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Candidatus Liberibacter asiaticus (CLas), a bacterium transmitted by the Asian citrus psyllid, Diaphorina citri, is the causal agent of citrus greening disease, or Huanglongbng (HLB). Currently, vector population suppression with insecticides and tree removal are the most effective strategies for managing the HLB pathosystem. In this study, we assessed the bactericidal capabilities of 2'-deoxy-2'-fluoro-D-arabinonucleic acid antisense oligonucleotides (FANA ASO) both in vitro and in vivo by (1) confirming their capacity to penetrate insect cells, (2) silencing bacterial essential genes, and (3) quantifying reductions in bacterial titer and D. citri transmission. We confirmed that FANA ASO are able to penetrate insect cells without the use of a delivery agent. Expression of an essential gene in the D. citri endosymbiont, Wolbachia (wDi), significantly decreased by 30% following incubation with a wDi-specific FANA ASO. Viability of isolated wDi cells also decreased in response to the FANA ASO treatment. Delivery of a CLas-specific FANA ASO to infected adult D. citri in feeding assays resulted in significant silencing of a CLas essential gene. CLas relative density and transmission were significantly lower among D. citri fed FANA ASO in diet compared to untreated insects. Root infusions of a CLas-specific FANA ASO into infected Citrus trees significantly reduced CLas titer during a 30-day trial. Our results suggest that FANA ASO targeting insect-transmitted plant bacteria or insect endosymbionts may be useful tool for integrated management of agricultural pathogens.

Purpose: The objectives of this study were to synthesize and characterize two types of cytarabine (Ara-C) lipid produgs and evaluate the prodrugs for sustained intraocular delivery after administration by intravitreal injection., Methods: Hexadecyloxypropyl cytarabine 5'-monophosphate (HDP-P-Ara-C) and hexadecyloxypropyl cytarabine 3',5'-cyclic monophosphate (HDP-cP-Ara-C) were synthesized starting from cytarabine (1-β-D-arabinofuranosylcytosine). Their vitreal clearance profile was simulated using a custom dissolution chamber, in vitro cytotoxicity was evaluated using cell proliferation assays, and in vivo ocular properties in rat and rabbit eyes were assessed using biomicroscopy, indirect ophthalmoscopy, tonometry, electroretinography, and histology., Results: HDP-P-Ara-C was cleared from the dissolution chamber (flow rate 2 µL/min) within 7 days. In contrast, HDP-cP-Ara-C, a much more insoluble prodrug, was still detectable 36 days after the dissolution process was started. HDP-P-Ara-C had a 50% cytotoxicity concentration of 52±2.6 μM in human retinal pigment epithelium (ARPE-19) and 32±2.2 µM in a rat Müller cell line, rMC-1. The 50% cytotoxicity concentration values for HDP-cP-Ara-C in ARPE-19 and rMC-1 cells were 50 µM and 25 µM, respectively. HDP-P-Ara-C was not detectable 2 weeks after the highest intravitreal dose (228 µg/rat eye) was injected, and no ocular toxicity was found. With HDP-cP-Ara-C, the drug depot was visible for 26 weeks following a single intravitreal injection (800 µg/rabbit eye). For both compounds, the electroretinogram, intraocular pressure, and other toxicity studies were negative except for the highest dose of HDP-cP-Ara-C (800 µg/eye), which had focal toxicity from the direct touch of the retina and decreased dark adapted a-waves and decreased flicker electroretinogram amplitudes (generalized estimating equations, p=0.039 and 0.01)., Conclusions: The cyclic monophosphate prodrug, HDP-cP-Ara-C, was found to have physiochemical properties better suited for sustained delivery of cytarabine to posterior segments of the eye. These properties included limited aqueous solubility, in vitro antiproliferative activity, and good tolerability after injection into rabbit eyes.

