Imiquimod Applied Topically: A Novel Immune Response Modifier

S. Tyring, MD, PhD

Departments of Dermatology and Microbiology/Immunology, The University of Texas Medical Branch at Galveston, Texas, USA

ABSTRACT

Imiquimod (S-26308, R-837) (1-(2-methylpropyl)-1H-imidazo[4,5-c]quinolin-4 amine), an immune response modifier, was approved as a 5% cream (Aldara, 3M Pharmaceuticals) by the US FDA in February 1997, for the treatment of genital and perianal warts. Drug activity results primarily from induction of interferon alpha (IFN-α) and other cytokines in the skin, which stimulate several other aspects of the innate immune response. Imiquimod also stimulates acquired immunity, in particular the cellular arm, which is important for control of viral infections and tumors. It is expected to be effective where exogenous IFN-α has shown utility, and where enhancement of cell-mediated immunity is needed. Recently presented Phase II clinical studies demonstrated efficacy in treating UV induced skin lesions, basal cell carcinoma, and actinic keratosis. Case studies have reported benefit when treating molluscum and in prevention of keloids after surgery.

Key Words:
imiquimod, immune response modifier, warts

The Th1 CMI response is very effective in most people for controlling viral infections and tumors. For example, chicken pox is almost universal and after an outbreak, the varicella zoster virus responsible is carried in the dorsal root ganglia for the rest of the individual’s life. Usually, no further lesions occur, except in those 20% of individuals who eventually develop herpes zoster. In another example, epidemiology studies report the Human Papillomavirus (HPV) is a frequently occurring infection with 50- 75% of sexually active adults having an antibody response to the virus.¹ About 15% of these individuals carry the virus and a severe outbreak of warts can occur if the cellular immune response is suppressed because of anti-graft rejection drugs following transplantation, anti-cancer chemotherapy, acquiring HIV infection, or in some cases, pregnancy.

Genital warts, the most common viral sexually transmitted disease, was chosen as the first clinical target for imiquimod because injectable interferon alpha (IFN-α) had demonstrated some benefit, and current therapies had not met the physicians’ or patients’ needs. Patient dissatisfaction was significant due to pain, tissue destruction, high recurrence rates, expense, and the time required for treatment. As well, current treatments only targeted the visible warts and did not treat the underlying HPV infection. Published results indicate that biopsies from these patients’ warts showed little immune recognition, but biopsies from warts undergoing spontaneous regression showed monocytic cellular infiltration and increased Th1 cytokine expression.^2,3 Similar results were seen in patients treated with interferon.⁴

Mechanism of Action

The exact biochemical mechanism of action for imiquimod is not known. However, studies have reported the following evidence:

• In human peripheral blood mononuclear cells (PBMCs), specifically monocytes, imiquimod at a low concentration of 1- 5µg/ml induces cytokine production including several subtypes of IFN-α, TNF-α, IL-1, IL-1RA, IL-6, IL-8, IL-10, IL-12 p40, granulocyte colony stimulating factor (G-CSF), granulocyte/macrophage colony stimulating factor (GM-CSF), macrophage inflammatory protein 1-α (MIP-1α), MIP-1ß, and macrophage chemotactic protein (MCP-1).^5,6
• Topical application of the 1% or 5% cream formulation of imiquimod to the skin of hairless mice increases IFN-α messenger RNA (mRNA) levels, and higher protein concentrations of IFN and TNF-α in the skin at the treatment site.^7,8
• Topical treatment of hairless mice with imiquimod causes Langerhans cells in the skin to enlarge, appear activated and migrate from the treatment site to the regional lymph node.⁹ These activated cells may enhance antigen presentation to Tcells.
• Imiquimod was shown to be effective in animal models against a number of viral infections and a variety of transplantable tumors.^10,11 The duration of antiviral activity lasts for 3-4 days after each oral imiquimod administration and correlates with elevation of oligoadenylate synthetase (2′,5′-AS) activity, though this increase is indirect through IFN-α production.¹² Elevated 2′,5′-AS was observed in the serum of mice, rats, guinea pigs, monkeys, and humans7 from 24-72 hours after oral treatment.
• Antitumor activity of imiquimod is also seen in a number of transplantable mouse tumor models¹³ Much of this antitumor effect is blocked by administration of antibodies to IFN-α.
• Imiquimod was effective at inhibiting growth of the human mammary tumor MCF-7 when transplanted into nude mice lacking T-cells, indicating that acutely, T-cells are not required for its antitumor effects.¹³
• Although it does not stimulate T-cells to divide, nor does it directly induce T-cell cytokines such as IL-2, IL-4 or IL-5, imiquimod can indirectly stimulate production of the T helper type 1 (Th1) cytokine, IFN-γ, in mouse splenic and bone marrow cultures, as well as human PBMC cultures. Production of IFN-γ in response to imiquimod is inhibited by antibodies to IL-12 and IFN-α, demonstrating the importance of these monocyte/macrophage cytokines.¹⁴ The mechanism of interaction between these cytokines has recently been defined.^15,16 Results show that IFN-α induces the IL-12 receptor β2 subunit on Th1 cells. These cells can then respond to IL-12 and produce IFN-γ. Thus, Th1 cells are the major source of IFN-γ. However, cytotoxic T-cells and natural killer (NK) cells are also able to produce IFN-γ in response to imiquimod.
• Imiquimod was reported to inhibit production of the Th2 cytokine IL-5 in both mouse and human cell systems, and as a result, it has also been found to inhibit both antigen and Sephadex induced eosinophilia in several animal models.¹⁷
• Results from a Phase I double-blind, randomized, parallel group study done in humans showed that all imiquimod treated patients had a ≥75% reduction in wart area.¹⁸ Imiquimod treatment stimulated significant increases in IFN-α as well as increases in TNF-α mRNAs, cytokines previously found to be induced by imiquimod in animal studies^7,10,19,20 and in human PBMC studies.^5,6

