Pharmacokinetic Properties of Orally Administered 4'-Cyano-2'-deoxyguanosine, a Novel Nucleoside Analog Inhibitor of the Hepatitis B Virus, in Viral Liver Injury Model Rats.

A nucleoside analog, 4'-cyano-2'-deoxyguanosine (CdG), which was developed as an inhibitor of the chronic hepatitis B virus (HBV), exhibited a superior antiviral activity against both wild-type and drugs-resistant HBV to marketed nucleoside analogs. In addition to previous pharmacokinetic studies of CdG in healthy rats, this study reports on an evaluation of the pharmacokinetic characteristics of CdG in a rat model of viral liver injury (VLI) induced by treatment with concanavalin A. Following an intravenous administration of CdG at a dose of 1 mg/kg, the plasma concentration profile of CdG in VLI model rats was found to be similar to that of healthy rats with no significant difference in kinetic parameters. However, when CdG was orally administered at a dose of 1 mg/kg, the maximum blood concentration was much lower in VLI model rats than in healthy rats. Interestingly, the amount of residual food in the stomachs in VLI model rats was significantly larger than that in healthy rats, indicating that the adsorption of CdG in the gastrointestinal tract was inhibited in the presence of food as well as other marketed nucleoside analogs. As observed in healthy rats, CdG was largely distributed to the liver compared to the kidney in the VLI model. These results suggest that liver pathology has only a minor effect on the pharmacokinetic properties of CdG, but the influence of food on CdG absorption needs to be considered.

6-Dithio-2'-deoxyguanosine analogs induce reactive oxygen species-mediated tumor cell apoptosis via bi-targeting thioredoxin 1 and telomerase.

Thioredoxin 1 (Trx1) and telomerase play key roles in the development and progression process of most tumors, and they both are promising drug therapy targets. We have, for the first time, discovered that Trx1 and telomerase had a dual-target synergistic effect. Based on that results, we designed a series of 6-dithio-2'-deoxyguanosine analogs (named as YLS00X) and verified whether they can inhibit Trx1 and telomerase simultaneously. TrxR1/Trx1 system activity and telomerase expression were significantly inhibited by 6-dithio-2'-deoxyguanosine analogs, especially YLS004. YLS004 can also cause ROS accumulation, and induce tumor cell apoptosis. The vitro antitumor activity of 6-dithio-2'-deoxyguanosine analogs using MTT assay on 11 different human cancer cells and found that human colon cancer cells(HCT116) and melanoma cells (A375) were the most sensitive cells to 6-dithio-2'-deoxyguanosine analogs treatment and vivo xenografts models also confirmed that. The serum biochemical parameters and multiple organs HE staining results of subacute experiments indicated that YLS004 might be mildly toxic to immune organs, including the thymus, spleen, and hematopoietic system. Besides, YLS004 was rapidly metabolized in the rats' blood. Our study revealed that YLS004, a Trx1 and telomerase inhibitor, has strong anti-tumor effects to colon cancer and melanoma cells and is a promising new candidate drug.

Pharmacokinetics studies of 4'-cyano-2'-deoxyguanosine, a potent inhibitor of the hepatitis B virus, in rats.

OBJECTIVES: 4'-cyano-2'-deoxyguanosine (CdG), a novel nucleoside analogue, has a high degree of antiviral activity against the chronic hepatitis B virus (HBV). The objective of this study was to develop an analytical method for quantitatively determining CdG levels in biological samples by liquid chromatography-mass spectrometry (LC/MS) and to investigate the pharmacokinetic properties of CdG in rats after intravenous and oral administration. METHODS: An analytical method using a UPLC system interfaced with a TOF-MS system was developed and validated. The pharmacokinetic properties after the intravenous and oral administration of CdG to rats were evaluated. In vivo pharmacokinetic interactions between CdG and entecavir were also investigated. KEY FINDINGS: A rapid, simple and selective method for the quantification of CdG in biological samples was established using LC/MS with solid-phase extraction. In vivo pharmacokinetic studies of CdG in rats demonstrated that CdG is highly bioavailable, is rapidly absorbed from the intestinal tract, is then distributed to the liver rather than kidney and is ultimately excreted via the urine in an unchanged form. The co-administration of CdG and entecavir led to pharmacokinetic interactions with each other. CONCLUSIONS: The data generated in this study provide support for the clinical development of CdG for use in the treatment of HBV.

