. While the optimal strategy is to prevent herpes labialis outbreaks via reduction of sun exposure, as well as through the use of sunscreen and oral anti-viral agents (especially in individuals experiencing frequent outbreaks), we recommend to our patients that they fill their prescriptions for ACHC as soon as possible and keep the cream at home, at work, and/or carry it with them while on vacation. At the onset of prodromal symptoms, therapy should be initiated immediately and no later than the appearance of the first sign of a recurrence.

Conclusion

Herpes labialis still lacks a cure, but several options are now available to limit inflammation and decrease healing time. The introduction of 5% acyclovir/1% hydrocortisone topical cream represents a forward step in understanding disease pathogenesis and targeting both the viral and immunogenic components of recurrent HSL.

References

1. Kuehl B. Cold sores – how to prevent and treat them
2. Spruance SL, Rea TL, Thoming C, et al. Penciclovir cream for the treatment of herpes simplex labialis. A randomized, multicenter, double-blind, placebocontrolled trial. Topical Penciclovir Collaborative Study Group. JAMA. 1997 May 7;277(17):1374-9.
3. Corey L, Nahmias AJ, Guinan ME, et al. A trial of topical acyclovir in genital herpes simplex virus infections. N Engl J Med. 1982 Jun 3;306(22):1313-9.
4. Hull CM, Levin MJ, Tyring SK, et al. Novel composite efficacy measure to demonstrate the rationale and efficacy of combination antiviral-antiinflammatory treatment for recurrent herpes simplex labialis. Antimicrob Agents Chemother. 2014 Mar;58(3):1273-8.
5. Xerese® prescribing information. Date of revision: January 2012. Coria Laboratories, a division of Valeant Pharmaceuticals North America LLC, Bridgewater, NJ.
6. Vestey JP, Norval M. Mucocutaneous infections with herpes simplex virus and their management. Clin Exp Dermatol. 1992 Jul;17(4):221-37.
7. Harmenberg JG, Awan AR, Alenius S, et al. ME-609: a treatment for recurrent herpes simplex virus infections. Antivir Chem Chemother. 2003 Jul;14(4): 205-15.
8. Nagafuchi S, Oda H, Mori R, et al. Mechanism of acquired resistance to herpes simplex virus infection as studied in nude mice. J Gen Virol. 1979 Sep;44(3):715-23.
9. Spruance SL. Herpes simplex labialis. In: Sacks SL, Straus SE, Whitley RJ, et al. editors. Clinical management of herpes viruses. Amsterdam, Washington DC: IOS Press. 1995;p3-42.
10. Strand A, Bottiger D, Gever LN, Wheeler W. Safety and tolerability of combination acyclovir 5% and hydrocortisone 1% cream in adolescents with recurrent herpes simplex labialis. Pediatr Dermatol. 2012 Jan-Feb;29(1): 105-10.
11. Hull CM, Harmenberg J, Arlander E, et al. Early treatment of cold sores with topical ME-609 decreases the frequency of ulcerative lesions: a randomized, double-blind, placebo-controlled, patient-initiated clinical trial. J Am Acad Dermatol. 2011 Apr;64(4):696 e1-11.
12. Evans TG, Bernstein DI, Raborn GW, et al. Double-blind, randomized, placebocontrolled study of topical 5% acyclovir-1% hydrocortisone cream (ME-609) for treatment of UV radiation-induced herpes labialis. Antimicrob Agents Chemother. 2002 Jun;46(6):1870-4.
13. Sacks SL, Thisted RA, Jones TM, et al. Clinical efficacy of topical docosanol 10% cream for herpes simplex labialis: A multicenter, randomized, placebocontrolled trial. J Am Acad Dermatol. 2001 Aug;45(2):222-30.
14. Tyring SK, Baker D, Snowden W. Valacyclovir for herpes simplex virus infection: long-term safety and sustained efficacy after 20 years’ experience with acyclovir. J Infect Dis. 2002 Oct 15;186 Suppl 1:S40-6.
15. Downing C, Moayyad J, Tamirisa A, et al. Acyclovir Lauriad((R)): a mucoadhesive buccal tablet for the treatment of recurrent herpes labialis. Expert Rev Anti Infect Ther. 2014 Mar;12(3):283-7.
16. Dougal G, Lee SY. Evaluation of the efficacy of low-level light therapy using 1072 nm infrared light for the treatment of herpes simplex labialis. Clin Exp Dermatol. 2013 Oct;38(7):713-8.
17. Raborn GW, Martel AY, Lassonde M, et al. Effective treatment of herpes simplex labialis with penciclovir cream: combined results of two trials. J Am Dent Assoc. 2002 Mar;133(3):303-9.
18. Spruance SL, Jones TM, Blatter MM, et al. High-dose, short-duration, early valacyclovir therapy for episodic treatment of cold sores: results of two randomized, placebo-controlled, multicenter studies. Antimicrob Agents Chemother. 2003 Mar;47(3):1072-80.
19. Morrel EM, Spruance SL, Goldberg DI. Topical iontophoretic administration of acyclovir for the episodic treatment of herpes labialis: a randomized, double-blind, placebo-controlled, clinic-initiated trial. Clin Infect Dis. 2006 Aug 15;43(4):460-7.
20. Spruance SL, Bodsworth N, Resnick H, et al. Single-dose, patient-initiated famciclovir: a randomized, double-blind, placebo-controlled trial for episodic treatment of herpes labialis. J Am Acad Dermatol. 2006 Jul;55(1):47-53.

