3.1. Imiquimod effect in immune system
Imiquimod ((IMQ) is a non-nucleoside heterocyclic amine which belongs to the class of 1H-imidazo-[4,5-c] quinolones (Garland, 2003; Gupta et al., 2002; Sauder, 2003; Suzuki et al., 2000). The precise mechanism of action of this synthetic molecule is unknown. However, preclinical studies revealed that imiquimod modifies the immune response by enhancing both the innate and adaptive immune system, in particular the cell-mediated pathways (Stanley, 2002). Generally, imiquimod acts as an immune response modifier, in an indirect manner, as it induces immune reactions and the secretion of many cytokines, which in turn stimulate T cells (Dahl, 2002; Gupta et al., 2002; Reiter et al., 1994). Moreover, it has been used as a potent antiviral and antitumor agent in different animal models (Dahl, 2002; Gupta et al., 2002; Reiter et al., 1994).
Innate immune system is based on the recognition of pathogens from the organism and activation of many cell types, which eliminate them. Imiquimod exerts its action in innate immune system by binding to cell surface receptors, such as toll like receptors (TLRs). There are 10 types of TLRs that recognize microorganisms or specific components derived from pathogens. Imiquimod exerts its biological action through TLR-7 and TLR-8 (Garland, 2003; Stanley, 2002; Schön and Schön, 2007; Vidal, 2006; Yoon et al., 2019). This interaction leads to activation of a signaling cascade, which promotes translocation of nuclear factor-kappa B (NF-κΒ). NF-κΒ binds to DNA and induces the expression of many pro-inflammatory cytokines from the peripheral blood mononuclear cells, such as interferon-α (IFN-α), tumor necrosis factor α (TNF-α), interleukin IL-1, IL-2, IL-6, IL-8, IL-12, granulocyte colony stimulating factor (GM-CSF), granulocyte macrophage colony stimulating factor (GM-CSF), as well as chemokines, such as CCL4 and CCL2 (Chosidow and Dummer, 2003; Dahl, 2002; Gupta et al., 2002; Reiter et al., 1994; Stanley, 2002; Schön and Schön, 2007; Vidal, 2006). The acute antiviral and antitumor effects of imiquimod are largely originating from its ability to induce innate immune responses, especially its ability to induce secretion of IFN-α and other cytokines, such as IL-6, IL-12 and TNF-α, which has been observed in many studies (Dahl, 2002; Reiter et al., 1994; Vidal, 2006; Yoon et al., 2019). Increased expression of IL-1 and IL-6 leads to the activation of T lymphocytes and IL-12, which is produced by macrophages, induces the production of IFN-γ by natural killer (NK) and T cells. Furthermore, imiquimod activates antigen presenting cells, such as dendritic cells, macrophages activated to secrete both cytokines and nitric oxide and B lymphocytes activated to proliferate and differentiate, which in turn activate the adaptive system (Chosidow and Dummer, 2003; Dahl, 2002; Gupta et al., 2002).
Another cell type that is activated and migrates towards the side of the infection, in response to IFN-α, IFN-γ and IL-12 are Langerhans cells, which in turn induce immune responses. These cells show increased mobility in the presence of imiquimod migrating to the regional lymph node and function as major antigen presenting cells (Chosidow and Dummer, 2003; Dahl, 2002; Gupta et al., 2002). Moreover, cytokines that are produced from innate immune responses, induce indirectly the production of IFN-γ from T helper cell type 1 (Th1), which are part of the adaptive immune system. IFN-α enhances the expression of IL-12 receptor B2 subunit on Th1 cells, which in turn respond to IL-2 and become the main source of IFN-γ. Moreover, IFN-γ in combination with IL-2 produced from Th1 cells, activates CD8 cells that are converted to cytotoxic T cells, in order to eliminate cells infected from virus and to provide the immune memory, which is necessary for a future contamination (Chosidow and Dummer, 2003; Gupta et al., 2002; Stanley, 2002; Vidal, 2006; Schön and Schön, 2007; Vidal 2006, 2006; Yoon et al., 2019). Furthermore, IFN-γ and IFN-α inhibit the production of cytokines IL-4 and IL-5 from Th2 cells (Vidal, 2006; Vidal, 2006). Overall, imiquimod has a unique mode of action as it does not directly kill the infected cells, but its strong antiviral and antitumor activity is due to its ability to enhance the production of many pro-inflammatory cytokines and to trigger an immune response (Chosidow and Dummer, 2003; Dahl, 2002; Gupta et al., 2002). The role of Imiquimod to stimulate innate immunity indicates its potential to treat viral infections, such as SARS-CoV-2 in early stages of the disease, where activation of innate immunity by a TLR-7 agonist is of vital importance. However, in late stage infected patients by SARS-CoV-2 Imiquimod could provoke further up-regulation of the “cytokine storm”, thus an alternative therapeutic intervention should be chosen for late stage infection, as described by Zhao et al., 2019.