Does Fluoroscopy Induce DNA Oxidative Damage in Patients Undergoing Catheter Ablation?

As no studies before now have thoroughly investigated the risk associated with the exposure to low-dose ionizing radiations in patients undergoing catheter ablation (CA), we aimed to evaluate the oxidative and DNA damage in 59 CA patients (10 of whom received N-acetylcysteine (NAC) before the procedure). A burst of oxidized/reduced glutathione ratio was observed 3 hours after procedure that was diminished by NAC administration. 8-Hydroxy-2'-deoxyguanosine (8-OHdG) concentrations, index of oxidative DNA damage, showed a peak 24 hours after CA. A positive correlation between 8-OHdG peak and fluoroscopy time and a negative correlation among 8-OHdG decrease (from the peak to 48 hours after CA) and all procedure parameters were found. Furthermore, DNA tail percentages (which reflect the extent and the number of DNA strand breaks) positively correlated with 8-OHdG concentrations. This study evaluates for the first time the kinetic of oxidative damage in patients undergoing CA procedure. Our findings raise the question of whether 8-OHdG can be used as a circulating biomarker of DNA oxidative damage induced by low-dose ionizing radiations in different clinical settings. Antioxid. Redox Signal. 28, 1137-1143.

Low-dose YC-1 combined with glucose and insulin selectively induces apoptosis in hypoxic gastric carcinoma cells by inhibiting anaerobic glycolysis.

This study aimed to establish a therapeutic strategy targeting hypoxic cancer cells in gastric carcinoma (GC). YC-1 is a HIF-1α inhibitor, and we revealed that low-dose YC-1 (10 µM) suppressed HIF-1α expression, and induced hypoxia-dependent apoptosis in the GC cell line 58As9. This hypoxia-specific apoptosis induction by YC-1 involved excessive reactive oxygen species (ROS) generation. The apoptotic effect of 10 µM YC-1 was enhanced by additional glucose (G) and insulin (I) treatments. RT-PCR demonstrated that 10 µM YC-1 reduced hypoxia-induced expression of HIF-1α targets involved in anaerobic glycolysis. Metabolic analysis showed that YC-1 shifted glucose metabolism in hypoxic cells from anaerobic glycolysis to oxidative phosphorylation (OXPHOS). Additional GI accelerated membranous GLUT1 translocation, elevating glucose uptake, and increased acetyl-CoA levels, leading to more ROS generation in hypoxic YC-1-treated cells. Finally, we evaluated the anti-cancer effect of low-dose YC-1 (1 mg/kg) + G (2 g/kg) and I (1 unit/3 g G) treatment in xenograft models. YC-1 + GI therapy strongly inhibited tumour growth. Immunohistochemical analysis demonstrated that YC-1 + GI reduced HIF-1α expression and pimonidazole accumulation in tumours. Conversely, YC-1 + GI increased intra-tumoral 8-OHdG and levels of apoptosis markers. Low-dose YC-1 + GI is a unique therapy targeting hypoxic GC cells that generates lethal ROS via forced activation of OXPHOS.

8-Hydroxy-2-deoxyguanosine ameliorates high-fat diet-induced insulin resistance and adipocyte dysfunction in mice.