¹Baylor College of Medicine, Houston, TX, USA
²Department of Dermatology, University of Texas Health Science Center at Houston, Houston, TX, USA
³Center for Clinical Studies, Houston, TX, USA

ABSTRACT
Molluscum contagiosum is a poxvirus infection of the skin that is commonly observed in children. The molluscum contagiosum virus (MCV) expresses several gene-products that are involved in its pathogenesis and evasion of the host immune system. MCV can be transmitted both to other sites of the body and to other individuals through direct physical contact as well as fomites. While diagnosis is generally straightforward clinically, management of molluscum contagiosum is controversial. Several treatment options are available for the destruction of individual lesions, but there is insufficient evidence for therapeutic intervention being any more effective than natural, spontaneous resolution. Complex cases, such as infection occurring in immunocompromised patients and in mucocutaneous sites, require an alternative approach to management. Molluscum contagiosum continues to represent a burden on children and parents worldwide.

Key Words:
molluscum contagiosum, viral infection, pediatric skin disease

Background

Molluscum contagiosum is a cutaneous viral infection that is commonly observed in both healthy and immunocompromised children. The infection is caused by a member of the Poxviridae family, the molluscum contagiosum virus (MCV). Molluscum contagiosum presents as single or multiple small white or flesh-colored papules that typically have a central umbilication. The central umbilication may be difficult to observe in young children and, instead, may bear an appearance similar to an acneiform eruption. The lesions vary in size (from 1 mm to 1 cm in diameter) and are painless, although a subset of patients report pruritus in the area of infection.¹ On average, 11-20 papules appear on the body during the course of infection and generally remains a self-limiting disease. However, in immunosuppressed patients, molluscum contagiosum can be a severe infection with hundreds of lesions developing on the body. Extensive eruption is indicative of an advanced immunodeficiency state.

