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Keywords:

  • Antiviral;
  • cytomegalovirus;
  • leflunomide;
  • transplantation

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

We previously reported that the immunosuppressive malononitrileamides leflunomide and FK778 exert antiviral activity against cytomegalovirus (CMV). In the current investigation, we tested the hypothesis that leflunomide exerts concurrent antiviral activity and immune suppression in CMV-infected cardiac allograft recipients. Lewis rats were transplanted with Brown Norway hearts and then inoculated with rat CMV. Plaque assay demonstrated that leflunomide (30 mg/kg/day) reduced viral loads by 4–6 logs, and that the reduction in viral load was unaffected by administration of uridine. Leflunomide was as effective as cyclosporine A (CsA) or tacrolimus in preservation of allograft integrity through day 28. These studies directly demonstrate the bifunctionality of leflunomide as concurrently immunosuppressive and antiviral, enhancing the promise of this agent as a clinical option for treatment of transplant recipients.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Cytomegalovirus (CMV) infection, although rarely of clinical consequence in the healthy immunocompetent population, remains clinically problematic among pharmacologically immunosuppressed organ transplant recipients. In these patients the CMV remains a source of diverse serious, often life-threatening complications including interstitial pneumonia, diffuse gastrointestinal mucosal ulceration, hepatitis and retinitis, as well as destructive inflammatory lesions in a variety of other locations (1). In addition, numerous studies have implicated the CMV as a contributing factor in allograft rejection (2,3).

Leflunomide, a malononitrileamide, is an anti-inflammatory drug approved for treatment of rheumatoid arthritis (Arava®, Aventis Pharmaceuticals, Inc., Bridgewater, NJ). Recent clinical studies have demonstrated that leflunomide possesses substantial immune suppressive potency in human renal and liver transplant recipients and may be safely dosed for more than 300 days (4). Following oral administration, leflunomide is rapidly metabolized to its active form, A77 1726 (5) which is known to inhibit protein kinase activity (6,7) and the activity of dihydroorotate dehydrogenase (DHODH), a key enzyme in the biosynthesis of pyrimidine nucleotide triphosphates (7,8). The specific contribution of each of these functions to the immunosuppressive activity of leflunomide in vivo remains to be fully resolved.

We have previously reported that A77 1726 exerts antiviral activity against CMV and herpes simplex 1 (HSV1) in vitro (9,10). The antiviral activity of this agent in vitro could not be accounted to pyrimidine depletion because addition of exogenous uridine, which restores pyrimidine nucleotide triphosphates to normal (or supranormal) levels, does not reconstitute infectious virus production. In contrast to ganciclovir, phosphonoformate and cidofovir, A77 1726 does not inhibit viral DNA synthesis, but rather disrupts virion assembly at the level of nucleocapsid tegumentation, and is thus equally effective against multi-drug-resistant CMV isolates in vitro (9).

We have also recently demonstrated that FK778 (Fujisawa Healthcare, Inc., Deerfield, IL), a related DHODH-inhibitory malononitrileamide compound with immunosuppressive properties, possesses antiviral activity against CMV in vitro and in vivo in a short-term acute infection study in radiation-immunosuppressed rats (11). However, while the maximum FK778-mediated reduction of virus yield in vitro was 1 order of magnitude, reductions achieved by A77 1726 treatment generally approached 3 orders of magnitude (9). In addition, the dosage of FK778 required to reduce viral loads in CMV-infected animals by 3 orders of magnitude (20 mg/kg/day) and, in separate experiments, to preserve cardiac allograft integrity in transplanted rats resulted in 20–28% mortality with survival times of 19–24.5 days (11). Although these reductions in viral loads reached statistical significance, historically substantially greater reductions seem necessary to achieve clinical impact.

