ISSN 19907508, Biochemistry (Moscow) Supplement Series B: Biomedical Chemistry, 2013, Vol. 7, No. 2, pp. 136–145. © Pleiades Publishing, Ltd., 2013. Original Russian Text © S.V. Bozrova, V.A. Levitsky, S.A. Nedospasov, M.S. Drutskaya, 2013, published in Biomeditsinskaya Khimiya.

Imiquimod: The Biochemical Mechanisms of Immunomodulatory and AntiInflammatory Activity S. V. Bozrovaa, b, V. A. Levitskya, c, S. A. Nedospasova, b, and M. S. Drutskayaa, c, 1 a

Engelhardt Institute of Molecular Biology RAS, ul. Vavilova 32, GSP1, Moscow, 119991 Russia tel.: +7 (499) 1359964; email: [email protected] bDepartment of Immunology, Biological Faculty, Lomonosov Moscow State University, Moscow, Russia c Oncology Department, Johns Hopkins University School of Medicine, Roche Glycart, Schlieren, 8952 Switzerland Received May 28, 2012

Abstract—Imidazoquinolines are a new group of compounds that recently have been introduced into clinical practice as antitumor and antiviral immunomodulators. Structurally, they are low molecular weight synthetic guanosinelike molecules. Although imiquimod, the most widely used imidazoquinoline, has been recom mended for treatment of several forms of skin cancer and papillomas, its molecular mechanisms of action are not fully understood. In particular, imiquimod is known as a specific agonist of Tolllike receptor 7 (TLR7) and this capacity is widely used in a large number of experimental studies and clinical trials. Nevertheless, detailed analysis of the published data suggests that the biological activity of imiquimod can not be explained only by its interaction with TLR7. Certain evidence exists that imiquimod directly interacts with adenosine receptors and other molecules that regulate synthesis of cyclic adenosine monophosphate. A detailed under standing of the biochemical basis of immunomodulating and antitumor effects of imiquimod will increase its clinical effectiveness and accelerate the development of new drugs with similar but improved pharmaceutical characteristics. This review summarizes the published data about effects of imiquimod on various intracellu lar biochemical processes and signaling pathways. Keywords: imidazoquinolines, imiquimod, tumor necrosis factor, antiinflammatory drug, cAMP DOI: 10.1134/S1990750813020042 1

INTRODUCTION

Imidazoquinolines are a new group of prescription drugs recently introduced into clinical practice as antitumor immunomodulators. Structurally, this class of low molecular weight synthetic compounds repre sents guanosine derivatives. Although imiquimod, the most widely used imidazoquinoline, has been recom mended for treatment of several forms of skin cancer and papillomas, its molecular mechanisms of action are not fully understood. For example, imiquimod is 1 To whom correspondence should be addressed.

Abbreviations used: AC—adenylate cyclase, APC—antigen pre senting cell; ATP—adenosine triphosphate, cAMP—cyclic adenosine monophosphate, CCPA—chloroN6cyclopentyl adenosine, CHO—Chinese hamster ovary, COX2—cyclooxy genase2, CNS—central nervous system, DC—dendritic cell, fMLP—formylmethionylleucylphenylalanine, GCSF— granulocyte colonystimulating factor, IFN—interferon, IL— interleukin, IP10—interferongamma induced protein 10, HPV—human papillomavirus, MIIC—compartment of major histocompatibility complex class II, MCP—monocyte chemoattractant protein, MHC—major histocompatibility complex, MIP1α, 1β—macrophageinduced protein 1α, 1β, NFκB—nuclear factor kappa B, NK—natural killer, OGF— opioid growth factor, OGFR—receptor of opioid growth factor, PDE—phosphodiesterase, PKA—protein kinase A, Th—Thelper, TCR—Tcell receptor, TLR—Tolllike receptor, TNF—tumor necrosis factor.

