Semiochemical Regulation of the Intraspecific and Interspecific Behavior of Tomicus yunnanensis and Tomicus minor during the Shoot-Feeding Phase.

Two pine shoot beetles, Tomicus yunnanensis and Tomicus minor, are the most destructive pests of Pinus yunnanensis in southwestern China. We investigated behavioral responses within and between these two species during the shoot-feeding phase using walking bioassays. We also identified the pheromonal aspects of beetles by static solid phase microextraction (SPME) and hindgut extraction following interactive communication by gas chromatography-mass spectroscopy (GC-MS). Both species were significantly attracted by their own species and the same sex, and attraction was inhibited by exposure to additional beetles or to the hindgut extracts of beetles which had shown interaction. Female and male T. minor and T. yunnanensis hindguts contained 0.19, 0.09, 0.22, and 0.05 ng/individual of (-)-trans-verbenol, respectively; following interaction with additional beetles, this increased to 16.74-292.71 ng/individual in T. minor females. Mean concentration of verbenone detected in the hindguts of female/male individuals of T. minor and T. yunnanensis under natural conditions were 0.16, 0.06, 0.03, and 0.05 ng/individual, respectively, but these correspondingly increased to 5.90, 2.43, 0.06, and 0.19 ng/individual after exposure to additional insects. In T. yunnanensis, the amounts of detectable (-)-trans-verbenol and verbenone extracted from hindguts were lower than those from T. minor. The levels of cis-verbenol and (-)-trans-verbenol most attractive to walking T. yunnanensis and T. minor were 0.1 and 1.0 ng/µl, respectively. Verbenone was not attractive at any of the concentrations tested. The addition of verbenone to cis-verbenol or (-)-trans-verbenol reduced the attraction responses. We conclude that the (-)-trans-verbenol produced by these two pine shoot beetles is attractive at low concentrations and repellant at high concentrations, thereby fostering intraspecific competition. Verbenone is produced to prevent overcrowding via interspecific inhibition, and to expel beetles during shoot-feeding.

Relative contributions of testosterone deficiency and metabolism syndrome at the risk of reduced quality of life: A cross-sectional study among Chinese mid-aged and elderly men.

Testosterone deficiency and metabolism syndrome (MetS) are universal among ageing males, and they have been suggested responsible for poorer quality of life (QoL). We aimed to evaluate the relative contributions of reproductive hormones and components of MetS at the risk of reduced QoL among Chinese mid-aged and elderly men. A cross-sectional study recruited 2,364 males aged 40-79 years, and 2,165 was included for analysis eventually. The Chinese version of ageing male symptoms scale, 36-item Short Form and Beck Depression Inventory were applied to assess QoL. Bivariate correlation analysis and multiple linear regression analysis were used to assess the relative contributions of reproductive hormones and components of MetS at the risk of reduced QoL. Testosterone deficiency and MetS contributed to poorer QoL, of which higher fasting blood glucose made the primary contribution, lower total testosterone mainly contributed to poorer physical functioning.

The rs5934505 single nucleotide polymorphism (SNP) is associated with low testosterone and late-onset hypogonadism, but the rs10822184 SNP is associated with overweight and obesity in a Chinese Han population: a case-control study.

Low testosterone is associated with late-onset hypogonadism (LOH) and obesity. Recently, studies have shown that four single nucleotide polymorphisms (SNPs), rs12150660, rs727428, rs5934505, and rs10822184, are associated with testosterone levels in populations of European descent. Therefore, we investigated whether the SNP loci are related to low testosterone, LOH, or obesity in a Chinese Han population. Ruling out co-morbidities, DNA was prepared from 409 men (aged 40-65 years) with low serum testosterone (defined as total testosterone <11.6 nmol/L) and 1 : 1 normal controls (matched age, body mass index (BMI), and the same living area) who were selected from 6898 males. According to the same standards, 310 men with LOH and 1 : 1 normal controls were selected from 6898 males. Excluding the cases with an unreliable sequencing result, genetic analyses were performed. The minor allele frequencies of the SNP loci rs12150660, rs727428, rs5934505, and rs10822184 were 0.1%, 44.6%, 18.7%, and 38.9%, respectively. rs5934505 was associated with the serum total testosterone and calculated free testosterone (CFT) levels (p = 0.045 and p = 0.021). rs5934505 (C>T) was associated with an increased risk of low total testosterone, low CFT, and LOH and adjusted for other factors, with an odds ratio (OR) of 2.01 (1.34-3.01), 2.14 (1.42-3.20), and 1.64 (1.04-2.58). rs10822184 was significantly correlated with weight and BMI (p = 0.035 and p = 0.027). rs10822184 (T>C) was associated with an increased risk of overweight and obesity. We adjusted for other factors, with odds ratios (ORs) of 1.94 (1.36-2.78) and 1.56 (1.00-2.43). In summary, our study provided convincing evidence that rs5934505 (C>T) was associated with the risk of low testosterone and LOH in Chinese populations. We were the first to find that rs10822184 (T>C) was significantly correlated with the risk of overweight and obesity in Chinese populations. However, further large and functional studies are warranted to confirm our findings.