The data generated by our FK778 experiments suggest that this compound possesses both antiviral and immunosuppressive properties. However, those studies also raise several important questions. First, considering the superior antiviral efficacy of A77 1726 in vitro, can leflunomide mediate greater reductions in viral load than FK778 in vivo at better tolerated doses? Second, the short duration of the acute infection studies of the antiviral activity of FK778 in vivo (10 days) raises questions as to the antiviral efficacy of malononitrileamides in the longer term. Finally, since the studies of the immunosuppressive and antiviral activities of FK778 were conducted in separate groups of animals under different conditions (i.e. animals were either transplanted or radiation-immunosuppressed and infected with CMV), whether or not malononitrileamide compounds can be concurrently immunosuppressive and antiviral remains to be confirmed.

In the current investigation, we have addressed these issues by assessing the antiviral efficacy of leflunomide in short-term acute infection studies, and by directly assessing the ability of this agent to exert concurrent antiviral activity and allograft-preserving immune suppression in the longer term in CMV-infected cardiac allograft recipients.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

All animal care and procedures were in compliance with “Principles of Animal Care” (National Society for Medical Research) and the “Guide for the Care and Use of Laboratory Animals” (Institute of Laboratory Animal Resources/NIH). All studies described herein were approved by the animal use committees of all institutions at which the work was performed (Ohio State University ILACUC protocol 98A0102, Rush IACUC protocol 98-030, University of Chicago IACUC protocol 71289).

Cells and virus

Rat embryo fibroblasts (REF) were isolated as described by Bruggeman et al. (12) (OSU ILACUC-approved protocol 98A0102) and propagated in MEM (Sigma Chemical Co., St. Louis, MO) supplemented with 10% fetal bovine serum (US Biotechnologies, Inc., Parker Ford, PA), 1% essential amino acids, 2% non-essential amino acids, 0.5% vitamins (Sigma), and 2 μg/ml bovine brain extract (Biowhittaker, Inc., Walkersville, MD). Cells were used for experiments at passages 5–8. Rat CMV (RCMV, Maastricht strain RA67; generously provided by Professor Cathrien Bruggeman, University Hospital Maastricht, The Netherlands) was propagated in vivo in radiation-immunosuppressed Lewis rats as previously described (12). Following euthanization at 4 weeks post-inoculation, the salivary glands were excised and homogenized as a 10% w/v suspension, and the supernatants cryopreserved at −80°C. Viral titers of cryopreserved salivary gland homogenates were determined by plaque assay on REF monolayers as previously described (12).

Acute infection protocol (Rush IACUC-approved protocol 98-030)

Lewis rats were inoculated with 1 × 106 PFU of RCMV i.p. following total body irradiation (6 Gy) as previously described (11). Groups of five animals each were treated by gavage with 20 or 30 mg/kg/day of leflunomide, or vehicle alone (1% carboxymethylcellulose). Additional groups were treated with 250 mg/kg of uridine twice daily, i.p. or with 10 mg/kg/day ganciclovir (Cytovene, Syntex, Morris Plains, NJ) i.p. on days 0–5. On day 10 post-inoculation ketamine-anesthetized animals were euthanized by exsanguination, followed by excision of salivary glands, lungs and spleens. Portions of tissues were processed for histology and immunohistochemistry or cryopreserved under sterile conditions for assay of viral load.

Cardiac transplantation protocol

Heart transplantation was performed using 125–175 g, 10–15 days old Brown Norway (BN) rats as donors and Lewis rats as recipients (Harlan, Walkersville, MD), following standard protocols as previously described (13). Animals were inoculated on the day of transplantation with 106 PFU of RCMV by i.p. injection and treated with 30 mg/kg/day of leflunomide or vehicle alone (1% carboxymethylcellulose) daily by gavage. Additional groups were treated i.p. with 20 mg/kg/day cyclosporine A (CsA, Sandoz, East Hanover, NJ), or 1 mg/kg/day tacrolimus (FK506, Fujisawa, Deerfield, IL). The recipients were monitored daily for 28 days or until death or rejection. On day 28, post-transplantation animals were euthanized and tissues were harvested as described above, including the heart allografts.