3.2. Imiquimod in clinical therapy
Imiquimod (IMQ), an immune response modifier, has shown to have antiviral and antitumor attributes. Specifically, imiquimod induces (2′–5′)-oligoadenylate synthetase, which confers an antiviral state and upregulates natural killer cells activity in vivo and in vitro. IMQ also enhances cell-mediated immunity (Miller et al., 1999). Lately Nerurkar et al., 2017, presented upregulated levels of chemokines in imiquimod treated mice presented three to five days after treatment. Fuertes et al., 2019 observed a higher IMQ efficacy for the treatment of anal condyloma compared to anal HSIL in HIV-infected individuals.
In in vitro studies against human peripheral blood mononuclear cells (PBMCs), imiquimod at 1–5 μg/ml induces the production of several cytokines 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) and macrophage inflammatory protein 1-α (MIP-1), MIP-1β and macrophage chemotactic protein (MCP-1) (Fuertes et al., 2019; Gibson et al., 1995) and at a low drug concentration about 0,5 μg/ml is found that IFN-a and IL-1RA are the only cytokines increased (Papadavid et al., 2007). Moreover, in in vivo studies that IMQ was administrated orally to mice (p.o.), stimulated a dose-dependent increase in serum levels of IFN-α, being active with doses as low as 3 mg/kg (Dahl, 2002). IFN-α levels were detectable 1 h after treatment, with peak levels occurring at 2 h after treatment. Optimal dosage of imiquimod ranged between 10 and 100 mg/kg, with higher dosages (150–250 mg/kg) inducing similar secretion levels. Multi-dose regimens were also tested, separated by 2 h, showing enhanced levels of IFN-α. Multiple doses of imiquimod on the same day caused augmented IFN-α levels, however high daily doses of IMQ to mice resulted in a hypo responsive state characterized by reduced cytokine induction, while separation of the doses by four or more days caused normal levels of cytokine induction. In rats, oral administration of imiquimod, in doses 2 mg/kg or more, induced increased serum levels of IFN-α and TNF-α and the kinetics of induction were similar to those seen in mice induction (Dahl, 2002).
Very recently, a thought-provoking analysis was put forward, demonstrating the effect of IMQ against influenza A virus infection (To et al., 2019). The authors provided with proof that delivery of imiquimod in mice, directly to the lungs via intranasal administration resulted in decreased expression in viral replication, airway inflammation, leukocytes levels (i.e. inflammatory cells such as macrophages, neutrophil, eosinophil), and pro-inflammatory cytokines/chemokines (IL-6, IFN-γ, IL-1β, GCSF, CCL3, CXCL2, TNF-α)) following influenza A virus infection. The effect was actually stronger in intranasal administration compared to an epicutaneous one. The therapy resulted in a near 5-fold elevation in Type I IFN-β in the lung tissue of mice after 3 days of treatment. Moreover, imiquimod significantly suppressed the mRNA levels of IL-6, CCL3, CXCL2 and IL-1β, otherwise increased in non-IMQ treated mice, whereas yielded a significantly higher antibody response in IgG1, IgG2a, IgE, IgM and total levels of IgG during infection. Additionally, TRL7 activation by IMQ was observed through a pronounced Type I IFN prompted response. Finally, triggered response of adaptive immune system was observed by efficient activation on the response of T lymphocytes (CD8+) and T helper cells (CD4+). In overall, IMQ effective treatment of influenza A virus was presented by suppression of inflammatory cells, cytokines/chemokines, activation of important type I IFN, and triggered adaptive system responses, while no significant lung dysfunction was observed as presented by tissue damping of naïve in comparison to influenza infected animals (To et al., 2019). Imiquimod, a potent TLR7 agonist, could be a strong primer of the immune response to infectious pulmonary viruses, such as CoVs ( ).
