Sustained viral response in a hepatitis C virus-infected chimpanzee via a combination of direct-acting antiviral agents.

Efforts to develop novel, interferon-sparing therapies for treatment of chronic hepatitis C (HCV) infection are contingent on the ability of combination therapies consisting of direct antiviral inhibitors to achieve a sustained virologic response. This work demonstrates a proof of concept that coadministration of the nucleoside analogue MK-0608 with the protease inhibitor MK-7009, both of which produced robust viral load declines as monotherapy, to an HCV-infected chimpanzee can achieve a cure of infection.

The nucleotide analogue 3-deazaadenosine prevents neointima-formation after balloon injury.

We have recently shown that 3-deazaadenosine (c3Ado) inhibits atherogenesis in mice. We studied whether its anti-inflammatory capacity would also affect neointima-formation after balloon injury. Sprague Dawley rats underwent balloon angioplasty. C3Ado was administered orally, starting 5 days prior to the balloon injury and continued for 2 weeks. Fourteen days after balloon injury the intima/media ratio in the c3Ado-treated group was reduced by 67% (p<0.001) and luminal stenosis by 50% (p<0.001). Neointimal cellular density was decreased by 25% (p<0.001) and the induction of c-Jun and ki67 was markedly lower. The reduction of the intima/media ratio was still observed 3 months after balloon injury. Furthermore, a c3Ado-dependent inhibition of PDGF-mediated ERK-activation and proliferation could be demonstrated. Short-term administration of C3Ado inhibits neointima-formation in rats for at least 3 months after injury. The present findings implicate that c3Ado may be useful as an inhibitor of restenosis-formation after balloon angioplasty in humans.

Robust antiviral efficacy upon administration of a nucleoside analog to hepatitis C virus-infected chimpanzees.

Hepatitis C virus (HCV) infects an estimated 170 million individuals worldwide and is associated with an increased incidence of liver fibrosis, cirrhosis, and hepatocellular carcinoma. Currently approved therapies to treat HCV infection consist of combinations of pegylated alpha interferon and ribavirin which result in a sustained viral response in 40 to 60% of patients. Efforts to develop improved therapies include the development of direct inhibitors of virally encoded enzymes such as the viral RNA-dependent RNA polymerase. A nucleoside analog, 2'-C-methyl-7-deaza-adenosine (MK-0608), has been shown to inhibit viral RNA replication in the subgenomic HCV genotype 1b replicon, with a 50% effective concentration (EC(50)) of 0.3 microM (EC(90) = 1.3 microM). To determine efficacy in vivo, MK-0608 was administered to HCV-infected chimpanzees, resulting in dose- and time-dependent decreases in plasma viral loads. In separate experiments, chimpanzees dosed for 7 days with MK-0608 at 0.2 and 2 mg per kg of body weight per day by intravenous administration experienced average reductions in viral load of 1.0 and >5 log(10) IU/ml, respectively. Two other HCV-infected chimpanzees received daily doses of 1 mg MK-0608 per kg via oral administration. After 37 days of oral dosing, one chimpanzee with a high starting viral load experienced a reduction in viral load of 4.6 log(10), and the viral load in the other chimpanzee fell below the limit of quantification (LOQ) of the HCV TaqMan assay (20 IU/ml). Importantly, viral load remained below the LOQ throughout the duration of dosing and for at least 12 days after dosing ended. The results demonstrate a robust antiviral effect on the administration of MK-0608 to HCV-infected chimpanzees.

Long-term administration of 3-deazaadenosine does not alter progression of advanced atherosclerotic lesions in apolipoprotein E-deficient mice.

