(S)-9-(3-hydroxy-2-phosphonylmethoxypropyl)adenine [(S)-HPMPA]: a purine analogue with trypanocidal activity in vitro and in vivo

The unique features of purine salvage systems of pathogenic haemoflagellates render them selectively susceptible to the cytotoxic effects of purine analogues. A series of acyclic nucleoside phosphonates were evaluated for activity against pathogenic haemoflagellates in vitro. One of the phosphonylmethoxyalkylpurines, namely (S)-9-(3-hydroxy-2-phosphonylmethoxypropyl)adenine [(S)-HPMPA], was active in vitro against bloodstream forms of Trypanosoma brucei rbodesiense, T. b. gambiense, multidrug-resistant T. b. brucei, T. congolense and T. evansi, but not against intracellular T. cruzi or Leishmania donovani. Cytotoxic effects against mammalian cells were observed at 4900-27 300-fold higher concentrations than those necessary to inhibit T. b. rbodesiense. (S)-HPMPA was able to eliminate T. b. rbodesiense and multidrug-resistant T. b. brucei in an acute rodent model with two administrations of 10 mg/kg each.     

Cidofovir for treating adenoviral hemorrhagic cystitis in hematopoietic stem cell transplant recipients

Adenovirus (AdV) infection is an important cause of morbidity and mortality in hematopoietic stem cell transplant (HSCT) recipients. We treated 16 patients with AdV hemorrhagic cystitis (HC) following HSCT with cidofovir (CDV; 1 mg/kg/day, three times weekly for 3 weeks). Patients included 10 males and six females with a median age of 50 years (range 10-62). Two of the 16 patients were unevaluable because of early death from nonadenoviral causes. CDV therapy cleared AdV from urine in 12 of 14 patients (86%). Of 14 patients, 10 (71%) showed clinical improvements in HC. Among 14 patients, seven (50%) had avoided renal damage, the most important CDV toxicity. One patient previously treated with foscarnet for cytomegalovirus (CMV) required hemodialysis, and CDV treatment was discontinued. In another patient, CDV treatment was discontinued because of grade 2 nephrotoxicity. Four patients became positive for CMV antigenemia while being treated with CDV, and two developed herpes simplex virus (HSV) stomatitis while being treated with CDV. CDV proved effective in treating AdV HC in transplant patients. However, CDV at 1 mg/kg/day given three times weekly failed to prevent breakthrough infection with CMV and HSV in some patients.     

Synthesis and early development of hexadecyloxypropylcidofovir: an oral antipoxvirus nucleoside phosphonate

Hexadecyloxypropyl-cidofovir (HDP-CDV) is a novel ether lipid conjugate of (S)-1-(3-hydroxy-2-phosphonoylmethoxypropyl)-cytosine (CDV) which exhibits a remarkable increase in antiviral activity against orthopoxviruses compared with CDV. In contrast to CDV, HDP-CDV is orally active and lacks the nephrotoxicity of CDV itself. Increased oral bioavailability and increased cellular uptake is facilitated by the lipid portion of the molecule which is responsible for the improved activity profile. The lipid portion of HDP-CDV is cleaved in the cell, releasing CDV which is converted to CDV diphosphate, the active metabolite. HDP-CDV is a highly effective agent against a variety of orthopoxvirus infections in animal models of disease including vaccinia, cowpox, rabbitpox and ectromelia. Its activity was recently demonstrated in a case of human disseminated vaccinia infection after it was added to a multiple drug regimen. In addition to the activity against orthopoxviruses, HDP-CDV (CMX001) is active against all double stranded DNA viruses including CMV, HSV-1, HSV-2, EBV, adenovirus, BK virus, orf, JC, and papilloma viruses, and is under clinical evaluation as a treatment for human infections with these agents.     