Eruption of molluscum contagiosum usually begins in a localized area of the skin, though the infection can be transmitted to other regions of the body – such as genital, perineal, pubic, and surrounding skin – through autoinoculation. Since MCV can be sexually transmitted, child abuse should be considered when the genital or perineal areas are infected, but autoinoculation is usually the most common source of genital infection.² Most cases of sexually transmitted molluscum manifest as papules localized exclusively to the genital area. A 2010 Spanish study reported that sexually transmitted molluscum contagiosum had increased three-fold in its regional hospital from 1987 to 2007.³ Rarely, molluscum contagiosum can also spread to the oral region – potentially from the genitalia during oral sex – as well as to the conjunctiva and cornea.^4,5 The latter scenario may result in chronic conjunctivitis or superficial punctate keratitis, which complicates the treatment of lesions in the orbital region. In atopic patients, eczema can develop around the papules approximately a month after onset. The eczema, which has also been observed in non-atopic children, occurs in upwards of 30% of patients and, importantly, increases the risk of autoinoculation since patients are more likely to scratch the eczematous region, spreading the viral particles to other areas of the body.⁶ The chronic conjunctivitis and eczema associated with molluscum contagiosum subside spontaneously when the lesions are eradicated. Pre-existing eczema may also predispose children to the infection; 62% of children with molluscum contagiosum in Australia reported a history of eczema.⁷

MCV is transmitted through close physical contact with an infected individual or with a fomite.⁸ There were an estimated 280,000 patient visits per year for molluscum contagiosum in the United States alone during the 1990s.⁹ While the data is limited, several studies have estimated the worldwide prevalence to be between 5% and 7.5% of children, but the number increases to 5%-18% within the human immunodeficiency virus (HIV) positive population and even reaches 30% among acquired immunodeficiency syndrome (AIDS) patients with a CD4+ count under 100/mL.^10-12 The infection is also observed at a higher frequency in certain geographic areas with tropical climates– such as Congo, Fiji, and Papua New Guinea – where the incidence can approach 20% in all children.¹³ It is not known whether this increased prevalence is due to a founder effect-associated genetic susceptibility of these populations to MCV infection or whether MCV becomes more virulent in tropical conditions.

Infection in immunocompetent patients is generally self-limiting and resolves on its own within 6 to 9 months. One study reported spontaneous resolution in 94.5% of patients within 6.5 months after initial infection; moreover, the same study reported that 23% of study participants were cured within one month after the first consultation with a dermatologist.¹⁴ It must be emphasized that spontaneous resolution primarily occurs in immunocompetent patients. In contrast, individuals with suppressed immune systems often suffer an increasing degree of recalcitrant molluscum contagiosum that is directly correlated with the level of immunodeficiency.

Biology and Pathogenesis of MCV

MCV is one of the largest human viruses. It is a member of the genus Molluscipoxvirus and contains a non-segmented single molecule of linear, double-stranded DNA 200-300 nm in length. The genome is covalently linked at both ends and encodes redundant, repeating sequences at both ends.¹⁵ Restriction endonuclease digestion is used to differentiate among the four subypes of molluscum contagiosum – MCV-1, MCV-1a, MCV-2, and MCV-3 – which all appear to cause very similar disease presentations.¹⁶ The genome of MCV-1 has been completely sequenced and several novel gene products involved in its pathogenesis and evasion of the immune system have been identified: MC54L, MC148, MC013L, MC159, and glutathione peroxidase.^17-19 The protein MC54L prevents inflammation by binding the pro-inflammatory cytokine human interleukin-18.20 MC148 promotes viral replication by preventing infected keratinocytes from differentiating.²¹ MC159 interacts with Fas (CD95; a member of the tumor necrosis factor receptor family), tumor necrosis factor (TNF), and TNF-related apoptosisinducing ligand (TRAIL) to inhibit apoptosis.²² MC80R, a major histocompatibility complex (MHC) class I homolog, interferes with the host presentation of peptides specific to MCV and inhibits cell-mediated cytotoxicity of infected cells.²³ Finally, infected cells express glutathione peroxidase to prevent leukocyte-mediated oxidative damage.¹⁷ Together, these viral gene products are able to maintain active infection until the host immune system gradually prevails.