Inflammatory mechanisms are involved in initiation and progression of atherosclerotic lesions. Previous studies demonstrated antiinflammatory and consecutive antiatherosclerotic effects of the adenosine analogue 3-Deazaadenosine (c(3) Ado) on early lesion development. The present study evaluated the effect of long-term administration of c(3) Ado in a mouse model of advanced atherosclerosis. Apolipoprotein E-deficient mice (age, 35 weeks; n = 31) with already established advanced atherosclerotic lesions were fed either a diet supplemented with c Ado or a regular chow diet for 21 weeks. Treatment resulted in a significant reduction of serum homocysteine levels. Lesion size and lesion morphology, such as frequency of intraplaque hemorrhage, size of necrotic cores, thickness of fibrous caps, and macrophage content within the plaque, were not different between the groups. Lesion calcification, expression of alpha-actin, and intercellular adhesion molecule-1, but not vascular cell adhesion molecule-1, were inhibited by treatment with c(3) Ado. We could not detect any effect on serum concentrations of interleukin-10 (IL-10) and interleukin-1beta (IL-1beta) or on soluble adhesion molecules intercellular adhesion molecule (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1). Electromobility shift assays of protein extracts isolated from aortas did not demonstrate different binding activities of nuclear factor-kappaB (NF-kappaB) and activator protein-1 (AP-1) after treatment with c Ado. Long-term treatment with the adenosine analogue 3-Deazaadenosine did not show significant effects on progression and stability of advanced atherosclerotic lesions in older apolipoprotein E-deficient mice. A potential antiatherosclerotic effect of c(3)Ado (eg, mediated through inhibition of adhesion molecules) might therefore be limited to prevention of early lesion formation and does not seem to play a relevant role in modifying advanced atherosclerotic disease.

Dietary supplementation with 3-deaza adenosine, N-acetyl cysteine, and S-adenosyl methionine provide neuroprotection against multiple consequences of vitamin deficiency and oxidative challenge: relevance to age-related neurodegeneration.

Folate deprivation induces neurotoxicity that is potentiated by additional nutritional and genetic deficiencies including vitamin E and apolipoprotein E deficiency. These deficiencies collectively induce oxidative damage, cognitive impairment, and compensatory alteration in glutathione generation. Treatment with agents that regulate distinct portions of the methionine cycle, including the S-adenosyl homocysteine hydrolase inhibitor, 3-deaza adenosine, the methyl donor S-adenosyl methionine, and the antioxidant N-acetyl cysteine, provide neuroprotection against various aspects of neurotoxicity in normal and apolipoprotein E-deficient mice and in cultured neuronal cells deprived of dietary folate and vitamin E and subjected to iron overload. Here it is demonstrated that simultaneous treatment with these agents provide superior neuroprotection by alleviating individual and overlapping neurotoxic consequences. These findings support combinatorial treatments with agents that compensate for differential insults in age-related neurodegenerative disorders.

Treatment of lethal Ebola virus infection in mice with a single dose of an S-adenosyl-L-homocysteine hydrolase inhibitor.

Ebola Zaire virus causes lethal hemorrhagic fever in humans, for which there is no effective treatment. A variety of adenosine analogues inhibit the replication of Ebola virus in vitro, probably by blocking the cellular enzyme, S-adenosyl-L-homocysteine hydrolase, thereby indirectly limiting methylation of the 5' cap of viral messenger RNA. We previously observed that adult, immunocompetent mice treated thrice daily for 9 days with 2.2-20 mg/kg of an adenosine analogue, carbocyclic 3-deazaadenosine, were protected against lethal Ebola virus challenge. We now report that a single inoculation of 80 mg/kg or less of the same substance, or of 1 mg/kg or less of another analogue, 3-deazaneplanocin A, provides equal or better protection, without causing acute toxicity. One dose of drug given on the first or second day after virus infection reduced peak viremia more than 1000-fold, compared with mock-treated controls, and resulted in survival of most or all animals. Therapy was less effective when administered on the day of challenge, or on the third day postinfection. Single or multiple doses of the same medications suppressed Ebola replication in severe combined immunodeficient mice, but even daily treatment for 15 consecutive days did not eliminate the infection.

Adenosine kinase inhibitors augment release of adenosine from spinal cord slices.

