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Effects of a novel topical immunomodulator, imiquimod, on keratinocyte cytokine gene expression.

Kono T, Kondo S, Pastore S, Shivji GM, Tomai MA, McKenzie RC, Sauder DN.

Division of Dermatology, University of Toronto, Canada.

A novel immunomodulator, imiquimod, has been shown to be an effective topical antiviral and antitumor agent in animal models. Imiquimod has been reported to induce interferon-alpha and other cytokines in animals and humans, but its precise role as an immunomodulator at skin sites has not been determined. We investigated its effect on cytokine gene expression in the human epidermal carcinoma cell line COLO-16 and human keratinocytes. COLO-16 cells were incubated with imiquimod (1 and 10 micrograms/ml) and human keratinocytes with 5 micrograms/ml for 6 or 24 h. Cytokine gene expression was analyzed by reverse-transcriptase PCR. In COLO-16 cells, imiquimod stimulated IL-6 mRNA levels 2.3- and 4.4-fold at 1 and 10 micrograms/ml after 6 h. IL-8 mRNA increased 4-fold at both 1 and 10 micrograms/ml. At 24 h, though IL-6 mRNA level at 1 micrograms/ml was further stimulated, enhanced expressions of IL-8 at 1 micrograms/ml and both IL-6 and IL-8 at 10 micrograms/ml were down-regulated. In human keratinocytes, 5 micrograms/ml of imiquimod stimulated IL-6 mRNA levels 1.4-fold at 6 h and 2.1-fold at 24 h, and IL-8 mRNA levels 1.7- and 2.0-fold at 6 and 24 h. IL-1 alpha mRNA levels in COLO-16 or keratinocytes were unchanged by either dose or incubation time. These results suggest that stimulation of IL-6 and IL-8 expression may be involved in the immunomodulating action of imiquimod.

Online source: www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=8061117&dopt=Abstract imiquimod Aldara



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Adjuvant effects of imiquimod on a herpes simplex virus type 2 glycoprotein vaccine in guinea pigs.

Bernstein DI, Miller RL, Harrison CJ.

James N. Gamble Institute of Medical Research, Cincinnati, Ohio 45219.

The adjuvant effects of imiquimod for a herpes simplex virus (HSV) vaccine were evaluated. Guinea pigs were immunized with 35 micrograms of lectin-purified HSV-2 glycoproteins 14 and 35 days before intravaginal HSV-2 inoculation. Immunizations were given either alone, with complete Freund's adjuvant, or with three different 5-day regimens of imiquimod. Although immunization alone decreased the severity of the acute disease and viral replication, in two separate experiments, the addition of imiquimod produced further decreases. The addition of subcutaneous imiquimod further decreased viral shedding by > 3 logs on day 1 after virus inoculation (P < .001). All groups that received immunization and imiquimod also developed significantly fewer HSV recurrent lesion days (P < .05-.001) compared to immunization alone. No recurrent lesions were detected in the group that received immunization and subcutaneous imiquimod. Imiquimod enhanced the effectiveness of HSV-2 immunization, especially in reducing recurrent HSV disease, and should be evaluated further as an adjuvant.

Online source: www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=8382722&dopt=Abstract imiquimod Aldara



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Effects of therapy with an immunomodulator (imiquimod, R-837) alone and with acyclovir on genital HSV-2 infection in guinea-pigs when begun after lesion development.

Bernstein DI, Miller RL, Harrison CJ.

James N. Gamble Institute of Medical Research, Cincinnati, OH 45219.

We evaluated the effects of the immunomodulator, imiquimod (R-837) applied topically, alone and in combination with i.p. acyclovir (ACV) on acute, recurrent and neural HSV-2 genital infection in guinea-pigs when 10 days of therapy was begun after HSV lesions developed. The combined therapy was most effective, significantly reducing the severity of the acute disease, as early as 2 days (P < 0.05), and vaginal viral shedding (P < 0.05) as early as 1 day after therapy was begun. The total lesion score for the acute disease was also significantly less in the group receiving imiquimod and ACV (5.4 +/- 0.5) compared to controls (13.1 +/- 1.2, P < 0.001) or imiquimod alone (9.8 +/- 1.2, P < 0.05). Therapy, however, had no significant effect on the number of days with recurrent lesions. Imiquimod increased the lymphoproliferative response to HSV (P < 0.01), while combined therapy reduced HSV antibody titers (P < 0.01) on day 28 compared to placebo and also reduced the effect of imiquimod alone on the lymphoproliferative response. The combination of this effective immunomodulator, imiquimod, and acyclovir appears to provide effective therapy for acute genital HSV-2 infection even when begun after lesion development.

Online source: www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=8457147&dopt=Abstract imiquimod Aldara



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Effect of a novel topical immunomodulator, S-28463, on keratinocyte cytokine gene expression and production.

Fujisawa H, Shivji GM, Kondo S, Wang B, Tomai MA, Miller RL, Sauder DN.

Division of Dermatology, University of Toronto, Sunnybrook Health Science Centre, Canada.

