点击显示 收起
University of Alabama School of Medicine, Birmingham
Veterans Affairs San Diego Healthcare System and the University of California at San Diego, La Jolla
The acyclic nucleoside phosphonate cidofovir (CDV) and its closely related analogue (S)-9-(3-hydroxy-2-phosphonylmethoxypropyl)-adenine ([S]-HPMPA) have been reported to have activity against many adenovirus (AdV) serotypes. A new series of orally active ether lipid-ester prodrugs of CDV and of (S)-HPMPA that have slight differences in the structure of their lipid esters were evaluated, in tissue-culture cells, for activity against 5 AdV serotypes. The results indicated that, against several AdV serotypes, the most active compounds were 152500-fold more active than the unmodified parent compounds and should be evaluated further for their potential to treat AdV infections in humans.
Adenovirus (AdV) infections are a serious problem worldwide, with respiratory illnesses, gastroenteritis, and ocular infections being the most common. Fifty-one distinct serotypes of AdV are associated with human infections. The most common serotypes13, 5, and 7cause respiratory infections, and serotypes 40 and 41 cause enteric infections. Although most AdV infections are usually mild or asymptomatic, they can cause significant rates of morbidity and mortality in immunocompromised hosts, often causing diarrhea, pneumonia, hemorrhagic cystitis, and disseminated disease. Serotypes 8, 19, and 37 are most commonly associated with ocular infections. Adenoviral ocular infections, such as keratoconjunctivitis, are highly transmissible, occur in epidemic proportions, and currently have no approved treatment. A vaccine to prevent febrile infections, which are most commonly associated with serotypes 4 and 7, has been developed for military personnel, but this vaccine is not available to the public and provides protection against only 2 serotypes. The absence of an approved antiviral agent to effectively combat AdV infections means that a variety of potentially serious conditions have no treatment, and this increases the possibility that an infection in an individual will spread to others and/or that different serotypes will arise.
The acyclic nucleoside phosphonate cidofovir (CDV) has activity against many herpesviruses, as well as activity against papovaviruses and poxviruses [1, 2]. A closely related analogue, (S)-9-(3-hydroxy-2-phosphonylmethoxypropyl)-adenine ([S]-HPMPA), has also been reported to have activity against many of these viruses, as well as activity against hepadnaviruses and iridoviruses [13]. Both nucleotide analogues also have activity against some AdV serotypes in vitro [46], and CDV has been evaluated in animal models of AdV diseases [5, 7, 8] and in clinical studies [911]. (S)-HPMPA has been tested against standard serotypes and clinical isolates, including AdV serotypes 18 and 19 [4, 6], and CDV has been tested against AdV serotypes 1, 5, 8, 14, 18, and 19 in vitro [5, 6]. The efficacy of topical CDV, used as either a prophylactic or a treatment for AdV 5 ocular infections, has been reported in animal models [5, 7, 8]. However, the activities of these compounds are serotype dependent, and, therefore, they may not be effective against all AdV serotypes [6]. This limitation increases the need for the development of more broadly active antiviral agents, particularly those that are active when given orally.
We have reported previously that the synthesis of ether lipid-ester prodrugs of CDV and of cyclic CDV can result in the formation of analogues that have enhanced activity in vitro and are orally active in mice infected with cytomegalovirus or orthopoxviruses [1215]. These new compoundshexadecyloxypropyl-CDV (HDP-CDV), octadecyloxyethyl-CDV (ODE-CDV), oleyloxyethyl-CDV (OLE-CDV), and oleyloxypropyl-CDV (OLP-CDV)had significantly better antiviral activity in tissue-culture cells than did the parent CDV [1214]. When given orally to animals, in experimental infections, they were as active as was CDV given parenterally [15].
The purpose of the present study was to assess the effects that different linkers and alkyl chain lengths have on antiviral activity, by determining the activity of these 4 analogues; of tetradecyloxypropyl-CDV (TDP-CDV) and eicosyloxypropyl-CDV (ECP-CDV), new analogues with an alkoxyalkyl structure; and of octyl-CDV (O-CDV), dodecyl-CDV (DD-CDV), eicosyl-CDV (EC-CDV), docosyl-CDV (DC-CDV), and tetracosyl-CDV (TC-CDV), alkyl esters with no linker moiety.