known as a specific agonist of Tolllike receptor 7 (TLR7) and this capacity is widely used in a large number of experimental studies and clinical trials. Nevertheless, certain evidence exists in the literature that the biological activity of imiquimod cannot be explained only by its interaction with TLR7. Experi mental data suggest that imiquimod directly interacts with adenosine receptors and other molecules that regulate synthesis of cyclic 3',5'adenosine mono phosphate. Understanding of the biochemical basis of immunomodulating and antitumor effects of imiqui mod will increase its clinical effectiveness and acceler ate the development of new drugs with similar but improved pharmaceutical characteristics. This review summarizes the published data about effects of imi quimod on various intracellular biochemical processes and signaling pathways. 1. MOLECULAR MECHANISMS OF IMIDAZOQUINOLINE ACTION Imiquimod was originally characterized as an anti viral drug that was able to induce synthesis of anti inflammatory cytokines, possibly, via Tolllike recep tors (TLR). It was demonstrated that imiquimod and other imidazoquinolines interacted with TLR7, while

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resiquimod (R848), a structural analogue of imiqui mod interacted with both TLR7 and TLR8 (Fig. 1). Recently, a problem of imiquimod penetration into cells has attracted attention of researchers [1]. Studies on plasmacytoid dendritic cells (DC), expressing TLR7 and TLR8 as the only molecules of the TLR family, have shown that imidazoquinolines are con centrated in the LAMP1+CD63+HLADR+ endo somes. LAMP1 and CD63 are markers of late endo somes and lysosomes characterized by acidic pH. The lysosomal compartment of plasmacytoid DC special izes in antigenic presentation by MHC II and so one may presume that imiquimod is located in the MIIC compartment. However, since imiquimod is a weak base that is capable to cross the cell membrane via pas sive diffusion, it has been suggested that initially imi quimod penetrates passively into the cell and then is accumulated in the MIIC compartment, where its protonation prevents its escape [1]. It should be noted that highly selective interaction of imiquimod with TLR7 is now questioned by results of recent studies [2–5] suggesting existence of alterna tive mechanisms realizing imiquimod effects. TLR7 is an intracellular endosome receptor, which recognizes nucleotides and nucleosides (including those of viral origin). Early studies demonstrate the MyD88depen dent mode of imiquimod interaction with TLR7 [6] because peritoneal macrophages deficient in this adaptor molecule were insensitive to induction by imiquimod or resiquimod, whereas wild type mac rophages responded to these compounds by high expression of proinflammatory cytokines. The MyD88 mediated signaling cascade results in activa tion of the transcriptional factor NFκB followed by subsequent transcription of proinflammatory cytok ines such as TNF, IL2, IL6, IL12, IFNalpha [7]. This induction of proinflammatory cytokines causes a marked local immune response, which may be directed also against tumor cells of skin melanomas or nonmelanoma skin cancers [8]. In addition, the effect of imiquimod is, possibly, associated with activation of antigen presenting cells (APC) and increased production of various lym

phokines; this results in more effective activation of Tcells specifically recognizing tumorassociated or viral antigens [6, 9]. It should be noted that the effect of imiquimod is preferentially directed to DC [6, 7, 10]. One study characterized the effect of imiquimod or resiquimod on plasmacytoid DC, the main source of interferon during viral infection. It was shown that in response to imiquimod or resiquimod DC pro duced interferonalpha and interferonomega. Besides induction of interferon (IFN), resiquimod increased production of other cytokines (including TNF and IL10), upregulated expression of costimu lator molecules, CCR7, and also increased viability of plasmacytoid DC [11]. The other study demonstrated that imiquimod was able to act as a mast cell activator interacting with TLR7 [12]. Mucosal and skin mast cells are involved in innate immunity and inflammatory processes. Mast cells activated via TLR7 stimulate migration of Langerhans cells into lymph nodes, increase cytotoxic immune response, and participate in skin inflamma tory processes [13]. This underlies perspectives of imi quimod as an adjuvant for subcutaneous vaccination. The effect of imiquimod and some other imidazo quinolines was also investigated on DC [14]. Activa tion of TLR7 and TLR8 by imidazoquinolines resulted in expression of CD40, CD83, CD86, CCR7 and production of IL6 and IL12p40. However, stim ulation of only TLR8 was accompanied by production of IL12p70 and expression of IL12p35 mRNA. That study demonstrated that activation of TLR7 and TLR8 resulted in positive regulation of the transcrip tion factors JNK and NFκB followed by subsequent expression of CCR7, CD83, CD86, CD40, IL6, and IL12p40. Although p38MAPK was involved in the positive regulation of these markers in response to the TLR7mediated signal, this kinase caused the inhibitory effect on CD40 expression and production of IL12 in the TLR8mediated cascade in DC. In addition, it was demonstrated that the Jak/STAT sig naling cascade was involved in the positive regulation of CD40 expression realized via TLR7 and inhibited