Adenovirus

Adenoviruses (AdV) are DNA viruses that typically cause mild infections involving the upper or lower respiratory tract, gastrointestinal (GI) tract, or conjunctiva. Rare manifestations of AdV infections include hemorrhagic cystitis, hepatitis, hemorrhagic colitis, pancreatitis, nephritis, or encephalitis. Adenovirus infections are more common in young children, owing to lack of humoral immunity. Epidemics of AdV infections may occur in healthy children or adults in closed or crowded settings (particularly military recruits). The disease is more severe, and dissemination is more likely in patients with impaired immunity (eg, organ transplant recipients, human immunodeficiency virus infection, congenital immunodeficiency syndromes). Fatality rates for untreated severe AdV pneumonia or disseminated disease may exceed 50%. More than 50 serotypes of AdV have been identified. Different serotypes display different tissue trophisms and correlate with clinical manifestations of infection. The predominant serotypes differ among countries or regions and change over time. Transmission of novel strains between countries or across continents and replacement of dominant serotypes by new strains may occur. Treatment of AdV infections is controversial because prospective, randomized therapeutic trials have not been done. Cidofovir is considered the drug of choice for severe AdV infections, but not all patients require treatment. Vaccines have been shown to be highly efficacious in reducing the risk of respiratory AdV infection but are currently not available.     

Brincidofovir clearance of acyclovir‐resistant herpes simplex virus‐1 and adenovirus infection after stem cell transplantation

Infections with adenovirus (AdV) and herpesviruses can result in considerable morbidity and mortality in pediatric hematopoietic stem cell transplant (SCT) recipients. Herpes simplex virus (HSV) reactivations are usually prevented by acyclovir (ACV) prophylaxis, whereas cidofovir (CDV) has been used off indication to manage AdV infections. We report a child with myelodysplastic syndrome undergoing multiple SCT, who experienced HSV‐1 disease including severe mucositis and herpetic whitlow, as well as high viral load AdV DNAemia. Both ACV and CDV were ineffective; however, viral loads were decreased with brincidofovir, resulting in viral clearance. A subsequent Epstein–Barr virus disease with relevant meningoencephalitis responded to rituximab.     

Development of CMX001 for the Treatment of Poxvirus Infections

CMX001 (phosphonic acid, [[(S)-2-(4-amino-2-oxo-1(2H)-pyrimidinyl)-1-(hydroxymethyl)ethoxy]methyl]mono[3-(hexadecyloxy)propyl] ester) is a lipid conjugate of the acyclic nucleotide phosphonate, cidofovir (CDV). CMX001 is currently in Phase II clinical trials for the prophylaxis of human cytomegalovirus infection and under development using the Animal Rule for smallpox infection. It has proven effective in reduction of morbidity and mortality in animal models of human smallpox, even after the onset of lesions and other clinical signs of disease. CMX001 and CDV are active against all five families of double-stranded DNA (dsDNA) viruses that cause human morbidity and mortality, including orthopoxviruses such as variola virus, the cause of human smallpox. However, the clinical utility of CDV is limited by the requirement for intravenous dosing and a high incidence of acute kidney toxicity. The risk of nephrotoxicity necessitates pre-hydration and probenecid administration in a health care facility, further complicating high volume CDV use in an emergency situation. Compared with CDV, CMX001 has a number of advantages for treatment of smallpox in an emergency including greater potency in vitro against all dsDNA viruses that cause human disease, a high genetic barrier to resistance, convenient oral administration as a tablet or liquid, and no evidence to date of nephrotoxicity in either animals or humans. The apparent lack of nephrotoxicity observed with CMX001 in vivo is because it is not a substrate for the human organic anion transporters that actively secrete CDV into kidney cells. The ability to test the safety and efficacy of CMX001 in patients with life-threatening dsDNA virus infections which share many basic traits with variola is a major advantage in the development of this antiviral for a smallpox indication.     

