3-(2-deoxy-ß-d-erythro-pentafuranosyl)pyrimido[1,2-α]purin-10(3H)-one deoxyguanosine adducts of workers exposed to asbestos fibers.

Asbestos is the commercial name for a group of silicate minerals naturally occurring in the environment and widely used in the industry. Asbestos exposure has been associated with pulmonary fibrosis, mesothelioma, and malignancies, which may appear after a period of latency of 20-40 years. Mechanisms involved in the carcinogenic effects of asbestos are still not fully elucidated, although the oxidative stress theory suggests that phagocytic cells produce large amounts of reactive oxygen species, due to their inability to digest asbestos fiber. We have conducted a mechanistic study to evaluate the association between 3-(2-deoxy-ß-d-erythro-pentafuranosyl)pyrimido[1,2-α]purin-10(3H)-one deoxyguanosine (M1dG) adducts, a biomarker of oxidative stress and lipid peroxidation, and asbestos exposure in the peripheral blood of 327 subjects living in Tuscany and Liguria, Italy, stratified by occupational exposure to asbestos. Adduct frequency was significantly greater into exposed subjects with respect to the controls. M1dG per 108 normal nucleotides were 4.0±0.5 (SE) in 156 asbestos workers, employed in mechanic, naval, petrochemical, building industries, and in pottery and ceramic plants, versus a value of 2.3±0.1 (SE) in 171 controls (p<0.001). After stratification for occupational history, the effects persisted in 54 current asbestos workers, mainly employed in building renovation industry (2.9±0.3 (SE)), and in 102 former asbestos workers (4.5±0.7 (SE)), with p-values of 0.033, and <0.001, respectively. A significant effect of smoking on heavy smokers was found (p=0.005). Our study gives additional support to the oxidative stress theory, where M1dG may reflect an additional potential mechanism of asbestos-induced toxicity.

Reproductive toxicity study with a novel deoxyguanosine analogue (Metacavir) in pregnant SD rats.

Our preliminary studies demonstrated that Metacavir has potential to become a new anti-HBV agent. The main targets of the toxic effects of Metacavir, in rhesus monkeys, were gastrointestinal tracts, liver, blood, and kidneys, which were not related to mitochondrial effects. In this study, the maternal toxicity, embryo-fetal developmental toxicity and teratogenicity were studied in pregnant Sprague-Dawley rats after intragastric administration of Metacavir (200, 100, 50, 0 mg/kg body weight) during the first 6-15 days of pregnancy. Slower weight gain was observed in 5 out of 21 rats subjected to a 200 mg/kg dose, as well as 2 out of 20 subjected to a 100 mg/kg dose. Compared with the solvent control group, the calibration weight gain in the 200 mg/kg and 100 mg/kg dosage groups respectively, during first 6-20 pregnant days were significantly different (P < 0.01, P < 0.05). Significant dose related adverse effects to other reproductive parameters were not seen in F0 and F1, but the number of stillbirths in high dose group showed notably difference compared with the control group (P < 0.05), while the litter incidence showed no difference. No Metacavir-associated pathological changes were observed. The present research indicated that at a dose of 200 mg/(kg·d) (i.e., 40 times the effective dose in rats), Metacavir shows some maternal toxicity to SD rats. The embryotoxicity in the 200 mg/kg group encompass decreased fetal body weight, and higher fetal mortality rates, compared with the control group. However, the litter incidence showed no statistical difference. All the treated rats displayed normal bone development, no teratogenicity and without adverse effects on fetal development, thus indicating that below a dose of 200 mg/(kg·d) there is no teratogenic side effects.

A nelarabine-resistant T-lymphoblastic leukemia CCRF-CEM variant cell line is cross-resistant to the purine nucleoside phosphorylase inhibitor forodesine.