Purpose: A long-lasting, slow-release, crystalline antiviral drug delivery system was initially reported using ganciclovir and cyclic cidofovir as the prototype compounds. The present study was undertaken to investigate the feasibility of applying this system to antiproliferative small molecules., Methods: The crystalline lipid prodrugs of hexadecyloxypropyl-arabinofuranosylguanine 5'-monophosphate (HDP-P-AraG), hexadecyloxypropyl 5-fluoro-2'-deoxyuridine cyclic 3',5'-monophosphate (HDP-cP-5-F-2dUrd), and hexadecyloxypropyl 5-fluoro-2'-deoxyuridine 5'-monophosphate (HDP-P-5-F-2dUrd) were synthesized from their parent compounds arabinofuranosylguanine (AraG) and 5-fluoro-2'-deoxyuridine (5-F-2dUrd). All three compounds were tested at escalating doses in rabbit eyes. Only one eye of each animal was injected with test compound, and the fellow eye was injected with 5% dextrose as the control. The injected eyes were monitored by slit lamp, a handheld tonometer, indirect ophthalmoscopy, electroretinography (ERG), and histology. The selected doses were used for efficacy study with the rat CNV model or the rabbit PVR model., Results: The highest nontoxic dose for HDP-P-AraG was 75 microg/eye, and was 70 and 210 microg/eye for HDP-P-5-F-2dUrd and HDP-cP-5-F-2dUrd, respectively. All compounds demonstrated a localized depot of crystalline aggregate in the vitreous with a clear view of vitreous and retina elsewhere. The drug depot of HDP-P-AraG was visible for 4 to 5 weeks; HDP-P-5-F-2dUrd, 8 to 10 weeks; and HDP-cP-5-F-2dUrd longer than 14 weeks. The treatment study showed HDP-P-AraG led to 33% reduction in CNV in the rat (P = 0.015), and HDP-cP-5-F-2dUrd provided 100% prevention of trauma-induced PVR in the rabbit (P = 0.046). The pretreatment study demonstrated a significant protection against intraocular proliferation compared with the 5-FU in a parallel study (P = 0.014)., Conclusions: The intravitreous injectable lipid prodrugs of AraG and 5-fluoro-2'-deoxyuridine could be long-lasting, slow-release, antiproliferative compounds to treat unwanted intraocular proliferation.

Causing damage to angiogenic vessels is a promising approach for cancer chemotherapy. The present study is a codification of a designed liposomal drug delivery system (DDS) for antineovascular therapy (ANET) with 2'-C-cyano-2'-deoxy-1-beta-D-arabino-pentofuranosylcytosine (CNDAC). The authors have previously reported that liposomalized 5'-O-dipalmitoylphosphatidyl CNDAC (DPP-CNDAC), a phospholipid derivative of the novel antitumor nucleoside CNDAC, is quite useful for ANET. DPP-CNDAC liposomes modified with APRPG, a peptide having affinity toward angiogenic vessels, efficiently suppressed tumor growth by damaging angiogenic endothelial cells. In the present study, the authors masked the hydrophilic moiety of DPP-CNDAC, namely, CNDAC, on the liposomal surface with APRPG-polyethyleneglycol (PEG) conjugate to improve the availability of DPP-CNDAC liposomes. The use of the APRPG-PEG conjugate attenuated the negative zeta-potential of the DPP-CNDAC liposomes and reduced the agglutinability of them in the presence of serum. These effects improved the blood level of DPP-CNDAC liposomes in colon 26 NL-17 tumor-bearing BALB/c male mice, resulting in enhanced accumulation of them in the tumor. Laser scanning microscopic observations indicated that APRPG-PEG-modified DPP-CNDAC liposomes (LipCNDAC/APRPG-PEG) colocalized with angiogenic vessels and strongly induced apoptosis of tumor cells, whereas PEG-modified DPP-CNDAC liposomes (LipCNDAC/PEG) did not. In fact, LipCNDAC/APRPG-PEG suppressed the tumor growth more strongly compared to LipCNDAC/PEG and increased significantly the life span of the mice. The present study is a good example of an effective liposomal DDS for ANET that is characterized by: (i) phospholipid derivatization of a certain anticancer drug to suit the liposomal formulation; (ii) PEG-shielding for masking undesirable properties of the drug on the liposomal surface; and (iii) active targeting to angiogenic endothelial cells using a specific probe.

Chronic neutrophilic leukemia (CNL) is a rare myeloproliferative disorder characterized by a proliferation mainly of mature neutrophils. The prognosis is generally poor and an optimal therapeutic strategy remains to be determined. Allogeneic hematopoietic stem cell transplantation (HSCT) is expected to be the only curative therapy so far. We report a 46-year-old male with progressive CNL who underwent bone marrow transplantation from an HLA-matched unrelated donor. After engraftment was achieved on day 35, relapse of CNL was confirmed on day 50. The progression of CNL was very rapid afterward and infiltration to the central nervous system was observed. The Janus Kinase 2 (JAK2) V617F homozygous mutation was detected from the peripheral blood or bone marrow samples throughout the clinical course. From comparison with reports of successful HSCT for CNL in the literature, it was inferred that HSCT should be performed in a stable status before progression. Furthermore, JAK2 V617F-positive CNL may contain an aggressive disease entity in contrast to previous reports. Accumulation of experiences is required to establish a definite role of HSCT in the treatment of CNL and a prognostic significance of JAK2 mutation in CNL., ((c) 2006 Wiley-Liss, Inc.)