Imiquimod may be useful in atopic diseases as well as other diseases where an increased Th1 response is needed. Wart regression by imiquimod is associated with an induction of local cytokines and cellular infiltrates that are involved with the generation of a cell mediated immune response.

Summary of Clinical Efficacy Trials

A Phase II study of 108 patients with genital warts compared topically applied 5% imiquimod cream to vehicle cream (see Table 1).²¹ The imiquimod group had 40% “complete wart clearance” compared to no “complete clearance” in the vehicle group. In addition, there was a median 90% reduction in wart area at the end of treatment among the imiquimod group, but no change in wart area in the vehicle treated group. Patients with totally cleared lesions entered a 10-week follow up period to observe wart recurrence and 81% of the imiquimod treated group remained wart free.

A Phase III multi-centered, randomized, double blind, placebo controlled trial compared the safety and efficacy of imiquimod 5% cream, 1% cream and vehicle.²² The main outcome measurements were the number of patients experiencing complete elimination of all baseline warts and wart recurrence. The reduction in baseline wart area, the duration of therapy required to eliminate warts, and the frequency and severity of adverse reactions were also monitored. Patients with total wart clearance entered into a 12-week follow up to monitor recurrence. The difference between the effectiveness of the 5% cream and vehicle was statistically significant (p<0.0001). The results using 1% cream were not significantly different from vehicle. The median time to clearance was 10 weeks, 12 weeks, and 12 weeks, respectively. Females had a higher clearance rate (77%, 46%, and 28%, respectively) than males (40%, 10%, and 6%, respectively), and females had a shorter median time to clearance (8 weeks) than males (12 weeks) in both imiquimod groups. The better response in females could be due to several factors including shorter duration of warts in females (3.4 months median) vs males (6.7 months median), better compliance for females, or better drug absorption in females. The treatment was well tolerated. Local erythema was the most common adverse reaction (67%, 26%, and 24%, respectively) but the majority of patients experienced no or only mild local inflammatory reactions.

In a second Phase III trial the difference between the effectiveness of the 5% cream and vehicle was also statistically significant (p<0.0001), though clearance in the 1% vs the vehicle group was not. Recurrence rates were 19% (9/48) for 5% imiquimod group, 17% (2/12) for the 1% group, and 0% (0/3) for the vehicle group. The low recurrence rate in the vehicle groups is not surprising since the mechanism of spontaneous clearance was shown to be due to immune recognition.2,3 Local skin reactions were more common and more severe with daily treatment, but there were no systemic adverse reactions. Results were again better in women. Both 3 times/week and daily treatment regimens were acceptable for safety and efficacy, however in the final analysis, most patients preferred the 3 times/week regimen.²³