Table 2
Characteristic examples of in vivo animal trials and clinical trials of Imiquimod administration with virus disease, such as influenza A, H1N1, H3N2 and influenza B virus.
| Disease |
Nr of patients |
Middle age of patients (years) |
Administration |
Period of treatment |
Outcome |
Side effects/adverse events |
Ref. |
| In vivo animal tests against influenza A virus and H1N1 |
| Influenza A virus |
– |
– |
directly to the lungs via intranasal |
– |
decreased expression in viral replication, airway inflammation, leukocytes levels |
– |
To et al. (2019) |
| H1N1 Influenza Virus |
– |
– |
intraperitoneal |
– |
stronger B cell responses to proliferate and differentiate into antigen specific IgM and IgG secreting antibodies with viral neutralizing activity |
– |
Li et al. (2018) |
| Clinical phase 2 b/3 trial with Vaccines against Influenza H1N1, H3N2 virus |
| H1N1 |
216 |
18–30 |
topical treatment with imiquimod immediately before intradermal influenza
vaccination |
Single dose |
Seroconversion, seroprotection, increased geometric mean titre (GMT) |
Fever, headache, malaise, myalgia, arthralgia, and severe adverse events and local symptoms included redness,
swelling, induration, ecchymosis, and pain |
Hung et al. (2016) |
Numerous clinical trials present imiquimod as a potent and versatile compound that can enhance cellular activity and innate immunity (Grimm et al., 2012; Kaspari et al., 2002; Kjaer et al., 1996; Kreuter et al., 2006; Mao et al., 2006; McCuaig et al., 2009; Schiffman et al., 1993). Especially, clinical trials in humans either with topical treatment or with suppositories revealed prevention of recurrences in anal canal condyloma, whereas in infants with infantile hemangioma the moderation in surface erythema was notably faster than in prior experience (Kaspari et al., 2002; McCuaig et al., 2009). Kreuter et al., 2006, further stressed the fact that application of imiquimod suppositories in intra-anal HPV Types 6 and 11 in HIV-Infected men after the surgical removal of intra-anal Condylomata Acuminata may yield better recurrences under the absence of sexual intercourse, while HIV-associated immunosuppression probably leads to a new increase in HPV-11 DNA load. Both Kaspari et al., 2002 and Kreuter et al., 2006 utilized 5% imiquimod (in cream Aldara) in suppositories anally. IMQ due to its antiviral and anti-inflammatory action has also been researched in its role for the treatment of HPV in the development of cervical intraepithelial neoplasia (CIN) (Kjaer et al., 1996; Mao et al., 2006; Schiffman et al., 1993). Specifically, in fifty-nine patients suffering from CIN an incremental administration was followed, in which administered dose was increased every two weeks until the maximum dosage of three vaginal suppositories (6.25 mg IMQ). CIN patients were divided in two groups one of placebo administration and the other of IMQ suppositories.66 The IMQ group presented higher histologic regression and remission in correlation to the placebo one. A 60% clearance of CIN was observed in the IMQ treated patients proving a well-established antiviral effect of imiquimod against HPV (Grimm et al., 2012).
In other trials the topical application of IMQ was researched (de Berker et al., 2017; Marks et al., 1988; Schwartz, 1996; Torres et al., 2007). Actinic keratosis, a common cutaneous, pre-cancerous neoplasm appearing as rough, dry, scaly lesions that occur primarily on chronic ultraviolet (UV) light exposure on skin of middle-aged and elderly people is a field that researchers took interest in (de Berker et al., 2017; Marks et al., 1988; Schwartz, 1996). Torres et al., 2007 through a double-blind, placebo-controlled, randomized study in 17 patients presented an increased expression of TLR3, TLR7, and TLR8, consistent with increased expression observed in human peripheral blood mononuclear cells upon treatment with imiquimod. The induction of several members of the cytoplasmic helicase innate immune pathway, as well as several TLRs, indicates that in addition to activation of the TLR7 pathway treatment with IMQ also results in priming of other innate pathways, which may augment other aspects of the innate immune response. Moreover, it is also stated the fact that the increase of CD8β, SELL, NT5E (CD73), LGALS2 (galectin 2), and LAIR1 (leukocyte-associated immunoglobulin-like receptor 1) receptors, as well as T-cell receptor (TCR) subunits TRD and TRG, TCR-signaling pathway genes (such as Fyn, Fyb and LCP2), genes associated with T-cell activation (such as HCK, CD69, PTPRC (CD45) SELL (CD62L, L-Selectin)), ITGA4 (antigen CD49D, alpha 4 subunit of VLA-4 receptor), and LAG3 are indicative of the stimulation of the adaptive immune system.70 Observations regarding the increase of T-cell activation were also provided in a fifty-two patient study with topical treatment and the same administration by van Seters et al., 2008.