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expression of cytokines and CD83. Thus, the consid ered study demonstrated that TLR7 and TLR8 acti vated similar signaling pathways, which, however, caused different effects on maturation of DC depend ing on particular receptor involved in signal transduc tion. Acting on B cells imiquimod and resiquimod increased their proliferation, and also expression of costimulator molecules and secretion of immunoglo bulins. Tomai et al. suggested that imidazoquinolines could act as the adjuvant for augmentation of the anti genspecific immune response [15]. The other study has shown that treatment of peripheral blood Bcells (CD19+) by imiquimod increased production of cytokines IL1α, IL1β, IL6, TNF, IL10, IL13, and chemokines MIP1α, MIP1β, MCP1, IP10, and IL8 [16]. Imiquimod may also act on keratinocytes [17]. Skin keratinocytes play an important role in innate immunity: they produce various chemokines, which attract leukocytes to the site of infection. Interestingly, TLR7 and TLR8 are not expressed on keratinocyte endosomes [18]. Imiquimod stimulated keratinocyte production of cytokines in a TLR7 independent man ner [2], possibly, acting via inhibition of adenylate cyclase (AC) and adenosine receptor signal transduc tion [3]. However, understanding of molecular mechanisms of imidazoquinoline action meets serious contradic tions. Fahey et al. reported that imiquimod and other TLR7 ligands exhibited different effects on Langer hans cells [19]. These APC located in epithelial tissues do initiate immune responses against various antigens (including human papillomavirus, HPV). HPV16 preferentially localized in the cervical mucosa is the major cause of cervical cancer [20]. Langerhans cells express endosomal TLR7 and TLR8. Imidazoquino lines induce specific antiviral response in the HPV16 pretreated Langerhans cells. Imidazoquinolines are simultaneous stimulators of both adaptive and innate immunity. The imidazoquinolineinduced innate immune response causes activation of adaptive immu nity via secretion of proinflammatory cytokines by activated macrophages and DC; this triggers the Th1 mediated response. Imidazoquinolines influence CCR7 and CCL21targeted migration of Langerhans cells to lymphoid tissues. This effect was demonstrated for resiquimod and 3M002 imidazoquinoline but not for imiquimod. Some time ago it was demonstrated that imidazoquinolines activated Langerhans cells functionally but no phenotypic differences were found between activated and nonactivated cells [10]. How ever, later Fahey et al. demonstrated that resiquimod caused phenotypic activation of Langerhans cells, which included expression of surface markers, proin flammatory cytokines and increased CCL21targeted migration of the cells. It was also found than in con trast to resiquimod, imiquimod insignificantly influ