Histologic analysis

Serial sections (5 μm) were prepared from OCT-embedded frozen tissue blocks and stained with hematoxylin/eosin for routine histology. To visualize the tissue distribution of virus, sections were stained with monoclonal antibody (mAb) specific for the 55 kDa RCMV DNA polymerase accessory protein encoded by the R44 gene (14) (RCMV mAb 8, generously provided by Professor Cathrien Bruggeman, University Hospital Maastricht, The Netherlands) by a modified avidin-biotin-peroxidase method as previously described (15). Graft infiltrating mononuclear leukocytes and antibody deposition were detected by immunohistochemistry using the following anti-rat primary mAbs: TCRαβ(R73), CD4 (OX38), CD8β (341), macrophage/monocyte (ED1), IgM (MARM-4) and IgG1(MARG1) (all from Serotec USA, Raleigh, NC).

Quantitation of viral load by plaque assay

Viral loads in salivary glands, lungs and spleens were quantitated by plaque assay of serial 10-fold dilutions of tissue homogenates as previously described (11). Mean plaque numbers derived from three replicate culture wells were corrected for dilution factor and expressed as mean PFU per gram of tissue ± 1 SD. Statistical significance of differences in viral load among treatment groups was determined by Student's t-test.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

We first sought to establish an antiviral dose response to leflunomide in vivo in radiation-immunosuppressed Lewis rats inoculated i.p. with 106 PFU of RCMV. The infected animals were euthanized 10 days post-inoculation, and the data presented in Figure 1 are representative of results of three independent experiments, two to three replicate plaque assays/experiment.

image

Figure 1. Plaque assay of homogenates of tissues recovered from Lewis rats 10 days post-inoculation. Animals were irradiated (6 Gy), inoculated i.p. with 106 plaque forming units of RCMV and treated with vehicle alone (CMV only), 500 mg/kg/day of uridine (Uridine), 20 (20 LEF) or 30 (30 LEF) mg/kg/day of leflunomide or 30 mg/kg/day of leflunomide plus 500 mg/kg/day uridine (30 LEF + Uridine). Animals were euthanized 10 days post-inoculation and tissues were homogenized and assayed for plaque formation on rat embryo fibroblast monolayers. The data points represent mean PFU per gram of tissue (from a single animal) calculated from three replicate culture wells and corrected for dilution factors. Error bars represent one standard deviation, and dashed lines represent mean PFU/g for each treatment group.

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As expected, inoculated animals treated with vehicle alone harbored high viral titers in salivary glands and lungs (∼106−108 PFU/g). Daily dosing of animals with 30 mg/kg of leflunomide significantly reduced viral load by ∼5 orders of magnitude in salivary glands (p = 0.0064) and by 5–6 orders of magnitude in lungs (p = 0.029). Viral titers measured in spleens of vehicle-treated animals were generally lower than those measured in salivary glands and lungs, and while 30 mg/kg/day of leflunomide reduced splenic viral loads by 3–4 orders of magnitude, this reduction did not reach statistical significance (p = 0.071). Doses of 20 mg/kg/day of leflunomide demonstrated antiviral activity as well; however, the reduction in viral load was in general several logs less than that induced by the 30 mg/kg/day dose, and the magnitude of responses among individual animals was much more variable. Consistent with our previous in vitro studies (9), the antiviral activity of leflunomide cannot be accounted to pyrimidine depletion since treatment of animals with exogenous uridine showed no significant impact upon leflunomide-mediated reduction of viral load. Viral titers in ganciclovir-treated animals were consistently below the detection limit of the assay (10 PFU/g).

Immunohistochemical visualization of the tissue distribution of RCMV antigen corroborated the data generated by plaque assay. Figure 2 demonstrates RCMV antigen distribution in salivary gland, lung and spleen of vehicle-treated animals. In contrast, RCMV antigen positivity was extremely rare in tissues of animals treated with 30 mg/kg/day of leflunomide.

image

Figure 2. Immunohistochemical visualization of RCMV antigen in tissues recovered from Lewis rats euthanized 10 days post-inoculation. Animals were treated as described in Figure 1. Tissue sections were stained with mAb specific for RCMV DNA polymerase accessory protein encoded by the R44 gene, as described in the Materials and Methods section.