A vehicle controlled safety and efficacy trial was done in HIVpositive genital wart patients.²⁴ The primary objective of this trial was to evaluate the safety of imiquimod 5% cream in HIVpositive patients. A secondary objective was to assess wart clearance and reduction in the wart area. No local skin reactions were seen in a majority of patients and only mild erythema was seen in most of the others. The difference between the effectiveness of the 5% cream and vehicle was not significantly different. However, there was a statistically significant difference between treatment groups for patients who achieved >50% reduction in wart area, 38% for imiquimod, and 14% for vehicle (p=0.013). This was a clinically meaningful reduction in wart area since wart area increases are frequently seen in these patients. These results suggest that in HIV patients, imiquimod induces the innate response that stops wart growth and causes wart area reduction and may, in part, be IFN-α mediated. However, the reduced total wart clearance in HIV patients compared to immunocompetent genital wart patients suggests a role for T-cell responses in initial wart clearance as well as in long term protection from recurrence. Imiquimod has an acceptable safety profile in HIV-positive and AIDS patients.

Imiquimod’s mechanism of action should also be effective for treating other chronic virus skin infections such as common warts, plantar warts, herpes simplex virus infection, and molluscum contagiosum, as well as skin tumors. Small studies have reported success in treating molluscum,^25,26 and a case report shows treatment of a patient with recalcitrant facial flat warts.²⁷ Results of a small pilot trial of imiquimod 5% cream in patients with Bowen’s disease showed that 14 of 16 patients cleared their lesions.²⁸ A pilot study showed success in treating basal cell carcinoma,²⁹ and Phase II results were recently reported (Geisse, personal communication, 2000 Oct). Phase II results were also reported for actinic keratosis (Stockfleth, personal communication, 2000 Oct). Other skin tumors that might respond include Kaposi’s sarcoma and cutaneous T-cell lymphoma since they have been reported to respond to interferon therapy.^30,31

Since imiquimod inhibits Th2 responses in preclinical animal models, atopic based skin inflammation such as atopic dermatitis might also benefit. Other conditions that have responded to topically applied imiquimod include alopecia areata (Stockfleth, personal communication, 2000 Oct) and keloids (Berman, personal communication, 2000 Oct).

Another possible use for these drugs is application with a vaccine for adjuvant activity. The imidazoquinolines are expected to enhance a Th1 response to the vaccine, which could be beneficial for virus or tumor vaccines. Drug application topically or transdermally could be explored with the injectable vaccine. On the other hand, skin inflammation due to excessive Th1 responses, such as psoriasis and contact dermatitis, might be worsened by topical treatment with imiquimod. Imiquimod is unique in being a topically active cytokine inducer and stimulant for the CMI response.

Conclusion

Wart regression by imiquimod is associated with an induction of local cytokines and cellular infiltrates that are involved with the generation of a cell mediated immune response. These results in humans are consistent with the preclinical results reported in animal models. Overall, imiquimod applied topically is an immune response modifier that would be a useful addition to the drugs that can be used to treat significant chronic conditions of the skin. As such, imiquimod applied topically, represents a new class of drug.