enced Langerhans cells. The other study also demon strated that the only significant effect of imiquimod on Langerhans cells consisted in their functional activa tion, characterized by induction of Tcell prolifera tion [10]. It is possible that differences in the effects induced by imiquimod and resiquimod are deter mined by activation of other signaling cascades, possi bly, not only via TLR. By acting on TLR8 imiquimod can induce an inhibitory effect [4]. Using imiquimod as the TLR8 agonist it was demonstrated that this drug inhibited TNF production in vitro in cultured human macro phages and synovial cells. These results may be poten tially important for treatment of rheumatoid arthritis (pathogenesis of this diseases is frequently associated with increased TNF production). Imiquimod also influences the central nervous sys tem (CNS). In astrocytes and microglial cells the inhibitory effect of imiquimod is realized via TLR9 [5]. Activation of these cells is associated with numer ous neurological diseases and they also play an impor tant role in the antiviral innate immunity of the CNS. Astrocytes originate from nerve cell precursors, while microglial cells have bone marrow origin [21]. Both cell types express TLR7 and TLR9, the receptors of innate recognition. Studies have shown that agonists of these receptors induce production of the similar set of cytokines in each cell type. However, significant dif ferences do also exist in the cytokine profile produced by each cell type. For example, microglial cells but not astrocytes produce the proinflammatory cytokine, IL10 and antiapoptotic cytokines IL9 and GCSF. This study also demonstrated that in both cell types imiquimod inhibited the TLR9dependent immune response and this effect did not depend on TLR7. Schon et al. demonstrated that imiquimod inter acted with adenosine receptors and adenylate cyclase (AC), the enzyme responsible for cAMP synthesis [3]. These authors also suggested that imiquimod acted as an adenosine receptor antagonist and AC inhibitor [3]. Further studies on imiquimod were performed in order to resolve the problems associated with incom plete understanding of molecular mechanisms of its action. For example, results of some pilot studies sug gest that imiquimod significantly increases cAMP lev els in various cells. 2. ANTIVIRAL AND ANTITUMOR EFFECTS OF IMIQUIMOD Originally, the antiviral activity of imiquimod was demonstrated in guinea pigs infected with influenza virus [22]. According to Hemmi et al. [6], high antivi ral activity of imiquimod may be attributed to stimula tion of type I IFN biosynthesis by plasmacytoid DC precursors. Imiquimod exhibited the most potent action on viruses sensitive to IFNalpha and IFN beta.

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Imiquimod and its structural analogue resiquimod were used to increase local or systemic immune responses in several experimental models of tumors and viral infections [23]. In most cases imiquimod stimulated tumordirected immune responses and suppressed viral replication. It is known that natural killer (NK) cells are important components of antitu mor and antiviral immunity. In this connection it is especially interesting that the effect of TLR7 agonists on NK results in induction of IL18 and IL12 [24]. Authors of this study ([24]) also demonstrated that NK activation by TLR7 ligands was accompanied by stimulation of CD69 expression, while activation of these cells by TLR8 ligands caused stimulation of CD69 expression and secretion of IFNgamma. Treatment of basalcell carcinoma with Aldara (a commercial preparation containing 5% imiquimoid, see below, Section 8, Clinical Application) increased carcinoma infiltration with NK cells [25], and treat ment of actinic keratosis was accompanied by increased NK activity in the skin lesions treated by Aldara. It is suggested that the antiviral function of imiquimod is determined by its apoptotic activity against infected cells [26]; this activity has been dem onstrated in experiments with keratinocytes and other epithelial human cells The other study has demonstrated that imiquimod participates in positive regulation of the opioid growth factor receptor (OGFR); this results in activation of the signaling cascade OGFOGFR [27], which trans duces the inhibitory signal to proliferating cells via cyclindependent kinases and this causes cell cycle arrest at G1S. Thus, imiquimod suppresses replica tion of cancer cells and this may represent one of pos sible mechanisms of its antitumor effect. The ability of Aldara cream to induce local inflam mation is widely employed in the murine model of psoriasis [54–56] for investigation of cellular and molecular mechanisms of this disease and elucidation of targets for its treatment. All the above described effects of imiquimod on various cell types are summarized in table. 3. OTHER EFFECTS OF IMIQUIMOD ANALOGUES Certain interrelationship exists between metabo lism of lipid mediators and activation of TLR8. During subsequent treatments with PDGF (platelet derived growth factor) or other substances (such as fMLP, A23187) imidazoquinoline resiquimod R848 increased formation of leukotriene B4, release of arachidonic acid and synthesis of prostaglandin E2 in polymorphonuclear leukocytes [28]. These cells are characterized by a full set of innate pattern recognition receptors except TLR3; they migrate to the focus of inflammation and play an important role in innate immunity. It was demonstrated that polymorphonu

