Genital warts treatment: Beyond imiquimod

ABSTRACT

Genital warts are one of the most common sexually transmitted diseases worldwide. The disease is a result of infection with low-risk types of human papillomaviruses, mostly type 6 and 11. Current therapies for genital warts are mainly ablative, or alternatively topical application of imiquimod cream and sinecatechin (polyphenon E) ointment to the warts. However, low patient compliance and high recurrence rate are significant problems for the treatment of genital warts by imiquimod and ablative therapies. We summarise recent literature in this area and propose combining imiquimod with other therapies to increase the efficacy of imiquimod.

KEYWORDS:

• genital warts
• imiquimod
• early protein 7
• interleukin 10

Infection with human papillomaviruses (HPV) results in diseases ranging from benign warts to cancers, with more than 180 subtypes reported.^1-3 The high-risk subtypes, especially HPV16 and 18, are associated with the development of cervical cancer and a subset of head and neck squamous cell carcinomas; the low-risk subtypes, such as HPV6 and HPV11, are responsible for a sexually transmitted disease, genital warts (condylomata acuminate (CA)).⁴

Genital warts are one of the most common sexually transmitted diseases worldwide.^5-7 Approximately 1% of the sexually active population has symptomatic genital warts.^8,9 More than 30 subtypes of HPV are able to infect the genital mucosa, with types 6 and 11 found in 90% of individuals affected.^10,11 The disease is transmitted mostly through sexual contact, although vertical transmission and autoinoculation have also been reported.^12,13

Following the initial clinical manifestation, genital warts may increase in number and size or, alternatively, undergo a spontaneous regression.¹⁴ In fact, approximately 30 percent of all warts will regress within the first four months of infection.¹⁵ However, the majority of genital warts will recur within three months of infection, even after undergoing appropriate treatments.¹⁵

Currently, treatment of genital warts focuses on removal of warty tissues, rather than eradicating the virus. Common treatments include 1) topical therapeutic agents; 2) physical removal of warts with cryotherapy, electrocautery or lasers; or 3) photodynamic therapy.^14,16,17 All these therapies, although effective to some extent, have high recurrence rates, and require long-term or repeat treatment. Recommended first-line therapies include topical application of imiquimod cream, an immunomodulatory agent, and sinecatechin (polyphenon E) ointment if the warts are small clusters <30cm². Second line therapies, using ablation methods such as laser/electrocautery, are recommended if the warts are large, or areas more than 30cm².^14,16,17

HPV uses many strategies to avoid the clearance of genital warts

HPV utilises multiple mechanisms to avoid being cleared by the immune system.^1,2 HPV infects keratinocytes (KC), but does not lyse of infected cells and causes minimal inflammation of the infected lesions. The maturation and antigen presentation function of Langerhans cells (antigen presenting immune cells of the skin) are inhibited by HPV infection.^18,19 HPV is able to down-regulate type I interferon (IFN) mediated antiviral immune responses,^20,21 and therefore antigen presenting cells are not properly activated for antigen processing and presentation. HPV early protein E7 also inhibits interferon-γ-mediated enhancement of keratinocyte antigen processing and inhibits HPV infected KCs lysis by T-cells. HPV E7 expressing KCs exhibit impaired IFN-γ-induced transcriptional upregulation of major histocompatibility complex (MHC) class I antigen processing and presentation-associated genes, and of membrane MHC class I restricted peptide/MHC class I complexes.²² Moreover, FOXP3+ regulatory T cells with a suppressive function have been observed to accumulate in large warts along with high expression of IL-10, TGFβ1 and low expression of IL-2 and IFNγ.²³ The accumulation of FOXP3+ regulatory T cells in large warts can be partly ascribed to the chemotaxis of CCL17 and CCL22, derived from the Langerhans cells and macrophages in warts.²³ HPV early protein E7 from HPV11, and HPV6 enhances IL-18 binding protein (IL-18BP) expression. The increased IL-18BP levels seen in E7-expressing KCs are capable of diminishing IL-18-mediated CD4 lymphocyte activation.²⁴ The increased IL-18BP production is through the CR3 region of E7 and this ability is shared among E7 proteins from both high and low -risk HPV subtypes. Furthermore, mutagenesis shows that increased IL-18BP production is mediated by a gamma-activated sequence (GAS) in the IL-18BP promoter. Recently, it has been shown that the levels of immune-tolerant indoleamine-2,3-dioxygenase (IDO+) increase in condyloma acuminate, and IDO+ antigen presenting cells are able to foster the generation of T regulatory T cells.²⁵ Taken together, these strategies, used by HPV, account for some of the difficulties inthe treatment of genital warts.