References

1. Koutsky L. Epidemiology of genital human papillomavirus infection. Am J Med 102(5A):3-8 (1997 May).
2. Tagami H, Oku T, Iwatsuki K. Primary tissue culture of spontaneously regressing flat warts. In vitro attack by mononuclear cells against wart-derived epidermal cells. Cancer 55(10):2437-41 (1985 May).
3. Coleman N, Birley HD, Renton AM, et al. Immunological events in regressing genital warts. Am J Clin Pathol 102(6):768-74 (1994 Dec).
4. Arany I, Tyring SK. Activation of local cell-mediated immunity in interferonresponse patients with human papillomavirus-associated lesions. J Interferon Cytokine Res 16(6):453-60 (1996 Jun).
5. Weeks CE, Gibson SJ. Induction of interferon and other cytokines by imiquimod and its hydroxylated metabolite R-842 in human blood cells in vitro. J Interferon Res 14(2):81-5 (1994 Apr).
6. Gibson SJ, Imbertson LM, Wagner TL, et al. Cellular requirements for cytokine production in response to the immunomodulators imiquimod and S-27609. J Interferon Cytokine Res 15(6):537-45 (1995 Jun).
7. Tomai MA, Birmachu W, Case MT, et al. Imiquimod: in vivo and in vitro characteristics and toxicology. In: Aly R, Beutner KR, Maibach H, Eds. Cutaneous Infection and Therapy. New York: Dekker 32:417-28 (1997).
8. Imbertson LM, Beurline JM, Couture AM, et al. Cytokine induction in hairless mouse and rat skin after topical application of the immune response modifiers imiquimod and S-28463. J Invest Dermatol 110(5):734-9 (1998 May).
9. Suzuki H, Wang B, Amerio P, et al. Imiquimod, a novel topical immune response modifier induces migration of Langerhans cells. (Abstr.) J Invest Dermatol 110(4):566 (1998 Apr).
10. Miller RL, Imbertson LM, Reiter MJ, Gerster JF. Treatment of primary herpes simplex virus infection in guinea pigs by imiquimod. Antiviral Res 44(1):31-42 (1999 Nov).
11. Kende M, Lupton HW, Canonico PG. Treatment of experimental viral infections with immunomodulators. Adv Biosci 68:51-63 (1988).
12. Bottrel RAT, Levy DE, Tomai M, Reis LFL. STAT-1 is a key target for imiquimod, an oral inducer of interferon. (Abstr.) J Interferon Cytokine Res 17(Suppl 2):S62 (1997).
13. Sidky YA, Borden EC, Weeks CE, Reiter MJ, Hatcher JF, Bryan GT. Inhibition of murine tumor growth by an interferon-inducing imidazoquinolinamine. Cancer Res 52(13):3528-33 (1992 Jul).
14. Tomai MA, Ahonen CL, Couture AM, et al. Effects of the imidazoquinolines, imiquimod and S-28463, on Th1 and Th2 cytokine responses in vitro. (Abstr.) J Invest Dermatol 110(4):651 (1998 Apr).
15. Rogge L, Barberis-Maino L, Biffi M, et al. Selective expression of an interleukin- 12 receptor component by human T helper 1 cells. J Exp Med 185(5):825-31 (1997 Mar).
16. Szabo SJ, Dighe AS, Gubler U, Murphy KM. Regulation of the interleukin (IL)- 12R b2 subunit expression in developing T helper 1 (Th1) and (Th2) cells. J Exp Med 185(5):817-24 (1997 Mar).
17. Hammerbeck DM, Tomai MA, Hupperts AM, McGurran SM, Reiter MJ, Wagner TL. The imidazoquinolines and related compounds as inhibitors of pulmonary eosinophilia. (Abstr.) Amer J Resp Crit Care Med 155(4 part 2):A624 (1997).
18. Tyring SK, Arany I, Stanley MA, et al. A randomized, controlled, molecular study of condylomata acuminata clearance during treatment with imiquimod. J Infect Dis 178(2):551-5 (1998 Aug).
19. Reiter MJ, Testerman TL, Miller RL, Weeks CE, Tomai MA. Cytokine induction in mice by the immunomodulator imiquimod. J Leukoc Biol 55(2):234-40 (1994 Feb).
20. Wagner TL, Horton VL, Carlson GL, et al. Induction of cytokines in cynomolgus monkeys by the immune response modifiers, imiquimod, S-27609 and S-28463. Cytokine 9(11):837-45 (1997 Nov).
21. Beutner KR, Spruance SL, Hougham AJ, Fox TL, Owens ML, Douglas JM. Treatment of genital warts with an immune-response modifier (imiquimod). J Am Acad Dermatol 38(2 Pt 1):230-9 (1998 Feb).
22. Edwards L, Ferenczy A, Eron L, et al. Self administered topical 5% imiquimod cream for external anogenital warts. HPV Study Group. Human Papilloma Virus. Arch Dermatol 134(1):25-30 (1998 Jan).
23. Beutner KR, Tyring SK, Trofatter KF, et al. Imiquimod, a patient-applied immuneresponse modifier for treatment of external genital warts. Antimicrob Agents Chemother 42(4):789-94 (1998a Apr).
24. Conant MA, Opp KM, Gilson RJC, et al, & HPV Study Group. A vehiclecontrolled safety and efficacy trial evaluating 5% imiquimod cream for the treatment of genital/ perianal warts in HIV-positive patients. (Abstr.) Presented at American Academy of Dermatology 56th Annual Meeting, Orlando, FL (1998).
25. Buckley R, Smith K. Topical imiquimod therapy for chronic giant molluscum contagiosum in a patient with advanced human immunodeficiency virus 1 disease. Arch Dermatol 135(10):1167-9 (1999 Oct).
26. Hengge UR, Goos M, Arndt R. Topical treatment of warts and mollusca with imiquimod. Ann Intern Med 132(1):95 (2000 Jan).
27. Schwab RA, Elston DM. Topical imiquimod for recalcitrant facial flat warts. Cutis 65(3):160-2 (2000 Mar).
28. MacKenzie-Wood A, de Launey J, Kossard S, Svans R, Fox T, Owens M. Safety and efficacy of imiquimod 5% cream for the treatment of Bowen’s Disease. (Abstr.) J Invest Dermatol 110(4):684 (1998 Apr).
29. Beutner KR, Geisse JK, Helman D, Fox TL, Ginkel A, Owens ML. Therapeutic response of basal cell carcinoma to the immune response modifier imiquimod 5% cream. J Am Acad Dermatol 41(6):1002-7 (1999 Dec).
30. Krown SE. Interferon and other biologic agents for the treatment of Kaposi’s sarcoma. Hematol Oncol Clin North Am 5(2):311-22 (1991 Apr).
31. Papa G, Tura S, Mandelli F, et al. Is interferon alpha in cutaneous T-cell lymphoma a treatment of choice? Br J Haematol 79(suppl 1):48-51 (1991 Oct).

