Immunopharmacology is an interdiscipline that studies the reciprocal interactions between the action of drugs and the effectiveness of various components of the immune network. A knowledge of these intricate relations is of paramount importance in understanding the mechanisms of how drugs can modulate the potential of the immune armoury to protect against foreign intruders and various pathologic conditions. Vice versa, immunopharmacology also is concerned with the action of drugs in dependence on the activity status of the immune system. One of the major practical aims of immunopharmacology is research and development of novel agents with prospective applications in immunostimulatory, immunosuppressive, immunorestoring, and immunoregulatory therapy.
Research at the Department of Immunopharmacology focuses on the following main topics:
- Immunobiological properties of antiviral acylic nucleoside phosphonates
- Immunostimulatory activity of acylic nucleoside phosphonates
- Upregulation of nitric oxide (NO) production by acylic nucleoside phosphonates
- Cytostatic activity of acylic nucleoside phosphonates
- Therapeutic effects of acylic nucleoside phosphonates in model diseases

      The prevailing research projects of the Department are focused on the possibilities of pharmacological modulation of soluble immune factors that play critical roles in defense mechanisms againstinfections. In this respect, major attention has been paid to acyclic nucleoside phosphonates (ANPs), developed in the Institute of Organic Chemistry and Biochemistry, Czech Academy of Sciences, by dr. A. Holy. Some of these agents are used in clinical practice against various infections, including hepatitis B (adefovir), HIV (tenofovir) and HIV-associated (cidefovir) infections. Our results demonstrate that ANPs are potent immunostimulators and immunomodulators. These effects can contribute to the outstanding antiviral activity of the compounds. A further aim of our studies is to contribute to the development of novel structures with enhanced and more specific immunobiological properties, with potential use in the pharmacology of infectious, cancer, and inflammatory diseases. The findings provide a rationale for the development of novel virostatics with combined antimetabolic and immunomodulatory modes of action.

      The major mechanism of antiviral action of ANPs is the inhibition of virus-induced DNA polymerases and/or reverse transcriptases. They suppress replication of both DNA-viruses and retroviruses, such as herpes simplex virus-1 and -2, cytomegalovirus, varicella zoster virus, Epstein-Barr virus, human herpes virus type 6, human papilloma virus, hepatitis B virus, visna virus, and immunodeficiency viruses HIV, SIV and FIV. The oral prodrug of prototype compound adefovir (PMEA, 9-[2-(phosphonomethoxy)ethyl]adenine), has been approved by the FDA for the treatment of hepatitis B; the oral prodrug of tenofovir [(R)-PMPA, 9-(R)-[2-(phosphonomethoxy)propyl]adenine] is used for the treatment of AIDS. Tenofovir also exhibits prophylactic activity in a simian model of immunodeficiency disease. We have found that it activates secretion of chemokines that are involved in the control of HIV entry into cells of the immune system. To date, we have screened for the immunostimulatory/immunomodulatory potential of more than 150 newly developed ANPs differing in the type of side chain or N8-substituents, encompassing mainly monoalkyl, dialkyl, cycloalkyl, alkenyl, or alkynyl derivatives of adenine (A) and 2,6-diaminopurine (DAP), substituted at the 6-amino group in the 9-[2-(phosphonomethoxy) ethyl] (PME) and enantiomeric (R)- or (S)-9-[2-(phosphonomethoxy)propyl] (PMP) series.
      A number of derivatives, mainly the N6-cycloalkyl derivatives of PMEDAP and (R)-PMPDAP series, were found to stimulate secretion of TNF-, IL-10, IL-1, and the chemokines RANTES and MIP-1. The most potent of them, activating secretion of chemokines at 2 - 5 mM concentration, are N⁶6-isobutyl-PMEDAP, N⁶-cyclopentyl-PMEDAP, N⁶-cyclooctyl-PMEDAP, (R)-N⁶-cyclopropyl-PMPDAP, (R)-N⁶-cyclopentyl-PMPDAP, and (R)-N⁶-(2-dimethylaminoethyl)-PMPDAP. Several of these ANPs exhibit higher effectiveness than the parent (R)-PMPA (tenofovir).
      The ability of ANPs to activate production of RANTES and MIP-1a is important from the point of view of HIV treatment. These chemokines are natural ligands for the chemokine receptors CCR5 and CXCR4, which are also used as co-receptors for the entry of HIV into the cells of the immune system, and in cooperation with the CD4 receptor ensure a productive infection. Blocking the appropriate - chemokine receptors on both macrophages and lymphocytes is presently considered to be a therapeutic approach against HIV. Also, other cytokines stimulated by ANPs have been implicated in virus replication. It is suggested that these novel compounds may be prospective candidates for exploitation in antiviral therapy.