BACKGROUND/AIM: Forodesine inhibits purine nucleoside phosphorylase, resulting in an accumulation of intracellular dGTP and consequently cell death. 9-ß-D-Arabinofuranosylguanine (ara-G) is an active compound of nelarabine that is intracellularly phosphorylated to a triphosphate form, which inhibits DNA synthesis. Both agents show cytotoxicity toward T-cell malignancies. In the present study, we investigated the cytotoxicity of forodesine in vitro using ara-G-resistant leukemia cells. MATERIALS AND METHODS: T-Lymphoblastic leukemia cell line CCRF-CEM and ara-G-resistant CEM variant cell line CEM/ara-G that we had previously established were used. RESULTS: A growth-inhibition assay demonstrated that CEM cells were insensitive to single-agent forodesine treatment. The cells were also insensitive to deoxyguanosine at a maximal concentration of 10 µM. CEM/ara-G cells were 80-fold more resistant to ara-G than were CEM cells, and the mode of sensitivity to forodesine and deoxyguanosine was similar to that of CEM cells. In the presence of 10 µM deoxyguanosine, forodesine effectively inhibited the growth of CEM cells but not that of CEM/ara-G cells. Flow cytometric analyses showed that combination of forodesine and deoxyguanosine induced apoptosis of CEM cells but not of CEM/ara-G cells. The addition of ara-G did not augment the cytotoxicity of the forodesine/deoxyguanosine combination towards CEM cells or CEM/ara-G cells. The combination index revealed antagonism between forodesine and ara-G. The intracellular production of ara-G triphosphate was reduced in the presence of forodesine. CONCLUSION: Nelarabine-resistant CEM/ara-G cells are insensitive to forodesine.

Six-month repeated dose toxicity of orally administered metacavir in rhesus monkeys.

OBJECTIVE: To explore the toxicities of metacavir, a novel deoxyguanosine analog with an anti-hepatitis B virus (HBV) potential, in a 6-month repeated dosing in rhesus monkeys. METHODS: Rhesus monkeys were divided into four groups with eight animals in each group. Metacavir or blank vehicles were given for up to 6-month, and then the animals were euthanized 3 and 6 months later. Biochemical and hematological parameters, general symptoms, ECG, serum antibodies, and tissue pathology were observed and recorded. RESULTS: No biologically meaningful influences on body weight, body temperature, ocular examination, ECG, and organ weight were observed. The main toxic effects included: obvious gastrointestinal toxicities were observed in metacavir 200 mg/kg group, in which animals experienced vomiting and decrease in food consumption. Along with the increase of dosing times, animals gradually tolerated the drug and all these effects gradually abated. Hematological damages included increased damage of red blood cells, decrease of red blood cell count and hemoglobin levels. Hepatic functions were also damaged at certain levels, including the decreases in total protein, albumin, and glucose and the fatty degeneration in hepatocytes. Mild stenosis and exfoliation of gastric and duodenal mucosa was observed. The mild necrosis and exfoliation of renal tubules epithelia was found 6 months after the start of dosing. All these toxic effects were dose dependent. CONCLUSION: The main target organs of the toxic effects of metacavir are gastrointestinal tract, liver, blood, and kidneys. The no-observed-adverse-effect-level (NOAEL) of metacavir for rhesus monkey were considered to be 50 mg/kg/day.

Synthesis of novel purine nucleosides towards a selective inhibition of human butyrylcholinesterase.

The search for new and potent cholinesterase inhibitors is an ongoing quest mobilizing many organic chemistry groups around the world as these molecules have been shown to treat the late symptoms of Alzheimer's disease as well as to act as neuroprotecting agents. In this work, we disclose the synthesis of novel 2-acetamidopurine nucleosides and, for the first time, regioselective N(7)-glycosylation with 2-acetamido-6-chloropurine, promoted by trimethylsilyl triflate, was accomplished by tuning the reaction conditions (acetonitrile as solvent, 65 degrees C, 5h) starting from 1-acetoxy bicyclic glycosyl donors, or by direct coupling of a methyl glucopyranoside with the nucleobase to obtain only N(7) nucleosides in reasonable yield (55-60%). The nucleosides as well as their sugar precursors were screened for acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) inhibition. While none of the compounds tested inhibited AChE, remarkably, some of the N(7) nucleosides and sugar bicyclic derivatives showed potent inhibition towards BChE. Nanomolar inhibition was obtained for one compound competing well with rivastigmine, a drug currently in use for the treatment of Alzheimer's disease. Experimental results showed that the presence of benzyl groups on the carbohydrate scaffold and the N(7)-linked purine nucleobase were necessary for strong BChE inactivation. A preliminary evaluation of the acute cytotoxicity of the elongated bicyclic sugar precursors and nucleosides was performed indicating low values, in the same order of magnitude as those of rivastigmine.