Cidofovir Activity against Poxvirus Infections

Cidofovir [(S)-1-(3-hydroxy-2-phosphonylmethoxypropyl)cytosine, HPMPC] is an acyclic nucleoside analog approved since 1996 for clinical use in the treatment of cytomegalovirus (CMV) retinitis in AIDS patients. Cidofovir (CDV) has broad-spectrum activity against DNA viruses, including herpes-, adeno-, polyoma-, papilloma- and poxviruses. Among poxviruses, cidofovir has shown in vitro activity against orthopox [vaccinia, variola (smallpox), cowpox, monkeypox, camelpox, ectromelia], molluscipox [molluscum contagiosum] and parapox [orf] viruses. The anti-poxvirus activity of cidofovir in vivo has been shown in different models of infection when the compound was administered either intraperitoneal, intranasal (aerosolized) or topically. In humans, cidofovir has been successfully used for the treatment of recalcitrant molluscum contagiosum virus and orf virus in immunocompromised patients. CDV remains a reference compound against poxviruses and holds potential for the therapy and short-term prophylaxis of not only orthopox- but also parapox- and molluscipoxvirus infections.     

Antiviral Screen against Canine Distemper Virus-Induced Membrane Fusion Activity

Canine distemper virus (CDV), a close relative of the human pathogen measles virus (MeV), is an enveloped, negative sense RNA virus that belongs to the genus Morbillivirus and causes severe diseases in dogs and other carnivores. Although the vaccination is available as a preventive measure against the disease, the occasional vaccination failure highlights the importance of therapeutic alternatives such as antivirals against CDV. The morbilliviral cell entry system relies on two interacting envelope glycoproteins: the attachment (H) and fusion (F) proteins. Here, to potentially discover novel entry inhibitors targeting CDV H, F and/or the cognate receptor: signaling lymphocyte activation molecule (SLAM) proteins, we designed a quantitative cell-based fusion assay that matched high-throughput screening (HTS) settings. By screening two libraries of small molecule compounds, we successfully identified two membrane fusion inhibitors (F2736-3056 and F2261-0043). Although both inhibitors exhibited similarities in structure and potency with the small molecule compound 3G (an AS-48 class morbilliviral F-protein inhibitor), F2736-3056 displayed improved efficacy in blocking fusion activity when a 3G-escape variant was employed. Altogether, we present a cell-based fusion assay that can be utilized not only to discover antiviral agents against CDV but also to dissect the mechanism of morbilliviral-mediated cell-binding and cell-to-cell fusion activity.     

Adenoviral infection in hematopoietic stem cell transplantation: early diagnosis with quantitative detection of the viral genome in serum and urine

Early diagnosis and prompt introduction of effective therapy are imperative to manage systemic, often fatal adenoviral (AdV) disease following hematopoietic stem cell transplantation (SCT). We evaluated the usefulness of real-time polymerase chain reaction (PCR) in the diagnosis of AdV disease in SCT recipients. Seven SCT recipients, including three with AdV disease, were retrospectively evaluated for AdV genome detection. In serum specimens, the AdV genome was detected at >10(3) copies/ml in the pre-SCT period in two of the five recipients studied. These two patients subsequently developed AdV disease. The three patients with AdV disease had high levels of >10(5) copies/ml during the 4-6 weeks post-SCT period. In none of these patients was the AdV genome detected in urine specimens in pre-SCT period. However, three recipients with detectable urinary levels during the period 1-2 weeks post-SCT subsequently developed AdV disease. Regarding the outcome, two of the three patients with AdV disease died of progressive renal failure. Our results suggest that quantitative determination of the AdV genome in serum and urine is useful to identify patients at high risk of developing AdV disease. Prospectively applied, these measures are expected to improve the dismal outcome of AdV disease in SCT recipients.     