8-Hydroxy-2-deoxyguanosine (8-OHdG), a marker of oxidative DNA damage, has been recently shown to exert anti-inflammatory effects through inhibition of Rac1. Inflammation in adipose tissue is a hallmark of obesity-induced insulin resistance, but the therapeutic potential of 8-OHdG in treatment of metabolic diseases has not been fully elucidated. The aim of this study was to examine the effect of exogenously administered 8-OHdG on adipose tissue and whole body metabolism. In cultured adipocytes, 8-OHdG inhibited adipogenesis and reversed TNFα-induced insulin resistance. In high-fat diet (HFD)-induced obese mice, 8-OHdG administration blunted the rise in body weight and fat mass. The decrease in adipose tissue mass by 8-OHdG was due to reduced adipocyte hypertrophy through induction of adipose triglyceride lipase and inhibition of fatty acid synthase expression. 8-OHdG also inhibited the infiltration of macrophages, resulting in amelioration of adipose tissue inflammation and adipokine dysregulation. Moreover, 8-OHdG administration ameliorated adipocyte as well as systemic insulin sensitivity. Both in vivo and in vitro results showed that 8-OHdG induces AMPK activation and reduces JNK activation in adipocytes. In conclusion, our results show that orally administered 8-OHdG protects against HFD-induced metabolic disorders by regulating adipocyte metabolism.

Investigation of the role of 8-OHdG and oxidative stress in papillary thyroid carcinoma.

The aim of this study was to determine levels of serum 8-hydroxy-2'-deoxyguanosine (8-OHdG) as an indicator of oxidant-induced DNA damage and oxidant status in patients with papillary thyroid carcinoma before and after surgery. This study included 25 patients with papillary thyroid carcinoma and age-matched 27 healthy controls. Total antioxidant status (TAS), total oxidant status (TOS), lipid hydroperoxide (LOOH), and 8-OHdG levels were measured. 8-OHdG levels were significantly higher in the preoperative papillary thyroid carcinoma (PTC) group compared with the healthy control group (p < 0.001) and were significantly lower after operation in patients with papillary thyroid carcinoma (p = 0.004). Oxidative stress index (OSI) levels were significantly higher in both preoperative and postoperative PTC patients compared with the healthy control group (p < 0.001 and p < 0.001, respectively). TOS levels were higher in the preoperative and postoperative PTC groups compared to the healthy control group (p < 0.001 and p < 0.001, respectively). TAS levels was lower in the preoperative PTC groups compared to the healthy control group (p = 0.011). Serum LOOH levels were higher in both preoperative and postoperative PTC groups compared to the healthy control group (p < 0.001 and p < 0.001, respectively). Correlation analysis yielded that serum 8-OHdG levels were positively correlated with OSI and LOOH levels in patients with PTC before surgery (r = 0.668, p < 0.001; r = 0.446, p = 0.025, respectively) and had a negative correlation with TAS levels (r = -0.616, p = 0.001). We have shown severe oxidative DNA damage and impaired antioxidant status in papillary thyroid carcinoma.

Differential cellular localization of antioxidant enzymes in the trigeminal ganglion.

Because of its high oxygen demands, neural tissue is predisposed to oxidative stress. Here, our aim was to clarify the cellular localization of antioxidant enzymes in the trigeminal ganglion. We found that the transcriptional factor Sox10 is localized exclusively in satellite glial cells (SGCs) in the adult trigeminal ganglion. The use of transgenic mice that express the fluorescent protein Venus under the Sox10 promoter enabled us to distinguish between neurons and SGCs. Although both superoxide dismutases 1 and 2 were present in the neurons, only superoxide dismutase 1 was identified in SGCs. The enzymes relevant to hydrogen peroxide degradation displayed differential cellular localization, such that neurons were endowed with glutathione peroxidase 1 and thioredoxin 2, and catalase and thioredoxin 2 were present in SGCs. Our immunohistochemical finding showed that only SGCs were labeled by the oxidative damage marker 8-hydroxy-2'-deoxyguanosine, which indicates that the antioxidant systems of SGCs were less potent. The transient receptor potential vanilloid subfamily member 1 (TRPV1), the capsaicin receptor, is implicated in inflammatory hyperalgesia, and we demonstrated that topical capsaicin application causes short-lasting mechanical hyperalgesia in the face. Our cell-based assay revealed that TRPV1 agonist stimulation in the presence of TRPV1 overexpression caused reactive oxygen species-mediated caspase-3 activation. Moreover, capsaicin induced the cellular demise of primary TRPV1-positive trigeminal ganglion neurons in a dose-dependent manner, and this effect was inhibited by a free radical scavenger and a pancaspase inhibitor. This study delineates the localization of antioxidative stress-related enzymes in the trigeminal ganglion and reveals the importance of the pivotal role of reactive oxygen species in the TRPV1-mediated caspase-dependent cell death of trigeminal ganglion neurons. Therapeutic measures for antioxidative stress should be taken to prevent damage to trigeminal primary sensory neurons in inflammatory pain disorders.