Imiquimod

Imiquimod is an immune modulator and directly activates innate immune cells through Toll-like receptor 7 (TLR-7).^26-29 Imiquimod elicits a robust pro-inflammatory response by dendritic cells in vitro, after binding to TLR7.³⁰ In addition, topical administration of imiquimod induces functional maturation of epidermal Langerhans cells in vivo, stimulates migration of these cells to regional lymph nodes; and promotes a Th1 biased and antigen-specific CD8+ T cell response.³¹ Imiquimod also facilitates antigen-specific CD8+ T-cell accumulation in the genital tract, and resulting in tumour growth inhibition through IFN γ.³² Imiquimod also interferes with adenosine receptor signaling pathways and the compound causes receptor-independent reduction of adenylyl cyclase activity.³³ Imiquimod induces apoptosis of tumour cells that involve caspase activation and at least in part depends on p53, Bcl-2 or A20 (Table 1).^33-35

Table 1. Enhancing the efficacy of imiquimod by combining with other therapies.

Molecule	Mode of Action	Synergy with Imiquimod	Reference
Imiquimod	Stimulates innate immune responses and induces apoptosis of HPV infected cells		^40^,^59
Interleukin 10 inhibitor	Interleukin 10 signalling blockade	Amplify the stimulation of innate immune responses by imiquimod	^47-49
Natural peptides	Inhibits the growth of HPV infected cells, stimulating inflammation signalling pathways	Both imiquimod and the natural peptides stimulate innate immune responses, cause apoptosis of HPV infected cells	^53
Therapeutic vaccine	Generates antigen specific T cells	Attracts more antigen specific T cells to the warts	^58^,^60

The 5% Imiquimod cream was first approved by the FDA in the late 1990s for the immunotherapeutic treatment of external anogenital warts with a regimen of once-daily application 3 times per week until complete clearance of warts or for a maximum of 16 weeks.³⁶ In 2010 the FDA approved a 3.75% Imiquimod cream.³⁷ This latter cream is available in a 7.5g pump that dispenses 0.235 g of cream per full actuation of the pump after priming.³⁷ Compared with 5% Imiquimod cream, 3.75% The primary cure rates are as high as 5% imiquimod. However, the 3.75% Imiquimod cream has improved patient compliance and fewer side effects to 5% cream.^37,38 Local inflammatory reactions are the most common adverse effect experienced with Imiquimod but these are generally well tolerated. The most common local inflammatory reaction reported in one study was erythema, occurring in 71 (67.0%) of 106 patients treated with 5% imiquimod cream.³⁹ Complete clearance of warts was observed in 45% to 56% in patients treated with the imiquimod, while in only 6% (3% to 8%) of patients with placebo cream. Recurrence occurred in 16% of those treated with imiquimod 5%, to (8%–22%) in those with placebo. Whether recurrences following treatment show a reduced occurrence and severity compared to untreated subject is not very well studied, although immune compromised patients show more frequent recurrences compared to those with normal immune function.⁴⁰

HPV E6 and E7 have been reported to strongly inhibit NFκB activation in response to imiquimod. Inhibition of imiquimod-induced cytokine production is dependent on residues in the CR1 and CR3 regions of E7 and results in reduced nuclear translocation and acetylation of the p65 subunit of NFκB.⁴¹

Taken together, the treatment of genital warts by local application of imiquimod cream remains lengthy and recurrence rates remain to be improved. Increasing the efficacy of imiquimod for the treatment of genital warts is t urgently needed.

Imiquimod and interleukin 10 (IL-10) inhibitor

IL-10 is a cytokine with multiple biological activities. The main biological function of IL-10 is to suppress the inflammatory response and regulate the differentiation and proliferation of T cells, B cells, natural killer cells, antigen-presenting cells, mast cells, and granulocytes. IL-10 acts on antigen presenting cells (APCs) by down-regulating the expression of MHC class II and co-stimulatory molecules and the production of reactive oxygen and nitrogen intermediates. IL-10 also acts directly on IL-10 receptor-expressing T cells to reduce their cytokine production and pathological effects.^42-44

Imiquimod acts on TLR 7 receptors and can stimulate antigen presenting cells to produce pro-inflammatory cytokines to boost adaptive immune responses, as well as enhance the secretion of IL-10 by antigen presenting cells to prevent excessive immune responses and avoid autoimmune disease. In a mouse tumour model, imiquimod induced high levels of IL-10 in addition to TNF-α and IFNγ. Elevated serum IL-10 appeared to be derived from dual cytokine secreting (IFNγ+ and IL-10+) CD4+ T cells. Blockade of IL-10, but not TGF-β, enhanced the anti-tumour effect of imiquimod by significantly prolonging the survival of tumour bearing mice.⁴⁵ Recently, it was demonstrated that mice depleted of B cells or interleukin (IL)-10-deficient B cells show a fulminant inflammation upon imiquimod exposure, whereas ablation of NFATc1 in B cells resulted in a suppression of imiquimod-induced inflammation. These data indicate a close link between NFATc1 and IL-10 expression in B cells.⁴⁶ Recently, two peptides designed through computer modelling have been shown to inhibit IL-10 mediated signaling both in vitro and in vivo.^47-49 Temporally blocking IL-10 signaling at the time of immunization in mice does not cause an excessive inflammatory response.⁵⁰ These results argue that imiquimod may achieve better efficacy against genital warts if used together with an IL-10 inhibitor. This would block the IL-10 production caused by imiquimod, therefore amplifying the pro-inflammatory responses by imiquimod with minimal side effects. However, whether there are any compensatory effects from other receptors or pathways to blocking the IL-10R is unclear.