The local anti-tumor effect of topical TLR7 agonist imiquimod 5% cream in breast cancer patients with skin metastases was investigated in a ten-patient study (de Berker et al., 2017). In this study, the variability of preexisting lymphocytic infiltrates within the cutaneous metastases and the lack of consistent quantitative changes of the infiltrate in biopsies were in contrast to the induction of a T-cell inflammatory infiltrate. The authors speculated that the effect of imiquimod may depend on the tumor microenvironment. Nevertheless, it is stated that IMQ can promote a pro-immunogenic tumor microenvironment with histological tumor regression based on the evidence of an immune-mediated response (Adams et al., 2012). Concerning Vulvar Paget Disease (VPG), there have also been attempts of imiquimod treatment (Cowan et al., 2016; Feldmeyer et al., 2011; Marchitelli et al., 2014; Sendagorta et al., 2010; van der Linden et al., 2012). van der Linden et al., 2012, have already performed a twenty patient study following treatment with 5% IMQ, topically, three times per week over a 16-week period. Other researchers and physicians have presented either case reports, where IMQ 5% was administered topically in 3 patients daily for 3 weeks and every other day for 3 following weeks (Sendagorta et al., 2010) or one patient 3 times per week (Feldmeyer et al., 2011) or greater in groups for every other day in 10 patients (Marchitelli et al., 2014) or 3 times per week in 8 patients (Cowan et al., 2016). A high clinical and histologic remission of the disease was achieved in high occurrence (75% in Cowan et al., 2016, 90% in Marchiteli et al., 2014), whereas Feldmeyer et al., 2011 confirmed the findings in his study as well. The above mentioned studies summarize the potency of IMQ in the handling of VPG.
There have also been interesting attempts to utilize IMQ as a vaccine adjuvant. Immunization of guinea-pigs with Herpes Simple Virus glycoprotein and imiquimod, reduced effectively virus recurrence in comparison with unimmunized controls (Gibson et al., 2002). In particular, even though the mechanism of action was not identified, the results indicated that the use of IMQ as an enhancer significantly diminished recurrent lesion days by 53–69% (Gibson et al., 2002). The effect of IMQ has also been examined against Influenza Virus, where Li et al., 2018 focused on the combination of imiquimod with H1N1/415742Md influenza virus particle. Specifically, a compound of IMQ with the virus (50 μg/10 μg, respectively) were intra-peritoneal administered in mice, whereas other treatment schemes were utilized as controls (such as, IMQ only group and H1N1 influenza only group). An upregulation of cytokines expressions was noted in both Th-1, Th-2 cytokines with elevated IL-10 secretion levels induced in the IMQ only group, while in the virus only group cytokine storm with increased levels of pro-inflammatory cytokines (IL-6, IFN-γ, IL-2, IL-4 and IL-5) was observed. The outcome revealed that the combination (IMQ and virus group) induced much stronger B cell responses to proliferate and differentiate into antigen specific IgM and IgG secreting antibodies with viral neutralizing activity. The main result was that imiquimod integrated with vaccine antigen can advance potent B cell activation and differentiation leading to accelerated viral specific antibody production, which contribute to the protection against imminent incoming pathogen (Li et al., 2018). In an another novel research, imiquimod has been administrated systemically in order to be tested as an adjuvant that improves immunogenicity of a tumor-lysate vaccine, inducing the rejection of a highly aggressive T-cell lymphoma (Papakostas, 2015). The results indicate that Tumor cell lysate vaccination using imiquimod as an adjuvant, enhanced the protection from tumor growth and induced a Th1-type as well as humoral immune responses against LBC cells. The research concluded that imiquimod administered alone significantly enhanced immune response to a tumor lysate vaccine and produced an elevated number of CD4+ T-cells and an IFN-γ Th1-type response along with specific antibodies (Papakostas, 2015).