Effects of 7,8-dihydro-8-oxo-deoxyguanosine on antigen challenge in ovalbumin-sensitized mice may be mediated by suppression of Rac.

BACKGROUND AND PURPOSE: Earlier we reported that 7,8-dihydro-8-oxo-deoxyguanosine (8-oxo-dG), an oxidatively modified guanine nucleoside, exerted anti-inflammatory activity through inactivation of the GTP binding protein, Rac. In the present study, the effects of 8-oxo-dG were investigated on responses to antigen challenge in sensitized mice, as Rac is also involved at several steps of the immune process including antigen-induced release of mediators from mast cells. EXPERIMENTAL APPROACH: Mice were sensitized and challenged with ovalbumin without or with oral administration of 8-oxo-dG during the challenge. Effects of 8-oxo-dG were assessed by measuring lung function, cells and cytokines in broncho-alveolar lavage fluid (BALF) and serum levels of antigen-specific IgE. Rac activity in BALF cells was also measured. KEY RESULTS: 8-oxo-dG inhibited the increased airway resistance and decreased lung compliance of sensitized and challenged mice to the levels of non-sensitized control mice and lowered the increased leukocytes particularly, eosinophils, in BALF. Furthermore, 8-oxo-dG suppressed allergy-associated immune responses, such as raised anti- ovalbumin IgE antibody in serum, increased expression of CD40 and CD40 ligand in lung, increased interleukin-4, -5, -13, interferon-gamma and tumour necrosis factor-alpha in BALF and mRNA levels of these cytokines in BALF cells, dose-dependently. The corresponding purine, 8-oxo-guanine, showed no effects in the same experiments. Finally, 8-oxo-dG, but not 8-oxo-guanine, inhibited the increased Rac activity in sensitized and challenged mice. CONCLUSION AND IMPLICATIONS: 8-Oxo-dG had anti-allergic actions that might be mediated by Rac inactivation. This compound merits further evaluation of its therapeutic potential in allergic asthma.

Oral administration of a synthetic agonist of Toll-like receptor 9 potently modulates peanut-induced allergy in mice.

BACKGROUND: Agonists of Toll-like receptor 9 have been shown to induce potent T(H)1-type immune responses and prevent and reverse ovalbumin-induced T(H)2-dominant allergic asthma in mice. OBJECTIVE: We examined oral administration of a synthetic agonist of Toll-like receptor 9 (immune modulatory oligonucleotide [IMO]) to modulate peanut-induced allergy in mice. METHODS: In the prevention model mice were sensitized 3 times by means of oral administration of peanut in the presence or absence of IMO. In a treatment protocol mice were sensitized orally with peanut on days 0 and 14 and treated 4 times with oral administration of IMO starting on day 21. RESULTS: In the prevention study mice that received the combination of IMO/peanut showed decreased IgE and increased IgG2a levels in the serum and intestinal tissue compared with mice sensitized with peanut only. In spleen cell recall experiments, production of IL-5 and IL-13 was inhibited and production of IFN-gamma was increased in mice immunized with the peanut/IMO combination compared with those sensitized with peanut only. In the treatment model IMO-treated mice showed decreased IgE, IL-5, and IL-13 levels and increased IgG2a and IFN-gamma levels in the serum, intestines, and spleen cells compared with PBS-treated mice. A reduction in local inflammation and restoration of normal structure in the intestines was found in the mice that received IMO in both models. CONCLUSION: These results indicate that IMOs can switch peanut-induced T(H)2 immune responses toward T(H)1 responses accompanied by reduced inflammation in the gastrointestinal tract and anaphylaxis in both the prevention and treatment models. CLINICAL IMPLICATIONS: IMOs might be suitable candidates for the management of peanut-induced allergy.