A pilot study of cidofovir in patients with kaposi sarcoma

A clinical trial was conducted to test the activity of cidofovir (CDV), a drug with in vitro activity against Kaposi sarcoma (KS)-associated herpesvirus (KSHV), in KS. Five patients with human immunodeficiency virus-associated KS (4 receiving antiretroviral therapy) and 2 patients with classical KS were administered CDV (5 mg/kg/dose) weekly for 2 weeks and then every other week. All 7 patients had progression of their KS at a median of 8.1 weeks (range, 5-27 weeks). Skin biopsy specimens of KS lesions showed no change in expression of latent or early lytic genes, but, in the 1 assessable patient, there was decreased expression of a late lytic gene. There was no decrease in the virus load of KSHV in peripheral blood mononuclear cells. This study does not provide proof of principle for the treatment of KS with CDV. However, it remains possible that antiherpesvirus therapy can be developed for herpes-induced tumors.     

Molecular monitoring of adenovirus in peripheral blood after allogeneic bone marrow transplantation permits early diagnosis of disseminated disease

Adenovirus (AdV) infection in the course of allogeneic stem cell transplantation (SCT) is associated with high transplant-related morbidity and mortality. Disseminated AdV disease is lethal in most instances. Early detection of AdV infection and identification of patients carrying a high risk of disseminated disease therefore remain a major challenge. In view of the large number of existing AdV types, we have established real-time polymerase chain reaction (PCR) assays permitting sensitive detection and quantification of all 51 currently known human AdV serotypes. In a series of 132 consecutive pediatric patients undergoing SCT, more than 5000 samples derived from peripheral blood (PB), stool, urine, and throat were screened for adenovirus infection by PCR during the posttransplantation period. Thirty-six patients (27%) tested positive by PCR, revealing AdV types of the subgenera A, B, C, D, and F. Except for enteritis in some patients with AdV positivity in stool, detection of the virus at sites other than PB was not associated with clinical signs of virus disease, and transplant-related mortality was not significantly different from AdV-negative patients. By contrast, 82% of patients who had detectable AdV in PB died from infectious complications (P <.001). Monitoring of PB specimens by real-time PCR permitted early diagnosis of invasive AdV infection in all instances. In patients who developed disseminated AdV disease, detection of the virus in PB preceded onset of clinical symptoms by a median of more than 3 weeks. The observation of AdV in peripheral blood may therefore serve as a basis for early initiation of preemptive antiviral treatment.     

Phase variation of the lpf operon is a mechanism to evade cross-immunity between Salmonella serotypes

Conventional wisdom holds that phase variation is a mechanism for immune evasion. However, despite fimbrial phase variation, mice previously exposed to Salmonella typhimurium are protected against a subsequent challenge. We evaluated whether lpf phase variation instead may be a mechanism to evade cross-immunity between Salmonella serotypes. Mice were immunized orally with S. typhimurium aroA mutants either that expressed the lpf operon (phase-on variant) or in which the entire lpf operon had been removed by deletion. During a subsequent challenge with virulent Salmonella enteritidis a selection against lpf phase-on variants was observed in mice previously exposed to S. typhimurium long polar fimbriae. Vaccination with S. typhimurium did not confer protection against challenge with S. enteritidis, presumably because lpf phase-off variants were able to evade cross-immunity. We propose that lpf phase variation is a mechanism to evade cross-immunity between Salmonella serotypes, thereby allowing their coexistence in a host population.     

Polyamines: agents with macrofilaricidal activity

There is a need for effective macrofilaricidal drugs. The polyamine metabolism of filarial worms has been recognized as a possible target for effective drug action. In an attempt to identify agents that might provide leads in developing an effective macrofilaricide, 78 polyamine compounds were selected from among > 250,000 structures that have been amassed by the Walter Reed Army Institute of Research, in the U.S.A. These thousands of agents have been chosen principally for drug-development programmes for other parasitic diseases. The 78 prospective drugs selected were evaluated for their macrofilaricidal activity against Brugia pahangi and Acanthocheilonema viteae, in male Mongolian jirds (Meriones unguiculatus). The animal models using these two parasites were designed to mimic, in so far as possible, human lymphatic filariasis and onchocerciasis, respectively. Thirteen of the compounds were found to be active although none of these has been previously reported to be macrofilaricidal. Two were suppressive for B. pahangi and 11 for A. viteae. These active agents may represent a nucleus around which highly effective drugs can be synthesised.     

