Dale A. Stringfellow

Comparation Interferon-Inducing and Antiviral Properties of 2-Amino-5-Bromo-6-Methyl-4-Pyrimidinol (U-25,166), Tilorone Hydrochloride, and Polyinosinic-Polycytidylic Acid

2-Amino-5-bromo-6-methyl-4-pyrimidinol (U-25,166), polyinosinic acid-polycytidylic acid [poly(I:C)], and tilorone HCl induced high levels of serum interferon in mice. Each consequently protected mice against infection with several viruses. After daily injection of inducer, mice developed a reduced interferon response (hyporeactivity) to each compound. However, hyporeactivity developed more slowly to U-25,166 and poly(I:C) than to tilorone HCl. After onset of hyporeactivity, 5 to 6 days without each inducer were required before normal serum interferon levels could be stimulated. Animals also developed a hyporeactive state as a consequence of Semliki Forest or encephalomyocarditis virus infections. By day 2 of either infection, mice had a suppressed interferon response to tilorone HCl, but remains responsive to poly(I:C) or U-25,166 until day 4. In vivo, poly(I:C) stimulated interferon production in a variety of cells and organs, whereas the tilorone HCl and U-25,166 responses involved a nonlymphoid component of the reticuloendothelial system. In vitro, poly(I:C) induced interferon in a variety of murine cells, U-25,166 was active in murine thymus and spleen organ cultures, and tilorone was inactive. These data indicate that U-25,166 is an interesting low-molecular-weight interferon inducer.

Antiviral and Interferon-Inducing Properties of 1,5-Diamino Anthraquinones

A series of anthraquinones with amino substituents at the 1,5 positions were found to induce interferon in mice. A prototype compound, 1,5-bis[(3-morpholinopropyl)amino]-anthraquinone (Ia), was an effective antiviral agent when administered either orally or parenterally. Peak interferon titers were found 12 to 24 h after drug treatment. The minimum oral dose of Ia required to induce serum interferon or to protect mice against a lethal virus infection was 62 mg/kg. Mice tolerated an oral dose of at least 30 times this minimum effective dose. A single dose of Ia given up to 6 days prior to infection had significant protective activity. Biological properties of Ia were compared with those of three other 1,5-diamino anthraquinones, which also induced interferon and demonstrated antiviral activity in mice. The most active compound was 1,5-bis[[2-(diethylamino)ethyl]amino]-anthraquinone (Ib), which protected mice against virus infection at a dose as low as 8 mg/kg (less than 1/60 its maximum tolerated dose). Mice developed hyporeactivity to interferon induction if the same inducer was injected daily, although by alternating between different inducers the loss of interferon responsiveness could be avoided.

Tilorone Hydrochloride: an Oral Interferon-Inducing Agent

Tilorone hydrochloride characteristically induces an unusually delayed and prolonged interferon response not commonly associated with other synthetic inducers. Maximum circulating interferon levels of 8,000 to 10,000 units/ml were detected 12 hr postinoculation and persisted for up to 30 hr after administration of tilorone. The fact that both oral and intraperitoneal injections of tilorone induce a similar response suggests that this delay is not based on the time required for adsorption from the gastrointestinal tract. In vitro, tilorone failed to induce detectable levels of interferon in either mouse peritoneal lymphocyte or macrophage cell cultures. Furthermore, interferon production could not be detected in the upper gastrointestinal tract after oral administration of tilorone. The striking suppression of interferon production in X-irradiated mice suggested that lymphatic tissue may be a source of interferon in response to tilorone. This concept was not supported, however, by experiments utilizing antilymphocyte serum (ALS). Tilorone induced comparable levels of interferon in both ALS-treated and control animals which had received normal serum, even though the ALS-treated mice had decreased spleen weights and peripheral lymphocyte counts. These data suggest that a radiosensitive cell population other than lymphocytes may be an important factor in the capacity of the host to produce interferon in response to tilorone. Mice which received repeated injections of tilorone developed a severe state of hyporeactivity. The degree of hyporeactivity was particularly striking when compared to that induced by polyinosinic acid:polycytidylic acid and emphasizes one of the major obstacles confronting interferon inducers as chemotherapeutic agents.