Inhibitory effect of 8-oxo-7,8-dihydro-2'-deoxyguanosine on the growth of KG-1 myelosarcoma in Balb/c nude mice.

We previously found that 8-oxo-7,8-dihydro-2'-deoxyguanosine (oh(8)dG) kills KG-1, a human myelocytic leukemic cell line with mutational loss of 8-oxoguanine glycosylase (OGG1) activity in vitro. This observation prompted us to investigate the cytotoxicity of oh(8)dG on KG-1 in vivo. This cytotoxicity was observed by administrating oh(8)dG (3.3-330mg/kgb.w./day) for 14 days into nude mice bearing a KG-1 myelosarcoma. The results were as follows; oh(8)dG inhibited the growth of KG-1 myelosarcoma dose-dependently in terms of tumor size and weight, but had no effect on the growth of myelosarcoma of U937, a human monocytic leukemic cell line possessing wild-type OGG1. 6-Thioguanine (6-TG), an anticancer drug inhibited the growths of KG-1 and U937 tumors. 2'-Deoxyguanosine (dG) had a statistically insignificant anti-growth effect on both tumors. The oh(8)dG-treated KG-1 tumor showed the increased expression of apoptosis-processing caspases 8, 9 and 3 together with DNA fragmentation, the increased expression of cell cycle inhibitors, p16 and p27, and the decreased expression of cell cycle accelerator, cyclins and cdks, indicating the nature of cytotoxicity is cell cycle arrest and apoptosis. The genomic DNA of oh(8)dG-treated KG-1 tumors showed an increase in OGG1 sensitive sites, which is consistent with an increase in the 8-oxo-7,8-dihydroguanine (oh(8)Gua) level in the DNA of KG-1 treated with oh(8)dG in vitro. Presumably an increased level of oh(8)Gua in DNA may trigger the cytotoxicity. These findings suggest that oh(8)dG is selectively cytotoxic to KG-1 or tumors that are OGG1-deficient.

Modulation of chemotherapy resistance in regional therapy: a novel therapeutic approach to advanced extremity melanoma using intra-arterial temozolomide in combination with systemic O6-benzylguanine.

This study investigated whether the therapeutic index of regional melanoma therapy using parenteral temozolomide could be improved by chemomodulation with O6-benzylguanine (O6BG), an inhibitor of the DNA repair enzyme O6-alkylguanine-DNA alkyltransferase (AGT). Using a nude rat s.c. human melanoma xenograft model of the extremity, tumors were analyzed for AGT level 2 to 3 hours after the i.p. injection of 3.5 to 70.0 mg/kg O6BG to inhibit AGT activity. Survival studies were conducted using animals that were treated with a 15-minute isolated limb infusion with 10% DMSO in PBS (control), temozolomide alone, or temozolomide in conjunction with single or multiple doses of i.p. O6BG. Tumor volume and toxicity level were monitored every other day. Administration of 3.5 mg/kg O6BG depleted tumor AGT activity by 93.5% (P < 0.01). Groups treated with regional temozolomide alone (350 mg/kg), systemic temozolomide with O6BG, or vehicle combined with O6BG showed no significant tumor responses compared with controls. Whereas use of regional temozolomide alone at a higher dose (750 mg/kg) showed some degree of tumor response, regional temozolomide given in conjunction with multiple dosages of O6BG showed a marked (P < 0.01) reduction in tumor growth with minimal toxicity. Our findings suggest that AGT modulation by the administration of O6BG in combination with temozolomide regional chemotherapy leads to a significant improvement in melanoma antitumor responses. Clinical trials using chemotherapy modulation may improve response rates in future regional infusion and perfusion drug trials.