Background: Treatment with interferon and subcutaneous cytarabine produces superior cytogenetic responses in chronic myeloid leukaemia (CML) than treatment with interferon alone, but at the expense of greater toxicity. Cytarabine ocfosfate (YNK01) is an oral precursor of cytarabine that may overcome some of the inconvenience and toxicities associated with subcutaneous cytarabine administration., Patients and Methods: We studied the efficacy and tolerability of combination therapy with interferon-alpha-2b and YNK01 in patients with newly diagnosed, untreated CML. Forty patients were treated with interferon-alpha-2b (5 MU/m2/day) plus monthly courses of YNK01 (600 mg/day for 10 days) for 1 year., Results: The 6-month complete haematological response rate was 63% and the 1-year major cytogenetic response rate was 30%, with 10% of cytogenetic responses being complete. With a median follow-up of 57 months, the estimated 5-year overall survival was 86% (95% confidence interval 70% to 94%). Treatment tolerability was poor, with toxicity leading to discontinuation of one or both drugs in 60% of cases. The median daily dose of interferon alpha-2b was 7.75 MU and the median dose of YNK01 was 600 mg/day for each 10-day treatment cycle., Conclusions: Interferon-alpha-2b and YNK01 produce cytogenetic responses comparable to those achieved with interferon-alpha-2b and parenteral cytarabine, although toxicity was excessive. Alternate dosing strategies may enhance the tolerability of YNK01.

Cytarabine ocfosfate (YNK01) is a prodrug analogue of cytarabine which is resistant to systemic deamination after oral administration. Following initial studies indicating significant anti-tumour activity of YNK01 a phase II trial was initiated in order to assess the tolerability and efficacy of a combination of this agent with interferon alpha-2b (IFN-alpha2b) in recently diagnosed chronic phase CML patients (n = 98). The treatment was subdivided into cycles consisting of 4 weeks of continuous administration of IFN-alpha-2b (3 MU/m(2)/day 1st week and then 5 MU/m(2)/day) and 14 days of oral YNK01 (600 mg/day 1st cycle). At the end of each cycle the dose of YNK01 was adjusted according to the blood count observed during the previous 4 weeks. The median time from diagnosis to inclusion in the trial was 2 months (range 6 days to 7.5 months). At 12 weeks, 62 patients (63%; 95% CI, 54-73) achieved a complete hematological response. At 24 weeks, of 98 patients, two achieved a complete cytogenetic response, 14 a partial response (16% major cytogenetic response rate; 95% CI, 9-24) and 34 a minor response; 19 patients were not evaluable for cytogenetic response. During the trial, 20 patients progressed to accelerated (6) or blastic phases (14). The median time to progression was 15 months (range 2-38 months). At 3 years the overall survival was 79% (95% CI, 70-88). Although the complete hematological response rate compared favorably with the 40% response rate previously obtained with the subcutaneous formulation of Ara-c, the cytogenetic response rate was less than expected. Most of the patients experienced side-effects and all permanently stopped YNK01. Although the combination seems attractive the initial dose of 600 mg per day is probably too high and should be reconsidered in further trials.

Background and Objectives: Subcutaneous Ara-C plus interferon (IFN) produces more cytogenetic responses than IFN in chronic myeloid leukemia (CML) but a greater toxicity. The objective of this study was to determine the efficacy and tolerance of IFN plus oral Ara-C ocfosfate (YNK-01) in IFN-resistant CML patients., Design and Methods: A phase II pilot study was conducted in 19 CML patients primarily resistant or with minimal cytogenetic response to IFN. Patients were scheduled to receive 6 monthly 14-day cycles of YNK-01 (500 mg/day), with progressive escalation if tolerated, in addition to IFN. Cytogenetic assessment was performed thereafter., Results: Of the first 7 patients, 5 had severe hematologic and 5 moderate gastrointestinal toxicity; IFN was reduced in 6, YNK-01 in 5, and treatment discontinued in 2; hematologic response was achieved in 2 of the 5 evaluable patients. In the following 4 patients the Ara-C was reduced to 300 mg: 2 had severe hematologic and 2 moderate gastrointestinal toxicity; IFN and Ara-C were reduced in 2 patients and treatment discontinued in 2 due to progression or toxicity; the other 2 achieved a minor cytogenetic response, progressing in one to a major response after 6 more cycles. In 8 patients the starting Ara-C dose was 200 mg: 5 had moderate-severe hematologic and 5 mild gastrointestinal toxicity; IFN was reduced in 5, Ara-C in 1, and treatment discontinued in 1; Ara-C was increased in 7 cases; hematologic response was obtained in 4 patients, 2 of whom attained a minor and 1 a major cytogenetic response., Interpretation and Conclusions: These results provide background for future studies aimed at ascertaining the role of oral Ara-C combined with IFN or STI571 in newly diagnosed CML.