Assessment of the genotoxic potential of nitric oxide-induced guanine lesions by in vitro reactions with Escherichia coli DNA polymerase I.

It has been suggested that carcinogenesis associated with chronic inflammation involves DNA damage by nitric oxide (NO) and other reactive species secreted from macrophages and neutrophils. The guanine moiety of DNA reacts with NO, yielding two major deamination products: xanthine (Xan) and oxanine (Oxa). Oxa reacts further with polyamines and DNA binding proteins to form cross-link adducts. In the present study, we characterized the structure of the cross-link adducts of Oxa with spermine (Oxa-Sp). Spectrometric analysis of Oxa-Sp adducts showed that they are ring-opened adducts of Oxa covalently bonded to the terminal amino (major product) and internal imino (minor product) groups of spermine. To assess genotoxic potential, Xan, Oxa, Oxa-Sp and an abasic (AP) site were site specifically incorporated into oligonucleotide templates. These lesions differentially blocked in vitro DNA synthesis catalyzed by DNA polymerase I Klenow fragment (Pol I Kf). The relative efficiency of translesion synthesis was G (1) > Oxa (0.19) > Xan (0.12) > AP (0.088) > Oxa-Sp (0.035). Primer extension assays with a single nucleotide and Pol I Kf revealed that non-mutagenic dCMP was inserted most efficiently opposite Xan and Oxa, with the extent of primer elongation being 65% for Xan and 68% for Oxa. However, mutagenic nucleotides were also inserted. The extent of primer elongation for Xan was 16% with dTMP and 14% with dGMP, whereas that for Oxa was 49% with dTMP. For Oxa-Sp, mutagenic dAMP (13%) was preferentially inserted. Accordingly, when generated in vivo, Xan and Oxa would constitute moderate blocks to DNA synthesis and primarily elicit G:C to A:T transitions when bypassed, whereas Oxa-Sp would strongly block DNA synthesis and elicit G:C to T:A transversions.

Synthesis and biological evaluation of 2- and 7-substituted 9-deazaadenosine analogues.

A series of 2-halogen and 7-alkyl substituted analogues of 9-deazaadenosine and 2'-deoxy-9-deazaadenosine was synthesized by new efficient methodology involving transformation of corresponding 9-deazaguanosine and 2'-deoxyguanosine, which in turn were synthesized by direct C-glycosylation of 1-benzyl-9-deazaguanine with 1-O-acetyl-2,3,5-tri-O-benzoyl-D-ribofuranose and methyl 2-deoxy-3,5-di-O-(p-toluoyl)-D-ribofuranoside, respectively. Deoxychlorination of C6 and diazotization/chloroor fluoro-dediazoniation of the sugar-protected 9-deazaguanosine, followed by selective ammonolysis at C6 and deprotection of the sugar moiety, gave 2-chloro- and 2-fluoro-9-deazaadenosine (6 and 9). Substitution of the 7-position of the dihalogen-intermediate with alkyl groups, followed by ammonolysis and deprotection, provided 2-chloro-7-alkyl-9-deazaadenosines (13a-e) and 2-fluoro-7-benzyl-9-deazaadenosine (13f). Catalytic hydrogenation of 13a-e gave 7-alkyl-9-deazaadenosines 14a-e. Similarly, 2-chloro-2'-deoxy-9-deazaadenosine (21), 2-chloro-2'-deoxy-7-methyl-9-deazaadenosine (25), 2'-deoxy-9-deazaadenosine (22), and 2'-deoxy-7-methyl-9-deazaadenosine (26) were prepared from sugar-protected 2'-deoxy-9-deazaguanosine. Among these compounds, 7-benzyl-9-deazaadenosine (14b) showed the most potent cytotoxic activity, with IC50 values of 0.07, 0.1, 0.2 and 1.5 microM, while both 7-methyl-9-deazaadenosine (14a) and 2-fluoro-9-deazaadenosine (9) also demonstrated significant cytotoxic activity with IC50 values of 0.4, 0.7, 0.3, and 1.5 microM, and 1.5, 0.9, 0.3, and 5 microM against L 1210 leukemia, P388 leukemia, CCRF-CEM lymphoblastic leukemia, and B16F10 melanoma cells, respectively.