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clear leukocytes do not express TLR7 [28]. This dem onstrates that TLR8 plays an important role in metab olism of lipid mediators involved in innate immune response to viral infections. The other study has shown that resiquimod R848 and two other imidazoquinolines 3M003 and 3M002 (as well as single stranded viral DNA) are potent inductors of TNF and IL12 expression in APC [29]. TLR8 agonists induced expression of the costimula tor molecule CD40 on myeloid DC; this caused phos phorylation of p38 MAPkinase and degradation of IκB. Based on these results it has been concluded that TLR8 agonists exhibit unique properties as activators of costimulator responses of APC and they may be used for potentiation of the immune response. 4. INTERACTION OF IMIQUIMOD WITH ADENOSINE RECEPTORS As it has been mentioned above, imiquimod activ ity is closely associated with the TLRdependent cas cade, causing stimulation of expression of proinflam matory cytokines [7]; this finally results in the stimu lation of the antitumor immune response. These observations were confirmed in experiments with var ious tumors such as melanoma and nonmelanoma skin cancers [8]. Nevertheless, the biological activity of imiquimod depends on and is determined by func tioning of other mechanisms such as induction of proinflammatory cytokines as it has been observed in the imiquimodtreated cells lacking TLR7 [3]. Taking into consideration similarities in the chemical struc ture of imiquimod and nucleosides it has been sug gested that human adenosine receptors are involved in realization of the imiquimod effects. It was demon strated that human adenosine receptors A3 and espe cially A1 and A2 possess some affinity to imiquimod [3]. In order to determine, whether imiquimod is an agonist or antagonist of adenosine receptors the effect of imiquimod on adenylate cyclase activity has been investigated. These experiments employed Chinese hamster ovary (CHO) cells transfected with a DNA construct containing genes of A1, A2a or A3 receptors. It was found that in the CHO cells expressing A1 ade nosine receptor, imiquimod did not inhibit forskolin activated AC, while 2chloroN6cyclopentyladenos ine (CCPA), the specific AC inhibitor, inhibited the enzyme activity by 40%. Although imiquimod had a minor effect on forskolinactivated AC, it slightly potentiated CCPAinduced inhibition [3]. The latter suggests that imiquimod causes receptorindependent inhibition of AC. In CHO cells expressing A2a receptors imiquimod did not stimulate AC activity and therefore it was not an A2a receptor agonist [3]. Since imiquimod causes some inhibition (by 20%) of the AC activity stimulated by CGS21680 (selective A2a receptor agonist) it is reasonable to suggest that it

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is an A2a receptor antagonist. The observed inhibition of forskolinactivated AC by imiquimod is consistent with the hypothesis of receptorindependent inhibi tion of AC by this compound. In the case of A3 receptor it was found that imiqui mod inhibited forskolinactivated AC by 20%, while A3 receptor agonist inhibited AC activity by 43%. These results suggest that imiquimod may be consid ered as a receptorindependent inhibitor of AC. Since all these experiments do not reveal any ago nist effect of imiquimoid on the CHO cells it has been suggested that imiquimod acts as an antagonist of all three types of receptors [3]. In addition it has been proposed that imiquimod may decrease cAMP pro duction by inhibiting adenosinereceptor independent AC activity. A new potential target of the imiquimod action suggests adenosine receptormediated induction of proinflammatory mediators. It was demonstrated that both activation of NFκB and expression of proin flammatory cytokines were similarly stimulated by imiquimod, TNF, and an A2a receptor antagonist [3]. These results suggest that induction of the synthesis of proinflammatory cytokines in TLR7 and TLR8 nega tive cells (of keratinocyte origin) by imiquimod involves adenosine A2a receptor (as A1 and A3 recep tors are absent in these cells) [3]. Subsequent studies revealed that imiquimod exhibited proapoptotic activ ity, while none of specific adenosine receptor ligands did not demonstrate such activity [3]. Interestingly, induction of the transcription factor NFκB and proinflammatory cytokines was achieved by imiqui mod concentraitons, which were too low for induction of apoptosis; in addition, incubation of cells with imi quimod in the presence of other adenosine receptor ligands did not influence the proapoptotic activity of imiquimod. The latter implies that the proapoptotic activity of imiquimod does not depend on the pres ence of adenosine receptors. All these events are sche matically shown in Fig. 2. Thus, studies by M.P. Schon and M. Schon have demonstrated that imiquimod binds to adenosine receptors with a micromolar affinity; since agonist activity has not been registered it appears that imiqui mod is an A2a receptor antagonist [3]. In addition, imiquimod causes receptorindependent inhibition of AC. 5. THE ROLE OF A2A RECEPTORS IN INFLAMMATION It is known that inflammation of damaged tissues is accopmanied by accumulation of extracellular ade nosine secreted by several cell types into the extracel lular space of inflamed tissues [30]. Binding of extra cellular adenosine to adenosine A2a receptor causes