We previously synthesized the 5'-O-diacylphosphatidyl derivative of 2'-C-cyano-2'-deoxy-1-beta-D-arabino-pentofuranosylcytosine (CNDAC), a novel antitumor nucleoside, and observed it to have a high antitumor activity. Since this compound is readily incorporated into liposomal membranes, we liposomalized the compound using a formulation for conventional and long-circulating liposomes, and investigated the antitumor activity of liposomal 5'-O-dipalmitoylphosphatidyl CNDAC (DPP-CNDAC). Long-circulating liposomes composed of DPP-CNDAC, dipalmitoylphosphatidylcholine, cholesterol and palmityl-D-glucuronide (PGlcUA) (2:2:2:1 as a molar ratio), as well as liposomes containing dipalmitoylphosphatidylglycerol (DPPG) instead of palmityl-D-glucuronide and those composed of only DPP-CNDAC, were injected intravenously into Meth A sarcoma-bearing mice. DPP-CNDAC showed suppression of tumor growth, whereas CNDAC did not at the same concentration, suggesting that 5'-phosphatidylation is useful to enhance therapeutic efficacy. Furthermore, liposomal DPP-CNDAC reduced the acute toxicity, and liposomes containing PGlcUA showed more enhanced activities of reducing tumor growth and increasing the lifetime of the mice than liposomes containing DPPG. To obtain a higher therapeutic efficacy, we injected long-circulating liposomal DPP-CNDAC 5 times. The tumor growth was suppressed to 13.2% (86.8% inhibition), and the survival time of the tumor-bearing mice increased to 128.5% with one completely cured mouse out of five. Next, the effect of DPP-CNDAC incorporation on the in vivo behavior of PGlcUA and DPPG liposomes was examined by a non-invasive method using positron emission tomography (PET). Liposomes were labeled with [2-(18)F]-2-fluoro-2-deoxy-D-glucose, and administered to tumor-bearing mice. PET images and time-activity curves indicated that DPP-CNDAC/PGlcUA-liposomes tended to accumulate in tumor tissues a little bit more than DPP-CNDAC/DPPG-liposomes, although the difference between the two kinds of liposomal distribution was not as marked as between PGlcUA and DPPG liposomes, suggesting that DPP-CNDAC incorporation partly affected the liposomal behavior in vivo but that the long-circulating character of PGlcUA-liposomes might not be fully abolished. Thus, the enhanced therapeutic efficacy of long circulating liposomalized DPP-CNDAC observed here may be due to passive targeting of DPP-CNDAC to the tumor tissue, making this formulation of DPP-CNDAC useful for cancer chemotherapy.

1-beta-D-Arabinofuranosylcytosine-5'-stearylphosphate (cytarabine ocfosfate, stearyl-ara-CMP) is a newly synthesized 5'-alkylphosphate derivative of 1-beta-D-arabinofuranosylcytosine (ara-C), which is lipophilic, resistant to inactivation by deamination, and orally active. Pharmacology of this drug was studied in patients with hematological malignancies. The concentrations of stearyl-ara-CMP, ara-C (its active metabolite), and 1-beta-D-arabinofuranosyluracil (ara-U, its inactive metabolite) were determined by radioimmunoassay. When six patients received a single p.o. dose of the drug (500 mg/m2), stearyl-ara-CMP, ara-C, and ara-U could be detected in the plasma for at least 72 h afterwards. The plasma disappearance curve of stearyl-ara-CMP corresponded to a one-compartment open model with first-order absorption kinetics. The peak plasma level (Cmax) was 322 +/- 218 nM, and the predicted time to reach Cmax (Tmax) was 6.5 +/- 4.5 h, while the elimination half-life (t1/2) was very long (32.0 +/- 8.4 h). The plasma ara-C level increased slowly to a Cmax of 26.3 +/- 12.7 nM (Tmax, 13.3 +/- 4.7 h) after stearyl-ara-CMP administration. This level was quite low compared with that achieved by low-dose s.c. ara-C therapy, but ara-C persisted longer in the plasma in the former case, and the area under the curve was similar for both regimens. For ara-U, the Cmax, Tmax, and t1/2 were 483 +/- 315 nM, 23.6 +/- 4.0 h, and 19.6 +/- 5.3 h, respectively. No stearyl-ara-CMP was detected in the urine, and only 8.0% of the administered dose was excreted as ara-C and ara-U within 72 h. The stearyl-ara-CMP concentration in the cerebrospinal fluid was below the limit of detection in three patients without meningeal involvement at 6 h. During clinical use of stearyl-ara-CMP, macrocytic anemia was observed, and some patients also developed megaloblastic change of their erythroblasts, suggesting a mild and persistent cytostatic effect. In conclusion, p.o. therapy with stearyl-ara-CMP achieved prolonged maintenance of the plasma drug level. Thus, the drug released a very low dose of ara-C over a long period in plasma and tissues and had a prolonged mild antineoplastic effect in patients with hematological malignancies.