Design, efficient synthesis, and anti-HIV activity of 4'-C-cyano- and 4'-C-ethynyl-2'-deoxy purine nucleosides.

Some 4'-C-ethynyl-2'-deoxy purine nucleosides showed the most potent anti-HIV activity among the series of 4'-C-substituted 2'-deoxynucleosides whose 4'-C-substituents were methyl, ethyl, ethynyl and so on. Our hypothesis is that the smaller the substituent at the C-4' position they have, the more acceptable biological activity they show. Thus, 4'-C-cyano-2'-deoxy purine nucleosides, whose substituent is smaller than the ethynyl group, will have more potent antiviral activity. To prove our hypothesis, we planned to develop an efficient synthesis of 4'-C-cyano-2'-deoxy purine nucleosides (4'-CNdNs) and 4'-C-ethynyl-2'-deoxy purine nucleosides (4'-EdNs). Consequently, we succeeded in developing an efficient synthesis of six 2'-deoxy purine nucleosides bearing either a cyano or an ethynyl group at the C-4' position of the sugar moiety from 2'-deoxyadenosine and 2,6-diaminopurine 2'-deoxyriboside. Unfortunately, 4'-C-cyano derivatives showed lower activity against HIV-1, and two 4'-C-ethynyl derivatives suggested high toxicity in vivo.

Synthesis and cytotoxicity of 9-(2-deoxy-2-alkyldithio-beta-D-arabinofuranosyl)purine nucleosides which are stable precursors to potential mechanistic probes of ribonucleotide reductases.

A series of 2[prime or minute]-thionucleosides, as potential inhibitors of ribonucleotide reductases, has been synthesized. Treatment of the 3[prime or minute],5[prime or minute]-O-TPDS-2[prime or minute]-O-(trifluoromethanesulfonyl)adenosine with potassium thioacetate gave the arabino epimer of 2[prime or minute]-S-acetyl-2[prime or minute]-thioadenosine which was deacetylated to give 9-(3,5-O-TPDS-2-thio-[small beta]-d-arabinofuranosyl)adenine in high yield. Treatment of the latter with diethyl azodicarboxylate-C(3)H(7)SH-THF gave 2[prime or minute]-propyl disulfide which was desilylated to give 9-(2-deoxy-2-propyldithio-[small beta]-d-arabinofuranosyl)adenine. Subsequent tosylation (O5[prime or minute]) and displacement of the tosylate with pyrophosphate afforded the 5[prime or minute]-O-diphosphate in a stable form as propyl mixed-disulfide, which upon treatment with dithiothreitol releases 9-(2-thio-[small beta]-d-arabinofuranosyl)adenine 5[prime or minute]-diphosphate. The arabino 2[prime or minute]-mercapto group might interact with the crucial thiyl radical at cysteine 439 leading to the inhibition of ribonucleotide reductases via formation of a Cys439-2[prime or minute]-mercapto disulfide bridge. The 2,6-diamino-, 2-amino-6-chloro- and 2-amino-6-methoxypurine ribosides were also converted to the corresponding 2[prime or minute]-deoxy-2[prime or minute]-propyldithio-[small beta]-d-arabinofuranosyl nucleosides, which might serve as convenient precursors to the arabino epimer of 2[prime or minute]-thioguanosine. Analogously, 2[prime or minute]-deoxy-2[prime or minute]-propyldithioadenosine was prepared from 9-([small beta]-d-arabinofuranosyl)adenine. The nucleoside disulfides show modest cytotoxicity in a panel of human tumor cell lines.