O6-benzylguanine suppression of O6-alkylguanine-DNA alkyltransferase in anaplastic gliomas.

The purpose of the study was to determine the dose of O(6)-benzylguanine (BG) that would suppress O(6)-alkylguanine-DNA alkyltransferase (AGT) activity to undetectable levels in > 90% of anaplastic gliomas, as measured 6 h after a 1-h BG infusion. Subjects who were scheduled for surgical resection of a known or presumed anaplastic glioma received a 1-h infusion of BG. Tumor tissue was surgically removed approximately 6 h after the end of the infusion and was analyzed for AGT activity. The BG dose was escalated until at least 11 of 14 subjects had no detectable AGT activity. An additional cohort of patients received the identified effective dose of BG approximately 18 h before tumor resection in order to compare our results with an earlier study using the longer time interval. In the 79 subjects who were enrolled, there was no significant toxicity that was attributed to the BG. A dose-response relationship was determined between the BG dose and the percentage of subjects with undetectable AGT. A dose of 120 mg/m(2) suppressed AGT to less than detectable levels in 17 of 18 patients when the drug-resection interval was 6 h. With an 18-h interval, only 5 of 11 subjects had undetectable AGT at the 120-mg/m(2) dose. We conclude that a BG dose of 120 mg/m(2) given 6 h before an alkylating drug would be effective in suppressing AGT and possibly potentiating the cytotoxic effects of the drug.

Synthetic unmethylated cytosine-phosphate-guanosine oligodeoxynucleotides are potent stimulators of antileukemia responses in naive and bone marrow transplant recipients.

Immunostimulatory cytosine-phophate-guanosine (CpG)--containing motifs in bacterial DNA are potent immune system activators. Depending on the bases flanking the CpG motif and on the DNA backbone, CpG oligodeoxynucleotides (ODNs) can induce relatively more B-cell activation or relatively more natural killer (NK)--cell activation. To evaluate their antitumor activities, an NK-optimized ODN (1585) and 2 B-cell--optimized ODNs (1826 and 2006) were compared for their ability to protect naive mice against a lethal acute myelogenous leukemia (AML) challenge. CpG 2006, but not CpG 1585, administered 2 days before the AML challenge, allowed mice to survive more than 100 times a lethal tumor dose. Cell depletion studies showed that protection did not require T or B cells but depended on NK cells and also on an NK-independent mechanism. CpG 2006 protected against AML challenge in both syngeneic and allogeneic bone marrow transplant (BMT) recipients at both early and late time points after transplantation. Although CpG 1585 had no protective effect on its own, it showed a striking synergy with CpG 2006 to induce prolonged survival to AML challenge in allogeneic recipients of T-cell-depleted marrow grafts, exceeding the survival benefit of donor lymphocyte infusion (DLI). When combined with DLI, a synergistic effect was observed in recipients of CpG2006 or 2006 + 1585 with 88% of mice surviving long-term. These data are the first to indicate that the systemic administration of CpG ODNs is a potent means of inducing therapeutic anti-AML innate immune responses in naive and BMT recipients. (Blood. 2001;98:1217-1225)

Effects of a hexameric deoxyriboguanosine run conjugation into CpG oligodeoxynucleotides on their immunostimulatory potentials.