The compounds were tested, by use of plaque-reduction assays, in human foreskin fibroblast cells infected with 5 different AdV serotypes, and the results were compared with those of assays with unmodified CDV. The AdV serotypes tested were AdV 3, 5, and 7, which are usually associated with respiratory infections; AdV 8, which is associated with ocular infections; and AdV 31, which causes enteric infections. Activity against these 5 AdV serotypes was evaluated in the CDV analogues and in (S)-HPMPA, hexadecyloxypropyl-(S)-HPMPA (HDP-HPMPA), and octadecyloxyethyl-(S)-HPMPA (ODE-HPMPA).
Materials and methods.
The structures and methods of synthesis for CDV, HDP-CDV, OLE-CDV, OLP-CDV, and ODE-CDV have been reported elsewhere [12, 14]. The analogues TDP-CDV, ECP-CDV, O-CDV, DD-CDV, EC-CDV, DC-CDV, and TC-CDV were prepared in a similar manner, and those details will be reported elsewhere. The lipid esters of (S)-HPMPA, HDP-HPMPA, and ODE-HPMPA were prepared by a novel direct synthesis method, and those details will be reported elsewhere.
AdV serotypes 3 (strain GB), 5 (strain adenoid 75), 7 (strain Gomen), 8 (strain Trim), and 31 (strain 1315/63) were obtained from the American Type Culture Collection. Virus pools were propagated in human foreskin fibroblast (HFF) cells incubated with Eagle minimal essential medium (MEM), 10% fetal bovine serum, and standard concentrations of L-glutamine, penicillin, and gentamicin.
Each AdV serotype's susceptibility to the various compounds was tested by use of a plaque-reduction assay. Two days before the assay, HFF cells were plated onto 6-well plates and incubated in 90% humidity and 5% CO2 at 37°C. On the day of the assay, compounds were diluted in 2× MEM at 2 times the desired final concentration. Compounds were serially diluted in 2× MEM at a concentration of 1 : 5 to yield 6 solutions varying in concentration from 100 g/mL to 0.03 g/mL. Each virus was diluted in MEM to a concentration that would yield 2030 plaques/well. The medium was aspirated from the wells, and 0.2 mL of solution containing the virus was added to each of the wells, in triplicate. Wells used to test the compounds' toxicity received 0.2 mL of medium, in triplicate. Plates were then incubated for 1 h and were shaken every 15 min. After incubation, 2 mL of overlayconsisting of equal amounts of 1% agarose and of the compounds diluted to the desired concentrations in 2× MEMwas added. Plates were incubated for 7 days, 1 mL of 2× MEM with 1% agarose without the compounds was added, and the plates were incubated for an additional 7 days. After this 14-day incubation, cells were stained with 0.02% neutral red in PBS and were incubated for 56 h, the stain was aspirated, and plaques were counted, at 10× magnification, by use of a stereomicroscope. The EC50 values were then calculated by use of MacSynergy II software (freeware available upon request from M. N. Prichard, University of Alabama School of Medicine, Birmingham, AL).
Results.
The activity of CDV, (S)-HPMPA, and the ether lipid-ester prodrugs against the AdV serotypes are summarized in table 1. Against AdV 3, 5, 7, 8, and 31, unmodified CDV had EC50 values of 0.52.0 mol/L. The most active compounds were HDP-CDV, ODE-CDV, and EC-CDV, which were 5>333-fold more active than CDV. OLP-CDV and OLE-CDV, the alkoxyalkyl analogues with a double bond in the alkyl chain, were generally less active than the saturated alkyl analogues. Alkyl analogues of CDV with chains of either 8, 12, 22, or 24 carbons had lower activity than did CDV. Although susceptibility somewhat varied from strain to strain, no remarkable differences between the 5 serotypes were noted. Two of the short-chain alkyl analogues, O-CDV and DD-CDV, were less active than CDV, which is consistent with the results of our other studies of CMV-infected cells [16].