Imiquimod has been evaluated in controlled phase 2 b/3 clinical trial as a topical pre-treatment agent in combination with trivalent influenza vaccination against influenza B, H1N1 and H3N2 viruses (Hung et al., 2016). By this study, improved protection against circulating strains of influenza viruses was provided with effective immunogenicity of influenza vaccination due to the topical imiquimod pretreatment before intradermal vaccination.
3.3. Imiquimod oral availability
Imiquimod, a TLR7/8 agonist that induces a potent anti-viral response, is characterized by the production of type I interferons (IFN), pro-inflammatory cytokines and chemokines (Gibson et al., 2002). A growing literature clearly points a systemic response following topical imiquimod treatment. The tissue response to Aldara treatment across a detailed time-course model determined the most likely mechanism driving systemic inflammation (Nerurkar et al., 2017a, 2017b). It has been shown that topical Aldara treatment induced a potent chemokine and cytokine response throughout the peripheral tissues and brain, with these responses being temporally distinct (Nerurkar et al., 2017a, Nerurkar et al., 2017b). IMQ binded to TLR7 on inflammatory cells, such as the Langerhans cells of the epidermis, dendritic cells and monocytes and induced cytokines secretion (IFN-α and TNF-α) (Papakostas, 2015). Specifically, the results also presented that IMQ was present in both plasma and brain as early as 4 h after treatment, suggesting that the primary mechanism of immune activation of topical Aldara treatment was through the direct ligation of IMQ with TLR7 receptors throughout the body (Nerurkar et al., 2017a, Nerurkar et al., 2017b). IMQ treatment could induce unintended medium grade systemic side effects that ranged in severity and frequency, including fever, fatigue, headaches, erythema, myalgia, application site inconvenience and raised erythrocyte sedimentation rate (Del Rosso et al., 2009; Gollnick et al., 2020; Kumar and Narang, 2011; Schwartz, 1996).
According to a clinical pharmacokinetic report conducted in order to assess the effect of food on the oral IMQ absorption, to characterize its pharmacokinetics, and to estimate its oral bioavailability on individuals that received a 100 mg oral dose of IMQ, the oral bioavailability was near 47%, with an absorption half-life of close to 1 h and independent of food consumption (Soria et al., 2000). The study suggested that food provided no effect on the rate, extent of absorption or bioavailability of oral imiquimod, and proved the suitability of IMQ for oral administration (Soria et al., 2000). In a phase I clinical study, tolerability, toxicity and biological effects of daily oral imiquimod administration were investigated in 21 patients with refractory cancer.88 Patients were treated with doses of 25 mg, 50 mg, 100 mg or 200 mg on a projected 112 day course, in which only three patients completed the course, all at the 50 mg dose. Treatment toxicities were dose related and mainly comprised flu-like symptoms, nausea and lymphopenia. Interferon production was not demonstrated within the first 24 h of the initial dose but, following repeated doses, ten of the patients developed detectable serum interferon concentrations with a maximum value of 5600 IU ml recorded. Daily oral administration of imiquimod presented dose-depented activation of the interferon production system but at higher doses resulted in flu-like side effect (Savage et al., 1996).
Παρόλα αυτά τα φιλοκυβερνητικά ΜΜΕ επιμένουν πως υπάρχουν ουρές έξω από τα εμβολιαστικά κέντρα, μάλιστα σύμφωνα να μελέτη του Ινστιτούτου Πολιτικής Υγείας, που διενεργήθηκε τον Οκτώβριο του 2020, σε τυχαίο και αντιπροσωπευτικό δείγμα 855 ενηλίκων άνω των 18 ετών από όλη την επικράτεια, και η οποία δημοσιεύθηκε την 1η Ιουνίου στο επιστημονικό περιοδικό «Journal of Evaluation in Clinical Practice» το 74% των Ελλήνων τάσσεται υπέρ του υποχρεωτικού εμβολιασμού, ενώ ένα 62% δηλώνει την πρόθεσή του να εμβολιαστεί έναντι της Covid-19!
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