CpG oligodeoxynucleotides (ODNs) are promising immunomodulatory agents for treating human diseases and vaccine development. Phosphodiester CpG ODNs were demonstrated to have poor immunostimulatory potentials for cytokine production. However, the conjugation of consecutive deoxyriboguanosine residues, called a dG run, at the 3' terminus of phosphodiester CpG ODNs significantly enhanced TNF-alpha and IL-12 production from mouse splenic dendritic cells (DCs). The optimal induction of cytokine production was achieved by the addition of a hexameric dG (dG6) run. In contrast, the existence of a dG6 run either at the 5' terminus of phosphodiester CpG ODNs or at the 3' terminus of phosphorothioate CpG ODNs diminished CpG-mediated cytokine induction, suggesting that the effects of a dG run depend on its location and the chemical property of the ODN backbone, respectively. In addition, we provided the evidence that the conjugation of a dG6 run caused the structural transformation of CpG ODNs, which facilitates their targeting into mouse APCs such as splenic DCs, B cells, and peritoneal macrophages with a scavenger receptor type A ligand specificity. Among primary APCs, DCs were the most potent for CpG ODN-mediated IL-12 production. Furthermore, we demonstrated that the conjugation of a dG6 run into the 3' terminus of phosphodiester CpG ODNs was crucial for their ability to generate Th1 immunity in vivo. Thus, the conjugation of a dG6 run into phosphodiester CpG ODNs would be an alternative way to optimize their immunostimulatory potentials in vitro and in vivo.

Eradication of human medulloblastoma tumor xenografts with a combination of O6-benzyl-2'-deoxyguanosine and 1,3-bis(2-chloroethyl)1-nitrosourea.

O6-Benzyl-2'-deoxyguanosine (dBG), a water-soluble inhibitor of O6-methylguanine-DNA methyltransferase (MGMT), potentiates the efficacy of 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) against MGMT-positive, BCNU-resistant Daoy human medulloblastoma tumor xenografts in athymic mice (S. C. Schold et al., Cancer Res., 56: 2076-2081, 1996). Such potentiation was comparable to that observed for O6-benzylguanine, the prototype MGMT inhibitor that is currently undergoing clinical trials. In this study, we optimized the therapeutic effect of the dBG and BCNU combination against brain tumor xenografts without inducing substantial toxicity in the host by adjusting the doses of both compounds. dBG was escalated from 133 mg/m2 to 200 and 300 mg/m2, whereas corresponding doses of BCNU were reduced from 25 mg/m2 to 17 and 11 mg/m2, respectively. The growth delays of 30.2, 38.4, and 22.3 days, respectively, observed for the above regimens suggest that the optimal drug combination is not achieved with maximum doses of dBG. In fact, the highest doses of dBG (300 mg/m2) contributed to more frequent BCNU-related toxicities, despite the reduced BCNU dosage, and a reduction of the therapeutic effect. Toxicity was related to the depletion of MGMT activity in the gut of host mice and was manifested by edema, inflammation, and hemorrhage in the bowel wall by subsequent BCNU administration. With additional dosage adjustments, we found that tumor suppression of >90 days without toxicity was observed at 200 mg/m2 dBG and 23 mg/m2 BCNU. At these doses, tumors were eradicated (regressed to an undetectable size for >90 days) in 8 of 12 animals. Thus, dBG is the first of the MGMT inhibitors to show a curative effect in combination with BCNU against a human central nervous system tumor xenograft in athymic mice.

Efficacy of the carbocyclic 2'-deoxyguanosine nucleoside BMS-200475 in the woodchuck model of hepatitis B virus infection.

Daily oral treatment with the cyclopentyl 2'-deoxyguanosine nucleoside BMS-200475 at doses ranging from 0.02 to 0.5 mg/kg of body weight for 1 to 3 months effectively reduced the level of woodchuck hepatitis virus (WHV) viremia in chronically infected woodchucks as measured by reductions in serum WHV DNA levels and endogenous hepadnaviral polymerase activity. Within 4 weeks of daily therapy with 0.5 or 0.1 mg of BMS-200475 per kg, endogenous viral polymerase levels in serum were reduced about 1,000-fold compared to pretreatment levels. Serum WHV DNA levels determined by a dot blot hybridization technique were comparably decreased in these treated animals. In the 3-month study, the sera of animals that had undetectable levels of WHV DNA by the dot blot technique were further analyzed by a highly sensitive semiquantitative PCR assay. The results indicate that BMS-200475 therapy reduced mean WHV titers by 10(7)- to 10(8)-fold, down to levels as low as 10(2) to 10(3) virions/ml of serum. Southern blot hybridization analysis of liver biopsy samples taken from animals during and after BMS-200475 treatment showed remarkable reductions in the levels of WHV DNA replicative intermediates and in the levels of covalently closed circular viral DNA. WHV viremia in BMS-200475-treated WHV carriers eventually returned to pretreatment levels after therapy was stopped. These results indicate that BMS-200475 should be evaluated in clinical trials for the therapy of chronic human hepatitis B virus infections.