Didemnins A and B. Effectiveness against cutaneous herpes simplex virus in mice

The antiviral activity of didemnin A and didemnin B against a lethal Semliki Forest virus (SFV) infection of mice and a cutaneous herpes type 1 infection in hairless mice was evaluated. Both compounds significantly decreased the severity of herpesvirus lesions if topical treatment with either didemnin A or didemnin B was started 2 days prior to infection. The survival rate was significantly greater (P = 0.03) in the didemnin B treated group than in controls. If initiation of treatment was delayed until 1 h after infection, no activity was obtained. The compounds were not active against cutaneous herpesvirus infection when injected intraperitoneally (i.p.). Didemnin B at concentrations as low as 1.5 micrograms, administered topically 3 times daily for 5 days, produced skin irritation. Eight times this level of didemnin A could be administered before similar toxicity was observed. The limited activity of didemnins A and B coupled with irritation at the treatment site limits their usefulness in treating cutaneous herpesvirus infection. Neither didemnin A nor B had significant activity in SFV-infected mice.

Antineoplastic properties of pyrimidinone interferon inducers

The interferon inducing, antiviral and antitumor activities of a new series of pyrimidinone compounds are compared and summarized in this report. An interesting split in mechanism of action appeared to exist in these compounds. One group represented by ABPP were very potent interferon inducing agents and the antiviral and antitumor activity of these agents correlated with their ability to induce interferon. The second group as exemplified by AIPP were not good inducers yet were just as active against virus infection and neoplastic disease. Presumably the split in antiviral and antitumor activity of these molecules was mediated by other host defense mechanisms as indicated by their ability to modulate various immune functions. Additional evidence for this has been reported by H. E. Renis et al. (24). They found that treatment of mice with antithymocyte serum along with AIPP or ABMP blocked the antiherpes simplex virus activity of the molecules. That is, that mice treated with AIPP or ABPP were effectively protected against virus infection but if a single dose of antithymocyte serum was given along with drug, the ability of the molecules to protect against virus infection was inhibited. These results suggest that the thymocyte played a critical role in mediating anti-Herpes virus and possible antitumor activity. This concept is further enhanced by the fact that each of the pyrimidinones did not have antiviral activity in vitro in various fibroblast cell culture monolayers. The antitumor activity of the pyrimidinones was very dependent upon tumor load. If too high a tumor load was injected, the animals were not protected against the lethal consequences of the neoplastic disease. We have reported similar observations with these compounds in mice where the antiviral activity of the molecules was overcome by increasing the level of virus injected (25). These observations suggest that the most efficient use of such molecules might be in combination chemotherapy with conventional cytotoxic or antiviral agents. Some clue as to why this is the case is given by studies in which mice were injected with malignant melanoma cells and the metastatic rate was monitored. In these studies the number of metastatic nodules in each mouse could be reduced but the number of mice without metastatic nodules was not significantly decreased indicating that the molecules could decrease the tumor load but not completely rid the animal of neoplastic cells. Although we presently do not know if interferon or interferon inducers will be effective in humans for the treatment of virus infections or neoplastic disease, ongoing studies should help answer these questions. The pyrimidinones are presently in preliminary clinical trials aimed at determining if they will induce interferon in humans and what effect they will have on various immune parameters. Results from these studies should provide answers as to the potential benefit of this interesting group of compounds.

Chemotherapy of viral infections.

SummaryAs indicated in Table 1, two approaches have been taken to the development of antiviral chemotherapeutic agents. Both have met with some success although the availability of effective chemotherapeutic agents for the treatment of virus infections is very limited. This situation should change in the near future as more specific, direct inhibitors of virus replication are identified and better ways are developed for stimulating specific and nonspecific host defense mechanisms. Although the discussion of host defenses centered upon the interferon system, there are a variety of other nonspecific host defense mechanisms that are also important including macrophages, lymphocyte subpopulations, and other as yet poorly defined soluble mediators.