Against the 5 AdV serotypes, the parent nucleoside phosphonate in the (S)-HPMPA series had an EC50 of 0.52.5 mol/L. Against these 5 serotypes, ODE-HPMPA exhibited activity that was 272,500-fold greater than that of unmodified (S)-HPMPA, whereas the activity of HDP-HPMPA was not significantly different from that of the parent compound. Although most compounds had similar activity against all AdV serotypes, several compounds had serotype-dependent activity. Overall, AdV 31 was the least sensitive to (S)-HPMPA and was also less sensitive to some of the CDV analogues than were other serotypes. Interestingly, CDV and (S)-HPMPA were more effective against AdV 5 than against any of the other serotypes, and HDP-CDV, ODE-CDV, and ODE-HPMPA were highly active.
The median cellular cytotoxicities (CC50) and selectivity indices (SI) of the compounds are summarized in table 2. For all analogues of CDV and (S)-HPMPA, toxicity was also greater than that of the parent compounds. The most toxic compounds were ODE-HPMPA and HDP-HPMPA, with CC50 values of 1.5 mol/L and 10 mol/L, respectively. However, they were nontoxic at the EC50 level, and the most active compoundsHDP-CDV, ODE-CDV, EC-CDV, and ODE-(S)-HPMPAhad SI values of 139>4333, 200>3000, 5503850, and 21882, respectively.
Discussion.
The immunocompromised host is susceptible to severe AdV infections, and mortality rates can reach 60% [10, 11]. Several antiviral drugs have been used to treat patients with severe disease. One of these drugs, ribavirin, has been used, with mixed results, to treat transplant patients [11]. CDV, which is already licensed for treatment of retinitis caused by cytomegalovirus infection in patients with AIDS, has also been used to treat AdV infections that arose after allogeneic hematopoietic stem-cell or bone-marrow transplants [10, 11]. Topical CDV for treatment of ocular infections was also effective in a pilot clinical study [9]. The low oral bioavailability and nephrotoxicity associated with CDV are reasons to find alternative therapies for AdV infections. The ether lipid-ester prodrugs of CDV and of (S)-HPMPA may not have some of the problems associated with the parent compounds, particularly the lack of activity when given orally [15]. The results of these studies suggest that several analogues of CDV and (S)-HPMPA have potent activity against many AdV serotypes.
In a previous study, it was reported that the antiviral activity that the ether lipid-ester prodrugs of CDV had against vaccinia and cowpox viruses was dependent on the number of carbon atoms in the alkyl or alkoxyalkyl chain, the linker moiety, and the presence of a double bond in the side chain linked to the phosphonate moiety [13]. Further analysis indicated that the most active compounds had carbon chains longer than 16 atoms and generally contained either an oxyethyl linker or an oxypropyl linker [13]. In general, the most active compounds had 20 carbon atoms in the lipid ester, with longer chains being somewhat less active. Similar relationships were noted with AdV and in other studies with cytomegalovirus.
Because of the alkoxyalkyl compounds' excellent in vitro activity, selectivity, and oral activity, these analogues should be evaluated for their potential to treat AdV infections in humans. In particular, HDP-CDV and ODE-CDVwhich, as we have reported, have enhanced activity in vitro against cytomegalovirus and orthopoxviruses and, in mice, are as active when given orally as CDV is when given parenterallywould appear to be the most promising candidates for such studies. Finally, ODE-HPMPA is also expected to be active when given orally and may be a good candidate for further study.