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formation of intracellular cAMP. Several studies have demonstrated that A2a receptor plays a key role in the regulation of acute inflammation [31]. Binding of extracellular adenosine to A2a receptor followed by AC activation and cAMP formation results in immu nosuppression. Binding of the adenosine A2a receptor antagonist, imiquimod, decreases AC activity. This effect may be potentiated by receptorindependent inhibition of AC. 6. PHYSIOLOGICAL EFFECTS OF cAMP According to modern concepts, cAMP production represents a converging point for several mechanisms of immunosuppression [32, 33]. It is possible that the regulatory CD4+ Tcells perform their inhibitory function by transferring intracellular cAMP to non regulatory Tcells via contact interactions [34]. More over, increased levels of adenosine under hypoxic con ditions and cell death in the tumor growth area may inhibit proliferation and activation of tumor infiltrating lymphocytes [35] by means of signaling via the Tcell adenosine A2a receptor, which induces AC activation [36]. Intracellular cAMP is an activator of protein kinase A (PKA) which causes a downstream activation of Cterminal Srckinase (Csk) [35]. The latter blocks signal transduction from TCR via consti tutive phosphorylation of the Cterminal inhibitory tyrosine residue (Y505) of proximal TCR phosphory lated Lck kinase [37]. This model is consistent with antiinflammatory effects of cAMP demonstrated in some other cell systems [38, 39] and a hypothesis that the inhibition of cAMP production should potentiate Tcell immunity. However, results of recent studies indicate, that cAMP production may also stimulate the Tcell response [40]. 7. cAMP AS A REGULATOR OF TNF PRODUCTION It is known that cAMP is a negative regulator of TNF production and exhibits immunoregulatory properties [33, 41]. In this context imiquimod attracts interest as a compound antiTNF property, for exam ple, in septic shock, in which TNF plays a key patho genic role. TNF overproduction exerts a strong patho logic effect in numerous inflammatory diseases [42], including Crohn’s disease [43], rheumatoid arthritis [44], multiple sclerosis [45]. During inflammatory injury the increase in the TNF level in the tissue microenvironment simultaneously induces two pro cesses which cause opposite physiological effects: one, extracellular, upregulating effect, which results in increased formation of TNF, and the other one, down regulating effect, causing a decrease of TNF produc tion. Both cycles converge around cAMP. In the upregulating cycle the effect of TNF results in inhibition of cAMP synthesis [46]. TNF increased activity of intracellular phosphodiesterase [46]

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Fig. 2. Summary of possible mechanisms of imiquimod action. This figure summaries all currently known mechanisms of imi quimod action. Being a TLR7 agonist imiquimod triggers the MyD88/NFkB cascade, which finally results in activation of proinflammatory cytokine transcription. Acting as a TLR8 antagonist imiquimod decreased transcription of proinflammatory cytokines. In addition to binding to various TLR imiquimod can interact with adenosine A2a receptor and thus block AC activity. This decreases cAMP production but increases expression of proinflammatory cytokines. Imiquimod can also modulate activity of the Tcell receptor and associated molecules. Imiquimod may also induce receptorindependent apoptosis and influence cell cycle by interacting with the opioid growth factor receptor (OGFR).

involved in catabolism of cAMP. The increased phos phodiesterase activity caused a decrease in the cAMP level and this caused an increase in the TNF produc tion (Fig. 3).