MCV likely enters the skin through small abrasions; this explains why eczema-prone and atopic individuals, who typically scratch inflamed areas on their skin, are more susceptible. Once MCV has penetrated the lower layers of the epidermis it begins to replicate, extending upwards, in the lower layers of the epidermis.²⁴ The estimated incubation period varies from 14 days to 6 months. When active infection commences, the epidermis hypertrophies, extending into the underlying dermis, and characteristic molluscum bodies (also known as Henderson-Paterson bodies) form inside cells of the stratum spinosum. As infection progresses, the molluscum bodies enlarge, causing the spinosa cells to migrate upwards while hyperplasia of the basal cell layer simultaneously replaces the migrating spinosa cells. Viral structures are rarely observed in the stratum basale, and the structure of the basal lamina remains intact. The hypertrophied epidermal cells project above the skin, which forms the characteristic tumor observed in molluscum contagiosum.²⁵ Interestingly, an inflammatory infiltrate is not observed until shortly before natural resolution of the lesion.²⁶

Diagnosis

The morphological appearance of molluscum contagiosum in most cases is sufficiently distinct to make a clinical diagnosis. However, some cases can be more challenging. Using a magnifying device to visualize the characteristic central umbilication often aids in diagnosis. If the magnifying lens does not yield a conclusive diagnosis, the clinician can biopsy the lesion and conduct either histopathological studies or polymerase chain reaction (PCR) methods. Histopathological analysis usually demonstrates epidermal hyperplasia producing a crater filled with molluscum bodies. Molluscum bodies, which are huge (up to 35 microns) discrete ovoid intracytoplasmic inclusion bodies, appear as large acidophilic granular masses, pushing the nucleus and numerous keratohyaline granules aside. Although identification of molluscum bodies is conclusive for diagnosis of molluscum contagiosum, molluscum bodies can be sparse and difficult to visualize in some densely inflamed lesions. Intact lesions display little inflammation while ruptured lesions show a mixed inflammatory response characterized by mononuclear cells, neutrophils, and multinucleated giant cells. Brick-shaped virions can usually be seen through negative-stain transmission electron microscopy.¹⁵

Molluscum contagiosum can bear similar clinical presentation to other disorders. Rarely, they may be confused with warts and, if located on the genitalia, may be mistaken for condyloma of a human papillomavirus infection.²⁷ Giant molluscum nodules can resemble basal cell epithelioma, keratoacanthoma, verruca vulgaris, condyloma acuminatum, or a warty dyskeratoma.²⁸

Clinical Management

In nearly all immunocompetent individuals, molluscum contagiosum is a self-limiting disease that will spontaneously resolve, usually without scarring. Allowing this natural resolution of infection without therapeutic intervention is an important and effective treatment strategy that also minimizes healthcare costs. Regardless of whether treatment is sought, it is imperative that the clinician advises the patient to avoid swimming pools, bathing with others, sharing of towels, and activities requiring physical contact with others.

Current therapeutic intervention in the treatment of molluscum contagiosum is intended merely to accelerate the eradication process. A systematic review in 2009 studied the efficacy of current therapeutic options for nongenital molluscum contagiosum and concluded that there was insufficient evidence to support the use of any treatment as being definitively effective.⁸ Nevertheless, for the rapid resolution of individual lesions, clinical experience advocates the use of curettage, cryotherapy, and some topical agents. Therefore, the patient may wish to seek treatment for a variety of reasons, including: alleviating pruritus; minimizing autoinoculation as well as transmission to others; addressing cosmetic concerns; and preventing scarring, secondary infection, or bleeding of the lesions.⁸ Patients with sexually transmitted molluscum contagiosum should receive early treatment to prevent the spread of infection to future sexual partners. Early treatment is also indicated for immunosuppressed patients whose infections can become severe. Prior to beginning treatment, the clinician should conduct a full-body skin examination to identify all lesions; failure to treat all lesions may lead to continued infection and autoinoculation.