Inhibitors of adenosine kinase, but not adenosine deaminase, produce antinociception when administered spinally. In this study, we evaluated the relative contribution of adenosine kinase and adenosine deaminase to the regulation of adenosine release into the extracellular space within the spinal cord by determining the effects of the adenosine kinase inhibitors 5'-amino-5'-deoxyadenosine and 5-iodotubercidin, and the adenosine deaminase inhibitor 2'-deoxycoformycin on adenosine release from spinal cord slices in an in vitro perfusion system. Both 5'-amino-5'-deoxyadenosine (5-50 microM) and 5-iodotubercidin (5-50 microM), but not 2'-deoxycoformycin (50 microM), augmented adenosine release. 5-Iodotubercidin was slightly more potent and effective than 5'-amino-5'-deoxyadenosine in augmenting release except at the highest concentration, where it was considerably more effective. Combinations of 2'-deoxycoformycin (50 microM) and minimally active concentrations of 5'-amino-5'-deoxyadenosine and 5-iodotubercidin (5 microM each) produced a synergistic enhancement of release. These results support a predominant involvement of adenosine kinase in regulating extracellular adenosine levels in the spinal cord, but adenosine deaminase also can play a significant role.

Pharmacokinetics of the antiviral agent carbocyclic 3-deazaadenosine.

The pharmacokinetics of carbocyclic 3-deazaadenosine (Cc3Ado), a competitive inhibitor of S-adenosyl-L-homocysteine hydrolase and novel antiviral agent, was investigated in female BALB/c mice. Mice were dosed either orally or intravenously with a single dose of 10 mg/kg (10 microCi [3H]Cc3Ado), and blood and tissue samples were taken at selected intervals for 24 hr. The plasma concentration vs. time data for Cc3Ado was best described by a two-compartment open model with first-order elimination. The apparent half-life was 23 and 38 min, for intravenously and orally administered Cc3Ado, respectively. Depending on route, tissue concentrations of Cc3Ado reached their maximum by 120 min, and concentrations of Cc3Ado were greatest in the liver, followed by the kidney, spleen, and stomach. By 24 hr, all tissues contained a similar amount of Cc3Ado. In the plasma, one major labeled metabolite was detected, which increased in concentration over time until about 45 min after dosing. None of the plasma Cc3Ado was protein bound, as assessed by in vitro protein binding analysis. Oral Cc3Ado was about 20% bioavailable. Data from this first investigation of the pharmacokinetics of Cc3Ado indicate that this antiviral agent is rapidly distributed and eliminated from the plasma and that distribution to tissues is widespread and rapid.

Combination therapy of Schistosoma japonicum by tubercidin and nitrobenzylthioinosine 5'-monophosphate.

Coadministration of nitrobenzylthioinosine 5'-monophosphate (NBMPR-P) with high doses of tubercidin by i.p. injection into Schistosoma japonicum infected mice beginning 5 weeks post-infection was highly toxic to the parasite but not the hose. Combination therapy resulted in a striking reduction in the number of worms, and the few worms that could be found were stunted. Combination therapy also caused a drastic reduction in the number of eggs in the livers (from 86,500 to 2,800 eggs/liver) and intestines (from 2,200 to 74 eggs/cm2), and 95% of eggs that were found were dead, indicating the termination of oviposition. Mice receiving the combination of tubercidin plus NBMPR-P appeared healthy and had normal size livers and spleens. These results demonstrate that by combining NBMPR-P with tubercidin high selective toxicity against S. japonicum can be achieved, as was shown previously with S. mansoni.

Combination therapy of schistosomiasis by tubercidin and nitrobenzylthioinosine 5'-monophosphate.