Phase I clinical trial and pharmacokinetic study of the spicamycin analog KRN5500 administered as a 1-hour intravenous infusion for five consecutive days to patients with refractory solid tumors.

PURPOSE: The spicamycin analogue KRN5500 is a nucleoside-like antibiotic with broad spectrum activity against human solid tumor models. It appears to possess a novel mechanism of action directed against the endoplasmic reticulum and Golgi apparatus with effects on protein processing. A Phase I trial was undertaken to determine the maximum tolerated dose (MTD), dose-limiting toxicities, and pharmacokinetic behavior of KRN5500 given as a 1-h i.v. infusion for 5 consecutive days every 3 weeks. EXPERIMENTAL DESIGN: Adult patients with refractory solid tumors, good performance status, and normal to near normal renal, hepatic, and hematological function were eligible for the study. At least three patients were evaluated at each dose level, and a modified Fibonacci algorithm was used for dose escalation. The MTD was based on the occurrence of severe toxicity during the first cycle of therapy. The plasma pharmacokinetics of KRN5500 was characterized during the first week of dosing. RESULTS: Characteristics of the 26 patients entered into the study were as follows: 13 males and 13 females; median age, 54.5 years (range, 40-70 years); and Eastern Cooperative Oncology Group performance status 0-1. A majority had refractory colorectal carcinoma (17 of 26 patients) with at least two prior regimens of therapy. The dose of KRN5500 was escalated from 0.8 to 4.9 mg/m(2)/day in five dose levels, and the MTD was 2.9 mg/m(2)/day. All dose-limiting toxicities were nonhematological and included pulmonary toxicities, hyperglycemia, fatigue, hepatotoxicity, and ataxia, with one fatality due to interstitial pneumonitis. Clinically significant toxicities occurring in multiple patients that were not dose-limiting included nausea/vomiting, diarrhea, fatigue, neurological symptoms, hyperbilirubinemia, hyperglycemia, lymphopenia, and thrombocytopenia. There were no objective responses, although 3 of 17 evaluable patients exhibited disease stabilization for 5-6 cycles. The pharmacokinetics for the first dose of KRN5500 was biexponential and linear across all five dose levels. Mean values of pharmacokinetic parameters were as follows: total plasma clearance, 6.15 +/- 2.37 liters/h/m(2); apparent volume of distribution at steady state, 6.56 +/- 1.98 liters/m(2); biological half-life, 1.29 +/- 0.37 h; and mean residence time, 1.07 +/- 0.31 h. Clearance was significantly lower (P = 0.011) in the eight patients who were at least 65 years old (4.6 +/- 1.6 liters/h/m(2)) as compared with the 18 younger patients (7.1 +/- 2.3 liters/h/m(2)). Peak plasma concentrations of KRN5500 in the cohort receiving the MTD ranged from 350 to 400 ng/ml. CONCLUSIONS: The MTD of KRN5500, when given as a 1-h i.v. infusion for 5 consecutive days, was 2.9 mg/m(2)/day. The only suggestion of therapeutic activity observed in this study was disease stabilization in three patients with chemorefractory colorectal cancer. Administering KRN5500 as a continuous i.v. infusion with the objective of prolonging systemic exposure to potentially cytotoxic concentrations of the drug should be considered.

Incorporation of the anticancer agent KRN5500 into polymeric micelles diminishes the pulmonary toxicity.