The Use of Photodynamic Therapy as an Antimicrobial Agent

There is increasing concern worldwide about bacterial resistance to antibiotic therapy. An alternative
approach may be the use of antimicrobial photodynamic therapy (APDT), which involves the use of light in
the presence of a photosensitizing agent to kill microbes.

B. Zeina, et al, in the British Journal of Dermatology1 reported that they used a combination of methylene
blue and visible light against a range of microbial species representative of those encountered on the skin
in health and disease. They determined the kill rates for these species (i.e., Staphylococcus aureus, S.
epidermidis, Streptococcus pyogenes, Corynebacterium minutissimum, Propionibacterium acnes, and
Candida albicans) and found that it was proportional to the light intensity used.

All the microbial test species were susceptible to APDT, but the eukaryotic species (C. albicans) was much
less susceptible than were the prokaryotic bacteria. The hypothesized that the reasons for this might be
due to the presence of a nuclear membrane, creating an additional barrier, or may also reflect differences
in cell size/volume. Candida species are about 25-50 times larger than the bacterial test species.

Other investigators studied the effect of PDT using light in the violet-blue range (407-420nm) on P. acnes in
35 patients.2,3 After 8 treatments, 80% of the subjects showed significant improvement in their acne. UV
was totally blocked and no side effects were noted.

These initial reports indicate that PDT could be used as a safe and effective procedure enabling the
successful treatment of control of a variety of microbe-associated skin diseases.

Reference

1. Zeina B, Greenman J, Purcell WM, Das B. Killing of Cutaneous microbial species by photodynamic therapy. Brit J Dermatol
   144:274-8 (2001).
2. Harth Y, Ellman M, Shalita AR. Acne phototherapy – 3 center clinical study. A Poster presentation at the 59th annual meeting of
   the American Academy of Dermatologists, March 2001, in Washington DC.
3. Harth Y, Ellman M, Shalita AR. Phototherapy for acne. A Poster presentation at the 59th annual meeting of the American
   Academy of Dermatologists, March 2001, in Washington DC.

National Registry Established for Patients with Alopecia Areata

Alopecia areata is an autoimmune disease that affects more than 4 million people in the US alone, and
whose hallmark symptom is unexplained hair loss. The National Institute of Arthritis and Musculoskeletal
and Skin Diseases (NIAMS) has established a national registry for patients to provide medical and family
history with three major forms of this disease:

• alopecia areata (patchy scalp hair loss)
• alopecia totalis (100% scalp hair loss)
• alopecia universalis (100% scalp and body hair loss).

Families with multiple affected members will be especially helpful in locating the gene or genes associated
with this condition.

The registry will serve as a liaison between affected families and investigators who are interested in
studying this disorder and serve as a central information source to facilitate research. Patient enrollment
is projected to begin in the fall of 2001, and is limited to residents of the US. The registry contact is:

Madeline Duvic, MD.
Phone: 713-792-5999
Fax 713-794-1491
e-mail: alopeciaregistry@mdanderson.org