Nitrobenzylthioinosine 5'-monophosphate (NBMPR-P) inhibits the transport of nucleosides, including tubercidin, in mammalian systems but not in Schistosoma mansoni. Administration of NBMPR-P with high doses of tubercidin (lethal doses if injected alone) by intraperitoneal injection into S. mansoni-infected mice was highly toxic to the parasite but not to the host. Combination therapy resulted in a striking decrease in the number and copulation of worms. The few worms that could be found were so stunted that it was difficult to identify their sex. Mice receiving the combination of tubercidin plus NBMPR-P appeared healthy and had normal-sized livers and spleens. Combination therapy also caused a drastic decrease in the number of eggs in the liver (from 32,500 to 1,800 eggs per liver) and in the intestine (from 1,295 to 2 eggs per cm2). All eggs found were dead, indicating the termination of oviposition. Very few granulomas were detected in livers of treated animals. Sections of these livers showed lesions containing dead worms and what appeared to be a process of regeneration of normal tissue around old granulomas. Thus, combination therapy reduced the number and the progress of the primary pathological lesions associated with schistosomiasis. These results demonstrate that through combination therapy, highly selective toxicity against a parasite can be achieved. The effectiveness, simplicity, and practicality of host protection afforded by this method may yield a promising chemotherapeutic approach for the treatment of schistosomiasis and other parasitic diseases.

Glucagonoma and its angiographic diagnosis.

Four patients with metastatic glucagonoma are described. Angiography demonstrated a small avascular primary tumor of the tail of the pancreas in one patient and large hypervascular tumors of the pancreatic head in the other three. Liver metastases, were hypervascular in all four. Including our 4 with 21 cases from the literature, glucagonomas show a 92% incidence of increased tumor vascularity--thus increasing the likelihood of successful angiographic diagnosis. The awareness of clinically subtle or atypical glucagonomas and use of plasma glucagon determination are important factors leading to early diagnosis of these neoplasms. Since angiography can localize the tumor, assess its extent, and detect hepatic metastases, it is essential to the detailed evaluation of glucagonomas.

Treatment of mouse neoplasms with high doses of tubercidin.

Previous studies from this laboratory demonstrated that a potent inhibitor of nucleoside transport, nitrobenzylthioinosine (NBMPR), protected cultured cells against cytotoxic nucleosides (nebularine, tubercidin, and toyocamycin). NBMPR and its 5'-monophosphate (NBMPR-P) also protected mice against potentially lethal dosage of these agents. This report describes protection of mice from potentially lethal dosages of tubercidin by administration of NBMPR-P and the use of combinations of these agents in treatments of mice bearing transplanted neoplasms. Treatment of mice bearing i.p. implants of the Ehrlich ascites carcinoma, leukemia L1210/TG8, and colon carcinoma 26 with potentially lethal dosages of tubercidin administered together with host-protecting dosages of NBMPR-P resulted in substantial kill of neoplastic cells and long-term survivors. In these experiments, therapeutic effects were achieved at optimal dosages of NBMPR-P, which protected host vital tissues but did not protect neoplastic cells in ascitic fluids (Ehrlich ascites carcinoma cells and leukemia L1210/TG8 cells). However, at supraoptimal dosages of NBMPR-P, the occurrence of therapeutic failures which were neoplastic deaths indicated that NBMPR-P also protected the neoplastic ascites cells against tubercidin cytotoxicity. Thus, the selectivity of tubercidin toxicity toward cells of the Ehrlich ascites carcinoma and leukemia L1210/TG8 was modified by NBMPR-P dosage.

Phospholipid derivatives of nucleoside analogs as prodrugs with enhanced catabolic stability.