KRN5500 is a highly active new semi-synthetic water-insoluble anticancer agent. The only mechanism of anticancer activity of KRN5500 described so far is an inhibitory effect on protein synthesis. At the time of writing, a phase I clinical trial is under way at the National Cancer Center Hospital, Tokyo, and at the National Cancer Institute in the USA. Although preclinical data did not indicate lung toxicity, some cases of severe pulmonary disorder were reported in the phase I clinical trials. This study has been conducted to examine whether incorporation of KRN5500 into polymeric micelles (KRN/m) could reduce the toxic effects caused by the current formulation of KRN5500. The in vitro and in vivo antitumor activities of KRN5500 and KRN/m were compared. Pulmonary toxicity of KRN5500 and KRN/m was studied using a bleomycin (BLM)-induced lung injury rat model. In BLM-rats, extensive pulmonary hemorrhage with diapedesis was observed with KRN5500 i.v. bolus injection at the dose of 3 mg/kg, which is equivalent to 21.0 mg/m2 (level 5) of the Japanese phase I trial. However, toxicity was not observed when rats were administered KRN / m at the equivalent dose to KRN5500 in potency. Electron microscopy of the lung treated with KRN5500 showed disruption of the alveolar type II membrane with release of lamellar debris. Furthermore, in vivo, KRN/m showed similar antitumor activity to KRN5500. These results indicate that KRN/m may be useful for reducing the pulmonary toxicity associated with the current formulation of KRN5500, while fully maintaining its antitumor activity.

The role of nucleoside analogues in the treatment of chronic lymphocytic leukemia-lessons learned from prospective randomized trials.

The newer purine nucleoside analogues (PNA), fludarabine (FAMP) and cladribine (2-chlorodeoxyadenosine, 2-CdA) have been synthesized recently and introduced into the treatment of chronic lymphocytic leukemia (CLL). The results of large phase II studies indicate that FAMP and 2-CdA are similarly active in CLL. Unfortunately, no prospective randomized study comparing the results of the treatment of CLL patients with FAMP and 2-CdA has been published so far. Significantly higher overall response (OR) and complete remission (CR) in patients treated initially with PNA than with chlorambucil or cyclophosphamide based combination regimens has been recently confirmed in five prospective multicentre randomized trials. These studies have also shown longer response duration in patients treated with PNA than with conventional chemotherapy. Overall survival progression free and events free survival were similar in patients treated with PNA and with chlorambucil or other alkylating agent based regimens. However, the majority of randomized trials were designed as cross over studies and most patients, treated with conventional chemotherapy were given PNA when refractory or in early relapse, which may influence the survival time. The results of a randomized study have shown a higher incidence of neutropenia and infections in patients treated with PNA than with chlorambucil. However. the frequency of autoimmune hemolytic anemia, pure red cell aplasia, secondary neoplasms and Richter's syndrome seems to be similar in patients treated with PNA and standard alkylating agents based chemotherapy. In conclusion, alkylating agents still have an important place in the routine management of the majority of CLL patients. They are in general safe, given on an out patients basis and significantly cheaper than PNA. PNA should be routinely used as second line treatment, and possibly as first line therapy in younger patients, who are candidates for potentially curative treatment such as stem cell transplantation and/or monoclonal antibodies.

KRN5500, a novel antitumor agent, induces apoptosis or cell differentiation in HL-60 cells.

BACKGROUND: KRN5500, a derivative of spicamycin, shows antitumor activity against a variety of tumor cell lines. However, the mechanism of cytotoxic action has remained unclear. METHODS: The viability of HL-60 human leukemic cells treated with KRN5500 was studied by the dye exclusion assay. Induction of apoptosis and effects on the cell cycle were investigated by flow cytometry: We measured cellular DNA content after extraction of fragmented DNA, and apoptosis-induced DNA strand breaks. Cell morphology was observed by light microscopy. DNA strand breaks at a nucleosomal unit were analyzed by electrophoresis. RESULTS: Our data demonstrated that KRN5500 caused inhibition of cell growth, and that apoptosis was the mode of cell death. G(1) phase cells were more susceptible to KRN5500 induced apoptosis. In addition, KRN5500 induced cell differentiation at lower concentration. CONCLUSIONS: It is anticipated that KRN5500 will be used clinically as an anti-leukemic agent. Its mechanism of antitumor action is to induce apoptosis or cell differentiation.