Oral activity of ether lipid ester prodrugs of cidofovir against experimental human cytomegalovirus infection

Infection with human cytomegalovirus (HCMV) can cause serious complications in bone-marrow and solid-organ transplant recipients, and current therapies are not optimal. We evaluated 2 orally active ether lipid ester analogues of cidofovir (CDV)--hexadecyloxypropyl-CDV (HDP-CDV) and octadecyloxyethyl-CVD (ODE-CDV)--in severe combined immunodeficient mice in which either human fetal retinal tissue or human fetal thymus and liver tissue had been implanted and was later infected with HCMV. Our results indicate that orally administered treatment with either HDP-CDV or ODE-CDV is 4-8-fold more active, on a molar basis, than is intraperitoneally administered CDV. These data suggest that HDP-CDV and ODE-CDV should be further evaluated as potential antiviral agents for treatment of HCMV infection.     

Psychotropic drugs as potential antitumor agents: a selective screening study.

Compounds with known psychotropic properties were tested for activity in murine ip L1210 leukemia and B 16 melanoma in a protocol designed to obtain leads for new antitumor agents which might also possess central nervous system (CNS) antitumor properties. Barbiturates and hallucinogenic compounds were the only compound types deliberately excluded. Representatives from most of the other known CNS agent classes were included among the 297 psychotropic drugs evaluated. Sixteen of these agents were reproducibly active against the L1210 tumor system with T/C values of 125%. Phenothiazines such as fluphenazine and butyrophenones such as triperidol were prominent among the confirmed active structural types. Dopamine, a beta-phenethylamine neurotrasmitter, was active. While reproducible B16 melanoma activity was not observed among the psychotropic drugs, most of the L1210 confirmed active agents were effective against the ip P388 tumor model and also were active in vitro against KB cells. Ic L1210 activity was not observed among the few compounds chosen for testing in that tumor system. The yield of ip L1210 confirmed actives from this group of psychotropic agents was 18 times that which would have been expected from the random screening of compounds.     

Ex vivo generation of cytotoxic T lymphocytes specific for one or two distinct viruses for the prophylaxis of patients receiving an allogeneic bone marrow transplant

Human adenovirus (AdV) infection and EBV-lymphoproliferative disease (LPD) are serious complications following allogeneic stem cell transplantation. In the healthy individual these viruses cause minor, self-limiting diseases but in the immunocompromised patient they are responsible for significant morbidity and mortality. The limitations of anti-viral drugs and a better understanding of the cellular immune response to viral pathogens have prompted interest in developing adoptive immunotherapy for transplant patients. Ex vivo expanded cytotoxic T lymphocytes (CTLs) specific for EBV have been used effectively both as prophylaxis against EBV-LPD and as treatment of established EBV+ lymphoma. To generate CTLs specific for AdV, we infected immature dendritic cells with virus, in the presence of lipid, and subsequently used these cells to stimulate PBMNCs. Cytotoxicity assays showed that the resulting CTLs specifically lysed AdV-expressing targets and that this was mediated predominantly by CD4+ T cells. To generate CTLs specific for both AdV and EBV, we developed a CD40 ligand co-culture system to infect B-lymphoblastoid cell lines (LCLs) with high efficiency. PBMNCs from healthy AdV-seropositive donors were stimulated weekly with autologous AdV+-LCLs. Chromium release assays demonstrated that the resultant CTLs had specificity against both EBV and AdV and that this was mediated by both CD4+ and CD8+ T cells. Our findings have potential implications for post-transplant AdV and EBV immunotherapy in recipients of allogeneic stem cell transplants.     