A new immunomodulating agent, imiquimod, has been reported to have antiviral and antitumor activities in animal models. S-28463 (4-amino-2-ethoxymethyl-alpha, alpha-dimethyl-1H-imidazo[4, 5-c]quinoline-1-ethanol), an analog of imiquimod, has more potent antiviral activity in animals than imiquimod. It has also been shown to be more potent at inducing cytokines in human blood in vitro. However, its precise role as an immunomodulator in the skin has not been determined. We investigated the effect of S-28463 on human keratinocyte (KC) production of interferon-alpha (IFN-alpha) and other proinflammatory cytokines, including interleukin (IL)-1alpha, IL-8, and tumor necrosis factor-alpha (TNF-alpha). Human KC were incubated with S-28463 at two concentrations (1 microgram/ml and 10 micrograms/ml) for 6 h. Cytokine gene expression was analyzed by reverse-transcriptase PCR. In human KC, S-28463 stimulated significant increases in IFN-alpha mRNA at both concentrations. IL-1alpha mRNA increased 1.4-fold at 10 micrograms/ml. IL-8 mRNA was upregulated 2.5-fold at 10 micrograms/ml. Twenty-four hours after treatment, IL-1 alpha, IL-8, and TNF-alpha protein were increased, but IFN-alpha was below the level of detection. These results suggest that in the skin, S-28463-induced-IL-1 alpha, IL-8, and TNF-alpha production may be involved in the immunomodulating action of S-28463.

Online source: www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=8836922&dopt=Abstract imiquimod Aldara



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Induction of cytokines in cynomolgus monkeys by the immune response modifiers, imiquimod, S-27609 and S-28463.

Wagner TL, Horton VL, Carlson GL, Myhre PE, Gibson SJ, Imbertson LM, Tomai MA.

3M Pharmaceuticals, Department of Pharmacology, 3M Center, 270-2S-06, St Paul, MN 55144, USA.

Imiquimod, S-27609 and S-28463 are imidazoquinolines known to have antiviral and antitumour properties mediated by the induction of cytokines, in particular interferon alpha (IFN-alpha). This study evaluated these compounds for their ability to induce cytokines and cytokine specific messenger RNAs (mRNA) in cynomologus monkeys (Macaca fascicularis). Peripheral blood mononuclear cell (PBMC) cultures from monkeys produced IFN, interleukin 1beta (IL-1beta), IL-6 and IL-8 after treatment with imiquimod, S-27609 and S-28463. Tumour necrosis factor alpha (TNF-alpha) was also increased in cultures stimulated with S-27609 or S-28463. Monkey PBMCs stimulated with imiquimod, S-27609 and S-28463 showed increased mRNA levels of IFN-alpha, IL-1alpha, IL-6 and the IFN inducible protein, MxA above those seen in untreated cultures. S-27609 and S-28463 also had higher TNF-alpha mRNA expression than cultures not receiving drugs. When compared to lipopolysaccharide (LPS), S-27609 was less effective at inducing IL-1beta, IL-6, IL-8 and TNF-alpha but induced higher concentrations of IFN. Similar results were seen when evaluating cytokine mRNA levels. Upon oral administration to monkeys, S-28463 stimulated a dose-dependent increase in serum concentrations of IFN, TNF-alpha, IL-1 receptor antagonist (IL-1Ra) and IL-6, while imiquimod induced increases in IFN and IL-1Ra concentrations. Finally, skin biopsies from monkeys treated topically with S-28463 had increases over baseline in mRNA for IFN-alpha, IL-1alpha, IL-6 and MxA protein. The data show that imidazoquinolines induce cytokines and cytokine specific mRNA in cynomolgus monkeys. These results demonstrate the usefulness of human amplimers and human ELISAs in the detection of cytokine specific mRNAs and proteins in cynomolgus monkeys. Copyright 1997 Academic Press Limited.

Online source: www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=9367544&dopt=Abstract imiquimod Aldara



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Attenuation of virus-induced airway dysfunction in rats treated with imiquimod.

Stokes JR, Sorkness RL, Kaplan MR, Castleman WL, Tomai MA, Miller RL, Lemanske RF Jr.

Division of Allergy and Immunology, University of Wisconsin, Madison, USA.

Viral respiratory infections cause acute airway abnormalities consisting of inflammation and physiological dysfunction in both animals and humans. It is likely that inflammatory cell products, such as cytokines, contribute substantially to viral-induced airway dysfunction. We hypothesized that imiquimod, an immune response enhancing agent that induces interferon-alpha, would attenuate the development of airway dysfunction during acute viral illness in rats. Adult Brown Norway rats were inoculated with parainfluenza type 1 (Sendai) virus or sterile vehicle, and treated with either imiquimod or water. Respiratory system resistance (Rrs), arterial oxygen tension (Pa,O2), lung viral titres and bronchoalveolar lavage (BAL) leucocyte counts were measured in anaesthetized, paralysed, ventilated rats. Virus-infected, water-treated rats had a significant decrease in Pa,O2 and had significant increases in leucocyte count and Rrs when compared to both the virus-infected, imiquimod-treated, (Pa,O2, p = 0.03; leucocyte count, p = 0.02; and Rrs, p = 0.009) and noninfected, water-treated rats (Pa,O2, p = 0.007; leucocyte count, p = 0.001; and Rrs, p = 0.01). In addition, imiquimod suppressed BAL eosinophils in both virus-infected (p = 0.02) and noninfected (p = 0.001) groups, and lowered overall virus titres (p = 0.03). Thus, both virus-induced airway inflammation and physiological dysfunction were attenuated significantly by imiquimod treatment in this animal model. By further delineating mechanisms by which infections induce airway dysfunction in animal models, more specific pharmacological interventions can be developed for the treatment of virus-induced asthma.

Online source: www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=9551732&dopt=Abstract imiquimod Aldara