Curettage, cryotherapy, and cantharidin are considered to be firstline treatment strategies due to their popularity and established efficacy for resolution of individual lesions. Curettage involves the physical removal of lesions with the use of a curette and is the preferred choice of treatment in the Netherlands.⁸ Risk factors for treatment failure include a high number of lesions and concomitant atopic dermatitis. Topical anesthetics can be used to reduce the pain and discomfort associated with curettage, which can be disturbing for some children. The development of small, depressed scars following curettage is possible and should be discussed with the parents or guardians prior to treatment. Cryotherapy entails the use of a cotton-tipped swab dipped in liquid nitrogen that is applied to individual lesions for 6 to 10 seconds each. A 2010 randomized trial comparing the efficacy of cryotherapy with the immunomodulator imiquimod found cryotherapy to be effective in completely curing all patients and yielding more rapid resolution than imiquimod.²⁹ However, cryotherapy also had an increased occurrence of adverse effects, such as pain, bullae, dyspigmentation, and mild scarring. Cantharidin – the most popular method of treatment among American dermatologists – is a topical blistering agent that is applied directly to the lesions, usually with the blunt end of a cotton swab.⁸ To prevent further autoinoculation or transmission, the site of treatment should then be covered with a bandage and washed with soap and water 2 to 6 hours after application. Treatments can be repeated every 2 to 4 weeks and are contraindicated for lesions located on the face, genitalia, or perianal regions. A retrospective study found 90% of children treated with cantharidin for molluscum experienced lesion clearance; the average number of patient visits to achieve complete resolution was 2.1.⁶ About 95% of parents of children participating in the study stated that they were satisfied with the treatment and would be willing to have their child treated again with cantharidin. Common side effects that were observed include transient burning, erythema, pain, and pruritus.

Several second-line therapies have been described, which include (but are not limited to): imiquimod, sinecatechins ointment, podophyllin, potassium hydroxide, salicyclic acid, topical retinoids, oral cimetidine, pulsed dye lasers, and silver nitrate. However, the efficacy of these treatment strategies in healthy patients is controversial. A 2009 review by van der Wouden et al and a 2006 review by Brown et al analyze comparative studies involving the aforementioned second-line treatments.⁸

Immunocompromised patients can develop severe, persistent infection by not only MCV but also opportunistic pathogens. Treatments that lead to wound formation, such as curettage, should be avoided since wounds elevate the risk of additional infection. Instead, imiquimod applied 3 nights per week is recommended.³⁰ For many HIV positive patients, recalcitrant lesions are sometimes resolved only after initiation of highly active antiretroviral therapy (HAART).³¹ Clearance of recalcitrant, refractory lesions in HIV positive patients has been achieved through the use of intravenous cidofovir, a nucleotide analog of deoxycytidine monophosphate.³² However, systemic cidofovir can be toxic on the kidneys, so topical cidofovir is currently being explored as a potential therapeutic agent. In Tyring’s investigative center, a therapy regimen of once daily 2% topical cidofovir ointment for 2 months successfully eradicated the facial lesions on an HIV patient. Furthermore, the authors used 0.05% ingenol
mebutate, which is most widely used to treat actinic keratoses (AK), to achieve complete
resolution of genital lesions in a renal transplant patient who was being treated concurrently for
AK. The proposed mechanism involves lesion necrosis and neutrophil-mediated, antibodydependent
cellular cytotoxicity.³³

Atopic dermatitis patients are at greater risk for scar formation with increasing number of lesions, so curettage is not advisable. Prior to treatment of the molluscum lesions, the physician should address the atopic dermatitis with corticosteroids and antihistamines. Other immunomodulatory agents may be useful in long-term therapy of atopic dermatitis, but these drugs may facilitate MCV infection.²⁶ In a recently published case report, Tyring et al demonstrated successful use of 0.015% ingenol mebutate gel resulting in complete resolution in
a child with atopic dermatitis.³⁴ Further work is necessary to determine the consistency of this
very favorable outcome.

Periocular lesions should be referred to an ophthalmologist for further treatment.