Fig. 1: Chemical structure of clinically employed acyclic nucleoside phosphonates

      NO is an important mediator and/or modulator of many physiological and pathophysiological phenomena, particularly within the cardiovascular and nervous systems. High-output NO production, controlled by a number of cytokines (mainly by IFN-g, TNF-a, IL-1a), is effectuated by the inducible isoform of NO synthase (iNOS). It is closely associated with the effectiveness of the immune system and is considered to mediate the inhibitory effects of IFN-g and/or other cytokines on the replication of a great many number of viruses, including poxviridae, herpetoviridae, retroviridae, and parvoviridae, e.g. hepatitis B virus, cytomegalovirus, Epstein-Barr virus, vaccinia virus, ectromelia virus, and HIV. Beside virustatic activity, NO possesses antibacterial and antiparasitic properties.
      More than twenty test ANP derivatives, including tenofovir, have been found to substantially augment production of NO triggered by IFN-g. The positive modulatory effect is mediated by enhanced expression of both TNF-g and IL-10. The NO-upregulatory activity may be one of the mechanisms of the inhibitory effects of ANPs on the replication of viruses.

      ANPs are inhibitors of DNA polymerases and DNA chain terminators. We investigated the effects of ANPs on the proliferation of mouse lymphocytes cultured in the absence or presence of T- and B-cell mitogens. The medial inhibitory concentrations (IC50) of N⁶-nonsubstituted compounds were found to range from 0.13 mM for PMEDAP to 354 mM for (R)-PMPA. Antiproliferative effects are more pronounced in the PME than in the PMP series; they are more prominent in 2,6-diaminopurine compared to adenine analogs. The highest cytostatic activity is exhibited by PMEDAP-N⁶-allyl (IC50 = 0.017 mM) and PMEDAP-N⁶-cyclopropyl (IC50 = 0.036 mM). The N⁶-substituted derivatives of (S)-PMPA are virtually devoid of cytostatic potential. They preferentially affect activated T lymphocytes, apparently in the S and/or M phase of the cell cycle. No correlation between antiproliferative and reported antiviral or immunostimulatory effects exists.

      We found that some ANPs interfere with processes involved in the etiopathogenesis of diseases of autoimmune origin. Thus, PMEA (adefovir), its bis-pivaloyloxymethyl ester bis (POM)-PMEA (adefovir dipivoxil), and PMEDAP, i.e. 9-[2- (phosphonomethoxy)ethyl] diaminopurine, almost completely inhibit the development of adjuvant arthritis in rats, a model of human rheumatoid arthritis.
      Another ANP derivative, 9-(S)-[3-hydroxy-2-(phosphonomethoxy)propyl]adenine [(S)-HPMPA] was found to be effective in a murine model of bilharsiosis caused bythe tropical parasite Schistosoma mansoni. The complete disappearance of immature eggs, along with a significant reduction up to a complete absence of mature eggs with 99 - 100 % dead eggs, was observed. No hepatic egg-induced granulomas were present in mice treated at the time of oviposition, and they were smaller in size in those treated before S. mansoni oviposition. In contrast to the effects of (S)-HPMPA, the commonly used drug of choice, i.e praziquantel (PZQ), is not ovicidal nor is it effective against young stages of the parasite.