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We next directly tested the hypothesis that leflunomide exerts effective concurrent antiviral and immunosuppressive activity in RCMV-infected cardiac allograft recipients. Heart grafts in animals treated with vehicle alone stopped beating by day 7 post-transplantation. The leflunomide dose was 30 mg/kg as determined by the in vivo experiments described above. Previous pharmacokinetic studies of Lewis and Brown Norway rats have shown this dose to result in serum A77 1726 levels of ∼100 μg/mL or 300 μM (16), levels which are generally well-tolerated in clinical studies of human liver and kidney transplant recipients (4). As shown in Figure 3, leflunomide, CsA and tacrolimus were equally effective in the preservation of allograft integrity through day 28 post-transplantation. T-cell infiltration was rare under all three immunosuppressive regimens (data not shown). Consistent with earlier studies (17), while vascular deposition of IgM and, to a lesser extent, IgG was apparent in grafts recovered from CsA- or tacrolimus-treated animals, none was detected in grafts of animals treated with leflunomide (data not shown). These observations suggest that the immunosuppressive activity of leflunomide was comparable to, if not greater than, the administered doses of CsA or tacrolimus.

image

Figure 3. Histology of cardiac allografts. Brown Norway rat hearts were heterotopically transplanted into Lewis rat recipients. Animals were inoculated i.p. with 106 PFU of RCMV, treated with 30 mg/kg/day of leflunomide, vehicle alone, 20 mg/kg/day of cyclosporine A or 1 mg/kg/day tacrolimus (FK506), and euthanized on day 28 post-transplantation. Sections cut from OCT-embedded frozen heart graft tissue blocks were stained with hematoxylin/eosin.

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The data generated by plaque assay of tissue homogenates presented in Figure 4 are representative of results of two independent experiments, two to three replicate plaque assays/experiment. These data show that leflunomide (30 mg/kg/day) significantly reduced viral loads by ∼4–5 orders of magnitude in salivary glands (p = 0.016), lungs (p = 0.041) and spleens (p = 0.05) of cardiac allograft recipients over a period of 28 days. Viral titers in tissues recovered from CsA- and tacrolimus-treated animals were generally 1–2 orders of magnitude greater than in those recovered from animals treated with vehicle alone; however, with the exception of salivary glands of CsA-treated animals (p = 0.015), these differences were not statistically significant. Again, immunohistochemical visualization of the tissue distribution of RCMV antigen corroborated the data generated by plaque assay (data not shown).

image

Figure 4. Plaque assay of homogenates of tissues recovered from Lewis rat cardiac allograft recipients. Animals were treated as described in Figure 3. Following euthanization on day 28 post-transplantation, tissues were homogenized and assayed for plaque formation on rat embryo fibroblast monolayers. The data points represent mean PFU per gram of tissue recovered from a single animal calculated from plaque numbers in three replicate culture wells and corrected for dilution factors. Error bars represent one standard deviation, and dashed lines represent mean PFU/g for each treatment group.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

We previously demonstrated potent antiviral activity of A77 1726 against CMV in vitro (9) and now have confirmed the antiviral activity of this agent in vivo and demonstrated its superior efficacy and lower toxicity as compared to the previously documented activity of FK778 (11). These current observations extend our earlier observations with FK778 and demonstrate that another member of the malononitrileamide family can exert both immunosuppressive and antiviral activities in vivo. Assessments of these activities with FK778 were conducted in separate groups of animals under different conditions (i.e. animals were either transplanted or radiation-immunosuppressed and infected with CMV) and over different periods of time (10 days for antiviral studies and 28 days for immunosuppression studies). Although the immunological consequences of total body irradiation and pharmacologic immunosuppression bear some similarities to one another, the mechanisms underlying the resultant immunosuppression and the global impact upon the internal milieu vary dramatically under these conditions. Hence in the current investigation, we have directly confirmed for the first time the concurrent bifunctionality of leflunomide as both an effective immunosuppressant and an effective antiviral drug in RCMV-infected cardiac allograft recipients over a period of 28 days, clearly demonstrating that the antiviral activity of leflunomide dominates over any enhanced viral load which may arise as a result of its immunosuppressive activity. Importantly, previous pharmacokinetic studies indicate that serum A77 1726 levels resulting from the leflunomide doses administered in these experiments were equivalent to generally well-tolerated levels observed in human liver and kidney transplant recipients (4,16).