The nucleoside 5'-diphosphate-L-1,2-dipalmitin derivatives of 1-beta-D-arabinofuranosylcytosine (ara-C), 9-beta-D-arabinofuranosyladenine (ara-A), and tubercidin have been synthesized, and their cytotoxicity has been evaluated against a mouse myeloma cell line (MPC-11) in vitro and against L1210 lymphoid leukemia both in vitro and in vivo. Sonication methods were utilized to solubilize these lipophilic derivatives in aqueous solution in order to facilitate such biological evaluation; the ara-A derivative resisted solubilization by several techniques. The nucleoside:phospholipid conjugates of ara-C and tubercidin both were cytotoxic towards the two cell lines, and detailed experiments were cytotoxic towards the two cell lines, and detailed experiments were carried out to show that the new derivatives (a) were not degraded in the medium prior to cellular uptake and (b) acted as prodrugs or molecular depots of the parent nucleoside analog. In addition, 1-beta-D-arabinofuranosylcytosine 5'-diphosphate'5'-L-1,2-dipalmitin was not a substrate for cytidine deaminase (cytidine aminohydrolase, EC 3.5.4.5), the primary enzyme responsible for the rapid catabolism of ara-C. In in vivo studies against L1210 lymphoid leukemia in mice, the 1-beta-D-arabinofuranosylcytosine 5'-diphosphate-5'-L-1,2-dipalmitin showed an increased efficacy (increased life span, 260%) relative to the parent ara-C (increased life span, 89%) regardless of treatment schedule used, whereas the tubercidin 5'-diphosphate-5'-L-1,2-dipalmitin appeared extremely toxic even at low dosages. That 1-beta-D-arabinofuranosylcytosine 5'-diphosphate-5'-L-1,2-dipalmitin was acting as a sustained release drug in vivo was demonstrated by utilizing a single dose administered on Days -1, 0, +1, and +2 relative to inoculation of the L1210 lymphoid leukemia cells on Day 0. Again, a much increased efficacy relative to the best treatment using ara-C was apparent. The potential advantages and the biochemical rationale for the development of these novel prodrugs are discussed.

5-FU versus combination therapy with tubercidin, streptozotocin, and 5-FU in the treatment of pancreatic carcinomas: COG protocol 7230.

From May 1972 until May 1976, 105 patients were entered on Central Oncology Group protocol 7230 to compare the combination of streptozotocin, tubercidin, and 5-fluorouracil (5-FU) versus 5-FU alone in the treatment of adenocarcinoma and islet cell carcinoma of the pancreas. Twenty-nine were not evaluable. Thirty-six evaluable cases received 5-FU, and 40 received the combination, with no significant difference in time to progression or survival. Toxicity in the two regimens was somewhat different but was essentially similar in magnitude. Results indicate no benefit in the treatment of adenocarcinoma of the pancreas with the three-drug combination over 5-FU alone. All of the islet cell tumor patients benefited from the combination by response or arrest of progression of disease. Further study should be directed toward the use of this combination in the treatment of functioning and non-functioning islet cell tumors of the pancreas.

Activity of tubercidin against immature Fasciola hepatica in mice.

HaM/ICR females mice (20 to 25 g), infected with from 3 to 5 Fasciola hepatica metacercariae per os, were treated with a single dose of thepurine nucleoside analog tubercidin (7-deaza-adenosine; Tu) 7, 14, 21, and 28 days postexposure.Tu was administered either by direct intravenous injection or intraerythrocytically. Most of the flukes were killed and host survival rates were markedly increased if treatment was started within 3 weeks postexposure. The minimal single intravenous dose of Tu that was maximally effective was between 10 and 20 mg/kg.

Treatment of schistosomiasis mansoni and japonica in baboons with tubercidin given by direct intravenous injection.

Baboons (Papio cyanocephalus and P. anubis), infected with Schistosoma mansoni or S. japonicum, were treated with single doses of tubercidin (7-deazaadenosine; Tu), 1, 3, and 5 mg per kg of body weight, administered by intravenous drip. Crystalline Tu was dissolved in sterile 0.9% NaCl solution (1 mg per ml), and the solution was delivered at a rate of 4 ml per minute. Detectable short-term host toxicity was limited to the 5 mg per kg dose, mainly in the form of reversible mild to moderate kidney damage. Only this 5 mg per kg dose administered to baboons with relatively heavy S. mansoni infections was capable of completely suppressing fecal egg excretion for 6 to 8 weeks, eliminating the female worms, and terminating active disease, as indicated by histopathological findings. Comparable effects were achieved following the administration of the 3 mg per kg dose to baboons with moderate to heavy S. japonicum infections.