In the downregulating cycle the increase in TNF causes induction of cyclooxygenase2. This enzyme catalyzes a ratelimiting step in formation of prosta glandin H, an intermediate from which all other pros

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Fig. 3. Mechanisms of TNF and cAMP interaction in the inflammatory injury. This figure shows two possible mechanisms of the interaction between TNF and cAMP. According to the first mechanism, the inflammation injuryinduced increase of TNF causes increased expression of cyclooxygenase2 (COX2). This, in turn, leads to increased production of prostaglandin E2 and con comitant increase of cAMP synthesis. This process thus forms socalled downregulating cycle, which decreases TNF expression in the cell due to increased synthesis of cAMP. The second mechanism includes the increase of phosphodiesterase (PDE) activity responsible for the decrease of intracellular cAMP and formation of the upregulating cycle, which increases TNF expression.

taglandins (including prostaglandin E) are synthesized [47]. The increase of prostaglandin E decreases TNF expression due to increased cAMP production (Fig. 3) [48]. In order to find a solution to the above mentioned contradiction it is necessary to investigate possible role of imiquimod in suppression of TNF production under pathological conditions and in the protective effect under acute liver failure and bacterial septic shock in various experimental models.

8. CLINICAL APPLICATION Imidazoquinolines are a new group of compounds that recently have been introduced into clinical prac tice as antitumor and antiviral immunomodulators. Imiquimod is the most widely used imidazoquinoline used for treatment of various skin cancers and other skin diseases such as papillomas and external genital warts [49]. In addition, imiquimod is characterized by marked antiviral activity against some viruses, for example, human papillomavirus (HPV) [50]. This drug successfully passed through clinical trials and was

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recommended by the US Food and Drug Administra tion (FDA no. 020723) for use as a cream for treat ment of skin cancer and papillomas under the trade mark Aldara [51]. Aldara is a cream for local application that contains 5% of the active substance (imiquimod). In clinical practice it is successfully used for treatment of such diseases as actinic keratosis, external genital warts, superficial basal cell carcinoma, and also some other skin diseases. In most cases patients well tolerate to this drug, however, sometimes local skin side reactions appear. These include skin itching, burning, papules, local erythemas and bleeding, rash, paresthesia. Sometimes hyper or hypopigmentation, and local alopecia appear. In some cases systemic side reactions have also been described. In some cases severe flulike symptoms [52], and also exacerbation of major immu nological symptoms such as psoriasis, eczema, spondyloarthropathy [53] were observed. However, all systemic adverse effects of imiquimod in patients are rather rare events. Generally, for many patients the treatment of skin diseases (induced by UV injury or HPV) by imiquimod is a good alternative to the surgi cal treatment [50]. Currently, reports describing the potential effect of imiquimod not only on various skin cancers but also on malignant diseases of other organs started to appear in the literature. For example, Kauffman et al. demonstrated that treatment of renal cell carcinoma by imiquimod increased local production of proin flammatory cytokines in the tumor and its infiltration by Tlymphocytes and caused a significant suppres sion of tumor growth [57]. There are some studies on the use of imiquimod for treatment of respiratory diseases [58, 59]. Bronchial asthma is frequently accompanied by chronic inflam mation, hypersensitivity and deformation of respira tory airways. Imiquimod significantly reduced chronic inflammation, hypersensitivity and deformation of respiratory airways in the experimental model of bron chial asthma in mice [59]. The same approach may be used for treatment of viral respiratory infections caus ing lung injuries [58]. Viral respiratory diseases fre quently appear in asthmatic patients. As it has been mentioned above, TLR7 plays an important role in the antiviral immune response. The highest level of TLR7 expression was found in lungs [60]. It is also known that bronchial asthma is associated with TLR7, but the functional importance of this polymorphism remains unclear. Imiquimod can induce bronchodilatation via TLR7dependent and TLRindependent mecha nisms. The TLR7dependent mechanism employs nitric oxide, while the TLR7independent mechanism involves prostaglandins and calciumactivated potas sium channels. This is very important as TLR7 is a therapeutic target for treatment of viral respiratory diseases. Despite significant success of the imiquimod use in clinical practice its molecular mechanisms of action