Imiquimod and natural peptides

Imiquimod activates dendritic cells and subsequently boosts the adaptive T cell response. Imiquimod also has the ability to induce apoptosis of viral infected cells and tumour cells. If more HPV infected KCs are made apoptotic when Langerhans cells are activated by imiquimod, more HPV related antigens will be taken up and processed by LCs, activating a more robust adaptive immune response.

During the last three decades, more than 200 host-defence peptides have been isolated and identified from skin secretions of Australian frogs and toads, using high-performance liquid chromatography-tandem mass spectrometry, and many of these peptides show antimicrobial activity; or neuropeptide-type activities, such as inhibiting the aggregation of Amyloid beta 42, which is the major precursor of the extracellular fibrillar deposits of Alzheimer's disease.⁵¹ The caerin 1 peptides have previously been shown to be potent membrane-active peptides stopping the formation of nitric oxide by neuronal nitric oxide synthase.^52,53 The caerin 1.1 peptide exhibits a helix-hinge-helix structure in membrane-mimicking solvents as confirmed by NMR studies, but shows a random structure in water.⁵⁴ It has been previously reported that caerin 1.1 has an anti-cancer effect against a number of human cancer cell lines (including leukemia, lung, colon, CNS, melanoma, ovarian, renal, prostate and breast cancers cell lines). Another peptide, caerin 1.9, has antimicrobial activity against a wide spectrum of Gram-positive and Gram-negative microbial strains.⁵⁵ It had been proposed that caerin 1-induced disruption of the cancer cell membrane was the major contributing factor to its activity, which leads to an excessive flux of ions and small molecules across the cytoplasmic membrane bilayer.⁵³ Recently, we demonstrated that these two peptides are able to inhibit the growth of HPV+ cell lines in vitro (unpublished data). Furthermore, their ability to inhibit HPV+ cell growth is significantly increased when imiquimod and the two peptides are present in combination. Moreover, the two peptides also inhibit HPV+ tumour growth in mice (unpublished data). Given that these peptides don't influence normal cells, this data suggests that these peptides interact with certain proteins on the HPV-infected cell membrane. Therefore, we propose that peptides caerin 1.1 and caerin 1.9 may: 1): lyse of HPV infected cells and/or ii) interact with particular receptors on the abnormal cell membrane. This subsequently caauses the release of more HPV related antigen for imiquimod activated antigen presenting cells to uptake and process, and activation of more T cells to kill HPV infected KCs, ultimately improving the efficacy of imiquimod.

Imiquimod and therapeutic vaccination

TLR ligands have been used to increase the immune response elicited by vaccination.^32,56,57 It is observed that CRT/E7 DNA vaccination in combination with imiquimod leads to an enhancement in the E7-specific CD8+ T cell immune response and a decrease in the number of myeloid-derived suppressor cells in the tumour microenvironment of tumour-bearing mice. Treatment with CRT/E7 DNA in combination with imiquimod also leads to significantly improved anti-tumour effects and prolonged survival in tumour bearing mice.³² In addition, the immunization leads to increased number of NK1.1+ cells and F4/80+ cells in the tumor microenvironment. In another study, topical treatment of melanoma metastases with the TLR7 agonist imiquimod plus administration of a multi-peptide cancer vaccine improved immune cell infiltration of melanoma metastases. Treatment of metastases with imiquimod plus vaccination leads to favorable gene signatures of the tumour.⁵⁸ Therefore, therapeutic vaccination, together with local administration of imiquimod may acquire better efficacy for the treatment of genital warts.

Summary and future directions

Genital warts are one of the most common sexually transmitted diseases worldwide. Current treatments incorporating the physical removal of warts and local administration of imiquimod are far from satisfactory, due to the length of time for successful treatment and high reoccurrence rate. Imiquimod activates innate and subsequent adaptive immune responses through activation of Toll-like receptor 7. Based on published literature, we propose that the efficacy of topical application of imiquimod for the treatment of genital warts may be increased if an IL-10 inhibitor is simultaneously used, or combined with natural peptides isolated from frog or toad. Finally, topical application of imiquimod may attract more T cells elicited by a therapeutic vaccine, and therefore may better eliminate HPV infected KCs of genital warts.

Disclosure of potential conflicts of interest

No potential conflicts of interest were disclosed.