Conclusion

Currently, there is no FDA approved therapy for molluscum contagiosum and there is no widely accepted standard of care. Many pediatricians favor spontaneous resolution over aggressive therapy. However, research should continue to be conducted in hope of finding an answer to this common, pediatric infectious disease. In addition, sexually transmitted molluscum should be treated not only for the benefit of the patient, but also to prevent spread to sexual partners. Most importantly, standardized therapy should be developed for immunocompromised patients, who may suffer both physically and psychologically from widespread molluscum, especially on the face and genitalia. Future studies should investigate outcome measures such as recurrence and transmission rates, disease-related quality of life, and scarring. A standardized outcome measure for each treatment would facilitate comparative assessment.

Acknowledgements

The authors would like to thank Dr. Marigdalia Ramirez-Fort and Dr. Farhan Khan for their
editorial contributions and creative insight.

References

1. Rogers M, Barnetson RSC. Diseases of the skin. In: McIntosh N, Helms P, Smyth R, et al. editors. Forfar and Arneil’s textboook of pediatrics. 5th ed. New York: Churchill Livingstone, (2008).
2. Bargman H. Is genital molluscum contagiosum a cutaneous manifestation of sexual abuse in children? J Am Acad Dermatol. 1986 May;14(5 Pt 1):847-9.
3. Villa L, Varela JA, Otero L, et al. Molluscum contagiosum: A 20-year study in a sexually transmitted infections unit. Sex Transm Dis. 2010 Jul;37(7):423-4.
4. Whitaker SB, Wiegand SE, Budnick SD. Intraoral molluscum contagiosum. Oral Surg Oral Med Oral Pathol. 1991 Sep;72(3):334-6.
5. Rao VA, Baskaran RK, Krishnan MM. Unusual cases of molluscum contagiosum of eye. Indian J Ophthalmol. 1985 Jul-Aug;33(4):263-5.
6. Silverberg NB, Sidbury R, Mancini AJ. Childhood molluscum contagiosum: experience with cantharidin therapy in 300 patients. J Am Acad Dermatol. 2000 Sep;43(3):503-7.
7. Braue A, Ross G, Varigos G, et al. Epidemiology and impact of childhood molluscum contagiosum: a case series and critical review of the literature. Pediatr Dermatol. 2005 Jul-Aug;22(4):287-94.
8. van der Wouden JC, van der Sande R, van Suijlekom-Smit LW, et al. Interventions for cutaneous molluscum contagiosum. Cochrane Database Syst Rev. 2009(4):CD004767.
9. Molino AC, Fleischer AB, Jr., Feldman SR. Patient demographics and utilization of health care services for molluscum contagiosum. Pediatr Dermatol. 2004 Nov-Dec;21(6):628-32.
10. Schwartz JJ, Myskowski PL. Molluscum contagiosum in patients with human immunodeficiency virus infection. A review of twenty-seven patients. J Am Acad Dermatol. 1992 Oct;27(4):583-8.
11. Stefanaki C, Stratigos AJ, Stratigos JD. Skin manifestations of HIV-1 infection in children. Clin Dermatol. 2002 Jan-Feb;20(1):74-86.
12. Koopman RJ, van Merrienboer FC, Vreden SG, et al. Molluscum contagiosum; a marker for advanced HIV infection. Br J Dermatol. 1992 May;126(5):528-9.
13. Kaye JW. Problems in therapy of molluscum contagiosum. Case report. Arch Dermatol. 1966 Oct;94(4):454-5.
14. Takemura T, Ohkuma K, Nagai H, et al. The natural history of molluscum contagiosum. Examination Treatment Dermatol Dis (Japanese). 1983;5(7): 667-70.