still require better understanding. This is an important problem due to wide applications of imiquimod in the clinical practice and a clear need to avoid possible side effects associated with its clinical use. Understanding of molecular mechanisms of the imiquimod action and also imiquimodmediated signaling cascades would help targeted application of this drug for treat ment of various pathologies and development of more effective preparations with similar mechanisms of action. Thus, imiquimod is a perspective preparation for treatment of various diseases; however its effective application in clinical practice still requires deeper understanding of mechanisms realizing its action as well as identification of particular molecular targets and biochemical mechanisms influenced by this drug. ACKNOWLEDGMENTS This work was supported by the Federal Contract no. 14.740.11.0931, the Russian Foundation for Basic Research (projects no. 110491320), a grant from the President of Russian Federation for Leading Scientific Schools (no. 2701.2012.4) and the Program “Molecu lar and Cellular Biology”. REFERENCES 1. Russo, C., CornellaTaracido, I., GalliStampino, L., Jain R., Harrington, E., Isome, Y., Tavarini, S., Sam micheli, C., Nuti, S., Mbow, M.L., Valiante, N.M., Tallarico, J., De Gregorio, E., and Soldaini, E., Blood, 2011, vol. 117, pp. 5683⎯5691. 2. Schon, M.P. and Schon, M., Br. J. Dermatol., 2007, vol. 157 Suppl 2, pp. 8⎯13. 3. Schon, M.P., Schon, M., and Klotz, K.N., J. Invest. Dermatol., 2006, vol. 126, pp. 1338⎯1347. 4. Sacre, S.M., Lo, A., Gregory, B., Simmonds, R.E., Williams, L., Feldmann, M., Brennan, F.M., and Fox well, B.M., J. Immunol., 2008, vol. 181, pp. 8002⎯8009. 5. Butchi, N.B., Du, M., and Peterson, K.E., Glia, 2010, vol. 58, pp. 650⎯664. 6. Hemmi, H., Kaisho, T., Takeuchi, O., Sato, S., Sanjo, H., Hoshino, K., Horiuchi, T., Tomizawa, H., Takeda, K., and Akira, S., Nat. Immunol., 2002, vol. 3, pp. 196⎯200. 7. Reiter, M.J., Testerman, T.L., Miller, R.L., Weeks, C.E., and Tomai, M.A., J. Leukoc. Biol., 1994, vol. 55, pp. 234⎯240. 8. Beutner, K.R., Geisse, J.K., Helman, D., Fox, T.L., Ginkel, A., and Owens, M.L., J. Am. Acad. Dermatol., 1999, vol. 41, pp. 1002⎯1007. 9. Gaspari, A.A., Cutis, 2007, vol. 79, pp. 36⎯45. 10. Burns, R.P., Jr., Ferbel, B., Tomai, M., Miller, R., and Gaspari, A.A., Clin. Immunol., 2000, vol. 94, pp. 13⎯23. 11. Gibson, S.J., Lindh, J.M., Riter, T.R., Gleason, R.M., Rogers, L.M., Fuller, A.E., Oesterich, J.L., Gorden, K.B., Qiu, X., McKane, S.W., Noelle, R.J., Miller, R.L., Kedl, R.M., FitzgeraldBocarsly, P., Tomai, M.A., and Vasilakos, J.P., Cell Immunol., 2002, vol. 218, pp. 74⎯86. 12. Heib, V., Becker, M., Warger, T., Rechtsteiner, G., Ter tilt, C., Klein, M., Bopp, T., Taube, C., Schild, H.,

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Translated by A. Medvedev Vol. 7

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