Treatment of human brain tumor xenografts with O6-benzyl-2'-deoxyguanosine and BCNU.

O6-Methylguanine-DNA methyltransferase (MGMT), a constitutively expressed DNA repair protein, removes alkyl groups from the O6-position of guanine in DNA. Tumor cells with high MGMT activity are resistant to nitrosoureas and other agents that form toxic O6-alkyl adducts. O6-Benzylguanine (BG) inactivates the MGMT protein and thereby enhances the sensitivity of tumor cells to alkylating drugs. However, the therapeutic potential of BG is limited by its poor solubility and its nonspecific inactivation of MGMT in normal tissues as well as in tumor tissues. Consequently, BG analogues are being developed to identify agents that have more favorable pharmacological characteristics. We evaluated O6-benzyl-2'-deoxyguanosine (dBG), the 2'-deoxyribonucleoside analogue of BG, for its ability to inhibit MGMT and to potentiate 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) in a MGMT-positive human brain tumor xenograft, Daoy. When given i.p. 1 h before BCNU (25 mg/m2) to animals bearing s.c. tumors, dBG (134 mg/m2) produced a growth delay of 24.7 days, compared to 21.6 days after treatment with an equimolar dose of BG (90 mg/m2) plus BCNU and -0.6 days after treatment with BCNU alone. The combination of dBG + BCNU also increased the survival of animals bearing intracranial tumors by 65%. By increasing the dose of dBG to 300 mg/m2 (the maximum dose that could be delivered i.p. in a standard treatment volume), the growth delay of s.c. tumors increased from -0.1 days with BCNU alone to 39.3 days. dBG suppressed both tumor and liver MGMT activity to less than 1.5% of baseline, and dBG + BCNU induced extensive perivascular apoptosis. Because dBG is a 10-fold less potent MGMT inhibitor than BG in HT-29 cell extracts, these results illustrate the capacity of BG analogues to potentiate BCNU toxicity, despite less in vitro activity than the parent compound, and emphasize the importance of in vivo evaluation of BG analogues.

Synergistic inhibition of human gastric carcinoma cell growth by 1-beta-D-arabinofuranosylcytosine and hydroxyurea or 2'-deoxyguanosine in vitro.

The cytotoxicity of 1-beta-D-arabinofuranosylcytosine (ara-C) in combination with hydroxyurea (HU) or 2'-deoxyguanosine (GdR) on human gastric carcinoma MK-1 cells and colon carcinoma HT-15 cells was studied. Synergistic interaction between ara-C and HU on MK-1 cells and HT-15 cells, or ara-C and GdR on MK-1 cells was shown using the combination index method. HU increased the accumulation of ara-C triphosphate (ara-CTP) in the acid-soluble pool and diminished the cellular deoxyCTP (dCTP) pool. HU had no effect on the incorporation of ara-C into DNA and RNA. These results indicate that HU-induced elevation in ara-CTP and decrease in dCTP are the basis for synergy among ara-C and HU in MK-1 cells. GdR diminished cellular dCTP slightly, but it decreased the accumulation of ara-CTP in the acid-soluble pool and did not increase the incorporation of ara-C into DNA. On the other hand, ara-C increased cellular deoxyGTP (dGTP) level in the presence of GdR. These results indicate that synergy between ara-C and GdR is mediated through increased cellular dGTP which might inhibit DNA synthesis directly.