The cutaneous application of antiviral agents was studied by iontophoresis, a process that increases penetration of most drugs 20- to 60-fold. Twenty-seven subjects with vesicular orolabial herpes were treated one time in a double-blind, placebo-controlled clinical study: nine received vidarabine monophosphate (ara-AMP), nine received acyclovir (ACV), and nine received NaCl. Ara-AMP-treated lesions yielded lower titers of virus after 24 hr compared with lesions treated with NaCl or ACV (P less than .05). Ara-AMP significantly decreased the duration of shedding of virus (P less than .05) and time to dry crust (P less than .05) compared with the other two agents. There was a trend toward decreased healing time after ara-AMP treatment.

The effects of 9-beta-D-arabinofuranosyladenine-5'-phosphate (ara-A-5'-P) on herpes simplex virus encephalitis in mice were studied. This drug is a monophosphate derivative of adenine arabinoside (ara-A). When administered by the intracerebral (ic) route, ara-A-5'-P was highly effective in virus-infected mice in the 17-20 g weight range provided therapy was begun before clinical symptoms of encephalitis appeared. The most effective dose schedule was found to be three daily ic injections totaling 400 micrograms/day (20 mg/kg) beginning on the third day after injection. Such treatment resulted in a survival rate of 50%-75% among infected mice after six months. In the early stages of many experiments, 90%-100% of the controls died whereas all of the treated animals survived.

The antiproliferative activity of 9-beta-D-arabinofuranosyladenine 5'-monophosphate against a cultured line of mouse leukemia cells (L1210/C2) was enhanced by addition of either 2'-deoxycoformycin or erythro-9-(2-hydroxy-3-nonyl)adenine. The activity of 9-beta-D-arabinofuranosyladenine 5'-monophosphate, alone or in combination with either of the two inhibitors of adenosine deaminase, was comparable to that of 9-beta-D-arabinofuranosyladenine (ara-A), apparently reflecting the rapid conversion of 9-beta-D-arabinofuranosyladenine 5'-monophosphate to ara-A by L1210/C2 cells. Several ara-A analogs were assayed for antiproliferative activity against L1210/C2 cells; of these, only 9-beta-D-arabinofuranosyladenine 5'-O-methylphosphate and 2'-deoxy-2'-amino-9-beta-D-arabinofuraosyladenine were active. Addition of 2'-deoxycoformycin to cell culture fluids enhanced the activity of 9-beta-D-arabinofuranosyladenine 5'-O-methylphosphate suggesting conversion to an adenosine deaminase-sensitive intermediate, presumably ara-A.

Several antiviral agents were applied topically or by iontophoresis to hairless mouse skin inoculated with herpes simplex virus type 1 (HSV-1), and the chemotherapeutic effectiveness was evaluated. Topical application of iododeoxyuridine, arabinoside A, and adenine arabinoside monophosphate (ara-AMP) moderately decreased the average lesion score, number of mice with paralysis, and number of mice dying in HSV-1-infected animals. Also, the mean survival time was moderately prolonged by topical application of those antiviral agents. When ara-AMP was applied by cathodal (-) iontophoresis to the HSV-1-infected skin, the average lesion score, number of mice with paralysis, and number of mice dying were greatly decreased. Furthermore, the mean survival time of mice was highly increased by cathodal (-) iontophoresis of ara-AMP. The therapeutic efficacy of ara-AMP iontophoresis was much superior to the topical application of iododeoxyuridine, arabinoside A, and ara-AMP. These data suggest that ara-AMP iontophoresis would be the method of choice for the management of HSV-1 skin lesions in hairless mice.

D-Galactopyranosyl residues were coupled to poly(L-lysine) and the antiviral agents arabinofuranosyladenine 5'-monophosphate (ara-AMP) and acyclovir were conjugated with this glycosylated polymer. In mice the ara-AMP conjugate accomplished a selective drug delivery to liver cells.