References
1. De Clercq E, Sakuma T, Baba M, et al. Antiviral activity of phosphonylmethoxyalkyl derivatives of purine and pyrimidines. Antivir Res 1987; 8:26172. First citation in article
2. De Clercq E, Holy A, Rosenberg L, Sakuma T, Balzarini J, Maudgal PC. A novel broad-spectrum anti-DNA virus agent. Nature 1986; 323:4647. First citation in article
3. De Clercq E. Vaccinia virus inhibitors as a paradigm for the chemotherapy of poxvirus infections. Clin Microbiol Rev 2001; 14:38297. First citation in article
4. Baba M, Mori S, Shigeta S, de Clercq E. Selective inhibitory effect of (S)-9-(3-hydroxy-2-phosphonylmethoxypropyl)adenine and 2-nor-cyclic GMP on adenovirus replication in vitro. Antimicrob Agents Chemother 1987; 31:3379. First citation in article
5. De Oliveira C, Stevenson D, LaBree L, McDonnell P, Trousdale M. Evaluation of cidofovir (HPMPC, GS-504) against adenovirus type 5 infection in vitro and in a New Zealand rabbit ocular model. Antiviral Res 1996; 31:16572. First citation in article
6. Gordon Y, Romanowski E, Araullo-Cruz T, et al. Inhibitory effect of (S)-HPMPC, (S)-HPMPA, and 2-nor-cyclic GMP on clinical ocular adenoviral isolates is serotype-dependent in vitro. Antiviral Res 1991; 16:116. First citation in article
7. Gordon Y, Romanowski E, Araullo-Cruz T, de Clercq E. Pretreatment with topical 0.1% (S)-1-(3-hydroxy-2-phosphonylmethoxypropyl)cytosine inhibits adenovirus type 5 replication in the New Zealand rabbit ocular model. Cornea 1992; 11:52933. First citation in article
8. Romanowski E, Gordon Y. Efficacy of topical cidofovir on multiple adenoviral serotypes in the New Zealand rabbit ocular model. Invest Ophthalmol Vis Sci 2000; 41:4603. First citation in article
9. Hillenkamp J, Reinhard T, Ross R, et al. The effects of cidofovir 1% with and without cyclosporin A 1% as a topical treatment of acute adenoviral keratoconjunctivitis: a controlled clinical pilot study. Ophthalmology 2002; 109:84550. First citation in article
10. Legrand F, Berrebi D, Houhou N, et al. Early diagnosis of adenovirus infection and treatment with cidofovir after bone marrow transplantation in children. Bone Marrow Transplant 2001; 27:6216. First citation in article
11. Ljungman P, Ribaud P, Eyrich M, et al. Cidofovir for adenovirus infections after allogeneic hematopoietic stem cell transplantation: a survey by the Infectious Diseases Working Party of the European Group for Blood and Marrow Transplantation. Bone Marrow Transplant 2003; 31:4816. First citation in article
12. Beadle JR, Hartline C, Aldern K, et al. Alkoxyalkyl esters of cidofovir and cyclic cidofovir exhibit multiple-log enhancement of antiviral activity against cytomegalovirus and herpesvirus replication in vitro. Antimicrob Agents Chemother 2002; 46:23816. First citation in article
13. Keith KA, Wan WB, Ciesla SL, Beadle JR, Hostetler KY, Kern ER. Inhibitory activity of alkoxyalkyl and alkyl esters of cidofovir and cyclic cidofovir against orthopoxvirus replication in vitro. Antimicrob Agents Chemother 2004; 48:186971. First citation in article
14. Kern ER, Hartline C, Harden E, et al. Enhanced inhibition of orthopoxvirus replication in vitro by alkoxyalkyl esters of cidofovir and cyclic cidofovir. Antimicrob Agents Chemother 2002; 46:9915. First citation in article
15. Quenelle DC, Collins DJ, Wan WB, Beadle JR, Hostetler KY, Kern ER. Oral treatment of cowpox and vaccinia virus infection in mice with ether lipid esters of cidofovir. Antimicrob Agents Chemother 2004; 48:40412. First citation in article
16. Wan WB, Beadle JR, Hartline C, Kern ER, Ciesla SL, Valiaeva N, Hostetler KY. Comparison of the antiviral activities of alkoxyalkyl and alkyl esters of cidofovir against human and murine cytomegalovirus replication in vitro. Antimicrob Agents Chemother (in press). First citation in article