Synthesis of 4'-ethynyl-purine nucleosides possessing anti-HIV activity.

Searching for more effective anti-HIV agents, we have prepared 4'-ethynyl-purine nucleosides. They were derived in several steps from 4-C-triethylsilylethynyl ribose, which was used as an intermediate in the synthesis of pyrimidine nucleosides. The adenine derivative exhibited significant anti-HIV activity and favorable cytotoxicity profile in vitro.

Synthesis and anti-HIV and anti-HBV activities of 2'-fluoro-2', 3'-unsaturated L-nucleosides.

The synthesis of L-nucleoside analogues containing 2'-vinylic fluoride was accomplished by direct condensation method, and their anti-HIV and anti-HBV activities were evaluated in vitro. The key intermediate 8, the sugar moiety of our target compounds, was prepared from 1,2-O-isopropylidene-L-glyceraldehyde via (R)-2-fluorobutenolide intermediate 5 in five steps. Coupling of the acetate 8 with the appropriate heterocycles (silylated uracil, thymine, N4-benzoylcytosine, N4-benzoyl-5-fluorocytosine, 6-chloropurine, and 6-chloro-2-fluoropurine) in the presence of Lewis acid afforded a series of 2'-fluorinated L-nucleoside analogues (15-18, 23-26, 36-45). The newly synthesized compounds were evaluated for their antiviral activities against HIV-1 in human peripheral blood mononuclear (PBM) cells and HBV in 2.2.15 cells. Cytosine 23, 5-fluorocytosine 25, and adenine 36 derivatives exhibited moderate to potent anti-HIV (EC50 0.51, 0.17, and 1.5 microM, respectively) and anti-HBV (EC50 0.18, 0.225, and 1.7 microM, respectively) activities without significant cytotoxicity up to 100 microM in human PBM, Vero, CEM, and HepG2 cells.

Reduction of the side effects of an antitumor agent, KRN5500, by incorporation of the drug into polymeric micelles.

For intravenous (i.v.) injection of a water-insoluble antitumor drug, KRN5500, we have successfully incorporated KRN5500 into polymeric micelles. In the present study, in vitro and in vivo anti-tumor activity against several human tumor cell lines and toxicity in mice of polymeric micelles incorporating KRN5500 (KRN/m) were evaluated in comparison with those of the prototype KRN5500. KRN/m was found to express similar antitumor activity to KRN5500 in the in vitro and in vivo systems. However, the vascular damage and liver focal necrosis observed with KRN5500 i.v. injection were not seen when KRN/m was administered i.v. Therefore, we expect that KRN/m will be superior to KRN5500 for clinical use and that the methodology of polymeric micelle drug carrier systems can be applied to other water-insoluble drugs.

Human thiopurine methyltransferase pharmacogenetics. Kindred with a terminal exon splice junction mutation that results in loss of activity.

Thiopurine methyltransferase (TPMT) catalyzes S-methylation of thiopurine drugs such as 6-mercaptopurine. Large variations in levels of TPMT activity in human tissue can result from a common genetic polymorphism with a series of alleles for low activity. This polymorphism is an important factor responsible for large individual variations in thiopurine toxicity and therapeutic efficacy. We now report a new variant allele, TPMT*4, that contains a G--> A transition that disrupts the intron/exon acceptor splice junction at the final 3' nucleotide of intron 9, the terminal intron of the TPMT gene. This new allele cosegregated within an extended kindred with reduced TPMT activity. We attempted to determine the mechanism(s) by which the presence of TPMT*4 might result in low enzyme activity. Although very few mature transcripts derived from allele TPMT*4 were detected, the mutation did lead to generation of at least two aberrant mRNA species. The first resulted from use of a novel splice site located one nucleotide 3' downstream from the original splice junction. That mRNA species contained a single nucleotide deletion and a frameshift within exon 10, the terminal exon of the gene. The second novel mRNA species resulted from activation of a cryptic splice site located within intron 9, leading to inclusion of 330 nucleotides of intron sequence. That sequence contained a premature translation termination codon. TPMT*4 is the first reported allele for low TPMT activity as a result of a mutation within an intron. These observations also provide insight into mechanisms of mRNA processing after disruption of a terminal exon splice junction.