In our previous in vitro investigation (9), we demonstrated that the antiviral activity of leflunomide is unique among traditional immunosuppressive agents. To this end, we measured virus yield from untreated CMV-infected cultured endothelial cells or fibroblasts, or infected cells treated with A77 1726, CsA or FK506. Neither CsA nor FK506 showed any antiviral effect. Unexpectedly, the virus yield from CsA-treated cells was reproducibly 2- to 3-fold greater than from untreated infected controls (9). These observations implied a direct, CsA-mediated enhancement of infectious virus production. Hence, it is of interest to note that viral loads measured in the current in vivo experiments were up to 2 orders of magnitude greater in the CsA-treated animals than in those treated with tacrolimus or vehicle alone. Although these increases did not reach statistical significance in lungs or spleens, the difference was significant in salivary glands (p = 0.015). While this phenomenon has not been reported in clinical studies, the direct impact of CsA upon CMV replication may be worthy of a closer examination in light of our in vitro and in vivo observations.

Our earlier in vitro studies demonstrated that the antiviral activities of A77 1726 and of the related malononitrileamide FK778 are not accountable to pryrimidine depletion (9,11). Likewise, administration of uridine to FK778-treated animals had no impact upon viral loads. The data generated in the current investigation show that the reduced viral loads measured in leflunomide-treated RCMV-infected animals were likewise unaffected by daily administration of exogenous uridine. Collectively these findings rule out pyrimidine depletion as an antiviral mechanism of these agents. In contrast, studies currently in progress have demonstrated that A77 1726 inhibits phosphorylation and perturbs intracellular localization of several viral tegument proteins which are essential components of the complete viral particle. These findings suggest that A77 1726-mediated kinase inhibition may be responsible for the disruption of virion assembly previously documented (9).

Likely as a consequence of this unique mechanism of action, A77 1726 is equally effective in vitro against viral isolates which are resistant to traditional anti-CMV drugs (9). Support for the effectiveness of leflunomide against resistant virus in vivo was provided by our recent report of an allogeneic bone marrow transplant recipient who developed CMV infection refractory to sequential therapy with ganciclovir, foscarnet, and cidofovir (18). The patient was ultimately treated with a combination of leflunomide and PFA, where upon the viral load decreased from 2.26 × 105 genome equivalents/mL of whole blood at initiation of leflunomide therapy to undetectable levels following 6 weeks of treatment. In addition, improvement in the patient's HSV oral ulceration corroborated our previous report of antiviral activity of A77 1726 against this herpesvirus (10). Additional support for the safety and antiviral efficacy of leflunomide was provided by John et al. (19) who recently reported the use of this agent in four renal transplant recipients in India who developed symptomatic CMV disease and were treated with leflunomide rather than ganciclovir for economic reasons. All patients cleared the virus and no major drug-related adverse events or decrease in graft function were observed.

In summary, we have directly demonstrated the concurrent bifunctionality of leflunomide as both immunosuppressive and antiviral in an allogeneic cardiac transplant model of RCMV infection, and have shown that this agent possesses superior antiviral efficacy and less toxicity compared to the related malononitrileamide FK778. Results from the present study, and the clinical studies summarized above (18,19), show great promise for leflunomide as an addition to the current battery of anti-CMV therapeutics and immunosuppressive drugs, and as a favorable option for the treatment of disease associated with viral isolates resistant to traditional antiviral agents.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

This study is funded by NIH/NIAID grant AI45881 (WJW). Portions of this work were presented at the American Transplant Congress, 2002, 2003.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References
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