Molecular epidemiology of adenovirus type 3 detected from 1994 to 2006 in Hyogo Prefecture, Japan

The molecular epidemiology of 126 adenovirus type 3 (AdV3) isolates obtained in Hyogo Prefecture (population: 5.5 million) from 1994 to 2006 was studied. The hexon-coding region, including 7 hypervariable regions (HVRs) (1,419 bp), was sequenced. We found 5 nonsynonymous nucleotide substitutions in the HVRs. The results are strongly suggestive of positive Darwinian selection. We classified the AdV3 strains analyzed here into 3 genome types: AdV3x (n=44), AdV3y (n=46), and AdV3z (n=36). AdV3x first appeared in 2001 in Hyogo Prefecture, and was detected predominantly during a large outbreak of AdV3 in 2003-2005. AdV3x was identical to a Korean strain responsible for a large outbreak of AdV3 in Korea in 1998-1999. We conclude that at least 3 genome types of AdV3 have circulated in Hyogo Prefecture, Japan, during the past 13 years (1994-2006). The findings also suggest that AdV3x was imported from Korea to Hyogo Prefecture in 2001.     

Assessment of disseminated adenovirus infections using quantitative plasma PCR in adult allogeneic stem cell transplant recipients receiving reduced intensity or myeloablative conditioning

OBJECTIVES: Disseminated adenovirus (AdV) infections following allogeneic stem cell transplantation (allo-SCT) are increasingly recognised, particularly in children. This study evaluated the clinical relevance of disseminated AdV infections in adult allo-SCT recipients, after different conditioning regimens. METHODS: In a cohort of 107 adult allo-SCT recipients, receiving either reduced intensity conditioning (RIC, n = 48) or myeloablative conditioning (MAC, n = 59), AdV DNA levels in plasma were determined retrospectively at 1, 3 and 6 months following transplantation. Results of this screening regimen were compared with a cohort of 58 paediatric allo-SCT recipients, in whom AdV DNA load was monitored prospectively, as part of a pre-emptive treatment strategy. In positive cases, the course of AdV DNA load and clinical outcome were assessed. RESULTS: AdV DNA levels > or =1000 copies/mL were detected in five adults (4.7%) and eight children (13.8%). Screening for AdV viraemia at 1, 3 and 6 months would have detected seven of eight paediatric patients. One adult, receiving MAC, died with disseminated AdV disease and in four (three RIC and one MAC) AdV viraemia was transient without clinical symptoms specifically attributable to AdV. Seven paediatric patients with AdV viraemia were pre-emptively treated with ribavirin or cidofovir and in three of them disseminated AdV infection was related to a fatal outcome. CONCLUSIONS: Disseminated AdV infections following allo-SCT was a rare event in the adults and cause morbidity in a minority of these patients. In four of five adult patients, spontaneous clearance of AdV viraemia occurred. Results did not differ between the conditioning regimens that were applied in the adult cohort.     

An experimental study of the antileprosy activity of a series of thioamides in the mouse

A series of substituted thioamides have been studied to establish whether their structure-activity pattern against Mycobacterium leprae is similar to that displayed against M. tuberculosis. Antileprosy activity was evaluated in the mouse foot pad using both the kinetic and continuous methods. Ethionamide and prothionamide were found to be the most active compounds and to be of approximately equal potency. Thioisonicotinamide was about five times less active. 2-t-Butyl-thioisonicotinamide, 2-dimethylamino-thioisonicotinamide, and pyrazine carbonic thioamide were inactive at the dosages tested. High-pressure liquid chromatographic methods were devised to study the potential influence of pharmacological factors on their in vivo activity. Fecal measurements suggested that all of the thioamides were well absorbed when fed in the diet. After intravenous administration, all of the thioamides were rapidly eliminated from the mouse. The differences in their elimination rates probably played only a minor role in affecting their relative antileprosy activities. It was concluded that the structural requirements for antileprosy and antituberculosis activity of the thioamides are probably similar.