In vivo gene therapy of cancer with E. coli purine nucleoside phosphorylase.

We have developed a new strategy for the gene therapy of cancer based on the activation of purine nucleoside analogs by transduced E. coli purine nucleoside phosphorylase (PNP, E.C. 2.4.2.1). The approach is designed to generate antimetabolites intracellularly that would be too toxic for systemic administration. To determine whether this strategy could be used to kill tumor cells without host toxicity, nude mice bearing human malignant D54MG glioma tumors expressing E. coli PNP (D54-PNP) were treated with either 6-methylpurine-2'-deoxyriboside (MeP-dR) or arabinofuranosyl-2-fluoroadenine monophosphate (F-araAMP, fludarabine, a precursor of F-araA). Both prodrugs exhibited significant antitumor activity against established D54-PNP tumors at doses that produced no discernible systemic toxicity. Significantly, MeP-dR was curative against this slow growing solid tumor after only 3 doses. The antitumor effects showed a dose dependence on both the amount of prodrug given and the level of E. coli PNP expression within tumor xenografts. These results indicated that a strategy using E. coli PNP to create highly toxic, membrane permeant compounds that kill both replicating and nonreplicating cells is feasible in vivo, further supporting development of this cancer gene therapy approach.

Antiproliferative activity in vitro and in vivo of the spicamycin analogue KRN5500 with altered glycoprotein expression in vitro.

The spicamycin analogue KRN5500 (NSC 650426; SPA) is derived from Streptomyces alanosinicus. The unique structure contains a purine, an aminoheptose sugar, glycine, and a tetradecadiene fatty acid. SPA potently inhibits the growth of certain human tumor cell lines in vitro (IC50 for growth <100 nM) and displays marked activity in vivo in Colo 205 colon carcinoma xenografts. Selective inhibition of labeled precursor incorporation was not evident at 1 or 4 h of exposure to the drug, but at 8 h, [3H] leucine incorporation was inhibited by approximately 40% at or below the IC50 for cell growth. Because of the structural similarity of SPA to inhibitors of glycoprotein processing, we examined the effect of SPA on indicators of glycoprotein synthesis and processing in HL60TB promyelocytic leukemia and Colo 205 colon carcinoma cells. Brief periods of exposure ( approximately 30 min) to SPA at the IC50 for growth increased incorporation of [3H]mannose. When examined by Western blotting after prolonged (40-48 h) incubation with lectins that target mannose-containing carbohydrates, Galanthus nivalis agglutinin and concanavalin A, a qualitative change in the pattern of mannose-containing glycoproteins was observed in HL60TB cells. Significant changes in the pattern of surface glycoprotein expression in intact cells were demonstrated by flow cytometry using fluorescence-labeled lectins. An increase in the number of cells binding G. nivalis agglutinin (indicating terminal mannose) was noted, but a decrease in the amount of lectin bound per cell was noted for wheat germ agglutinin (detecting sialic acid and terminal beta-N-acetyl glucosamine residues). Electron microscopy revealed loss of microvilli, and the Golgi apparatus appeared inflated. Our findings, therefore, raise the possibility that cells exposed to SPA have altered glycoprotein processing after exposure to low concentrations of drug, prior to the occurrence of overt cytotoxicity. These effects are consistent with a prominent early effect of SPA on the enzymatic machinery or organelles important for proper glycoprotein processing and emphasize the novelty of this agent's likely mechanism of action.