15. Damon IK. Poxviruses. In: Knipe DM, Howley PM, editors. Fields virology. 5th ed. Philadelphia: Lipincott Williams & Wilkins, p2963-5 (2007).
16. Nakamura J, Muraki Y, Yamada M, et al. Analysis of molluscum contagiosum virus genomes isolated in Japan. J Med Virol. 1995 Aug;46(4):339-48.
17. Senkevich TG, Bugert JJ, Sisler JR, et al. Genome sequence of a human tumorigenic poxvirus: prediction of specific host response-evasion genes. Science. 1996 Aug 9;273(5276):813-6.
18. Xiang Y, Moss B. IL-18 binding and inhibition of interferon gamma induction by human poxvirus-encoded proteins. Proc Natl Acad Sci USA. 1999 Sep 28;96(20):11537-42.
19. Bertin J, Armstrong RC, Ottilie S, et al. Death effector domain-containing herpesvirus and poxvirus proteins inhibit both Fas- and TNFR1-induced apoptosis. Proc Natl Acad Sci USA. 1997 Feb 18;94(4):1172-6.
20. Agromayor M, Ortiz P, Lopez-Estebaranz JL, et al. Molecular epidemiology of molluscum contagiosum virus and analysis of the host-serum antibody response in Spanish HIV-negative patients. J Med Virol. 2002 Feb;66(2):151-8.
21. Chen N, Baudino T, MacDonald PN, et al. Selective inhibition of nuclear steroid receptor function by a protein from a human tumorigenic poxvirus. Virology. 2000 Aug 15;274(1):17-25.
22. Garvey TL, Bertin J, Siegel RM, et al. Binding of FADD and caspase-8 to molluscum contagiosum virus MC159 v-FLIP is not sufficient for its antiapoptotic function. J Virol. 2002 Jan;76(2):697-706.
23. Smith KJ, Yeager J, Skelton H. Molluscum contagiosum: its clinical, histopathologic, and immunohistochemical spectrum. Int J Dermatol. 1999 Sep;38(9):664-72.
24. Pierard-Franchimont C, Legrain A, Pierard GE. Growth and regression of molluscum contagiosum. J Am Acad Dermatol. 1983 Nov;9(5):669-72.
25. Shelley WB, Burmeister V. Demonstration of a unique viral structure: the molluscum viral colony sac. Br J Dermatol. 1986 Nov;115(5):557-62.
26. Brown ST, Nalley JF, Kraus SJ. Molluscum contagiosum. Sex Transm Dis. 1981 Jul-Sep;8(3):227-34.
27. Ha SJ, Park YM, Cho SH, et al. Solitary giant molluscum contagiosum of the sole. Pediatr Dermatol. 1998 May-Jun;15(3):222-4.
28. Brown J, Janniger CK, Schwartz RA, et al. Childhood molluscum contagiosum. Int J Dermatol. 2006 Feb;45(2):93-9.
29. Al-Mutairi N, Al-Doukhi A, Al-Farag S, et al. Comparative study on the efficacy, safety, and acceptability of imiquimod 5% cream versus cryotherapy for molluscum contagiosum in children. Pediatr Dermatol. 2010 Jul-Aug;27(4):388-94.
30. Liota E, Smith KJ, Buckley R, et al. Imiquimod therapy for molluscum contagiosum. J Cutan Med Surg. 2000 Apr;4(2):76-82.
31. Calista D, Boschini A, Landi G. Resolution of disseminated molluscum contagiosum with Highly Active Anti-Retroviral Therapy (HAART) in patients with AIDS. Eur J Dermatol. 1999 Apr-May;9(3):211-3.
32. Meadows KP, Tyring SK, Pavia AT, et al. Resolution of recalcitrant molluscum contagiosum virus lesions in human immunodeficiency virus-infected patients treated with cidofovir. Arch Dermatol. 1997 Aug;133(8):987-90.
33. Javed S, Trying SK. Ingenol mebutate for the therapy of molluscum contagiosum. J Am Acad Dermatol. In press 2014.
34. Rosen RH, Gupta AK, Tyring SK. Dual mechanism of action of ingenol mebutate gel for topical treatment of actinic keratoses: rapid lesion necrosis followed by lesion-specific immune response. J Am Acad Dermatol. 2012 Mar;66(3):486-93.