Synthesis and antimicrobial activity of 9-o-substituted palmatine derivatives.

A series of new palmatine derivatives with alkyl or alkyl with N-heterocyclic structures were designed and synthesized at C-9-O according to the principle of association. These compounds were characterised by (1)H NMR, (13)C NMR, ESI-MS and elemental analysis, and tested for their antimicrobial activity in vitro to evaluate structure-activity relationships. The results indicated that 9-O-substituted palmatine derivatives exhibit varying degrees of antimicrobial activity. Antibacterial activities of compounds (3a-f) against Gram +ve bacteria increased 2- to 64-fold than that of palmatine. The compounds (3a-f) possessed relatively weaker inhibitory effects against Gram -ve bacteria and fungi than that against Gram +ve bacteria. Antimicrobial activities of compounds (5a-e) are lower than that of compounds (3a-f). Compound 3d showed the highest antimicrobial activity of all the compounds. The LD50 values of compounds (3a-f) decreased as the alkyl side chain was elongated. Compound 3f showed least toxicity.

The ageing males' symptoms scale for Chinese men: reliability,validation and applicability of the Chinese version.

In this study, the ageing males' symptoms (AMS) scale was translated into Chinese following methodological recommendations for linguistic and cultural adaptation. This study aimed to confirm the reliability, validation and applicability of the simplified Chinese version of the scale (CN-AMS) in older Chinese men, a free health screening for men older than 40 years was conducted. All participants completed a health questionnaire, which consisted of personal health information, AMS scale, the generic quality of life (QoL) instrument SF36 and the Beck Depression Inventory (BDI). The fasting blood samples of participants were collected on the day of completing the health questionnaire. Serum total testosterone (TT), albumin and sex hormone-binding globulin levels were measured and the level of free testosterone was calculated (calculated free testosterone, CFT). A total of 244 men (mean age: 52 ± 7.3 years, range: 40-79 years) were involved in the investigation and provided informed consent before their participation. The reliability of CN-AMS was analysed as internal consistency reliability (Cronbach's alpha was 0.91) as well as a 4-week-interval test-retest stability (Pearson's correlation was 0.83) and found to be good. The validation of CN-AMS was analysed as the internal structure analysis (Pearson's correlation between total score and each item score r = 0.48-0.75), total-domain-correlation (among the three domains r = 0.47-0.68, p < 0.01; domains with the total score r = 0.81-0.88, p < 0.01), and cross-validation with other scales (with SF36 r = -0.59, p < 0.01; with BDI r = 0.50, p < 0.01). Androgen deficiency (AD) was defined as the presence of three sexual symptoms (decreased frequency of morning erections, sexual thoughts and erectile dysfunction) in combination with TT < 11 nmol/L and CFT < 220 pmol/L, and the sensitivity and specificity for CN-AMS was 68.8 and 6.8% respectively. The CN-AMS had sufficient sensitivity in screening AD of older men, but the low specificity made it unsuitable to be adopted as the diagnostic criteria. The scanning capability of AMS scale for AD has the downward trend with ageing and a hypothesis is proposed to give a possible reason for the new finding.

[Effect of ketanserin on arterial baroreflex-blood pressure control in conscious hypertensive rats].

The effect of ketanserin on arterial baroreflex-blood pressure control (ABR-BP) were studied in conscious freely-moving spontaneously hypertensive rats (SHR) and renovascular hypertensive rats (RVHR). The ABR-BP was measured by using a new method comparing with the pressor responses (in area) to angiotensin II before and after blocking the baroreflex efferent pathway by guanethidine and methyl atropine. It was found that ketanserin enhanced markedly the ABR-BP in both groups of hypertensive rats (SHR: 51% to 74%; RVHR: 59% to 77%). This suggests that the enhancement of ABR-BP may be involved in the anti-hypertensive effects of ketanserin.

Preclinical pharmacology and pharmacokinetics of the anti-hepatitis virus agent 2'-fluoro-5-ethyl-1-beta-D-arabinofuranosyluracil in mice and rats.

The preclinical pharmacology and pharmacokinetics of 2'-fluoro-5-ethyl-1-beta-D-arabinofuranosyluracil (FEAU), a selective inhibitor of herpesvirus and hepatitis virus replication, were investigated in the mouse and rat. Following intravenous (i.v.) or oral (p.o.) administration, FEAU was cleared from the plasma primarily unchanged, with a terminal half-life of 58 to 80 min in the mouse and 63 to 78 min in the rat. The steady-state volumes of distribution times bioavailabilities of FEAU were approximately 2.1 and 3.4 times the total body water volumes after p.o. administration of 10 mg of drug per kg of body weight in mice and rats, respectively. A comparison of the area under the concentration-time curve after i.v. and p.o. FEAU administration indicated that the p.o. dose was completely absorbed in both species. When tritiated FEAU was used in mice, 35.0% of the i.v. dose and 33.5% of the p.o. dose were excreted in urine as unchanged FEAU, 8.1% (i.v. dose) and 9.2% (p.o. dose) were excreted as tritiated water, and 15.6% (i.v. dose) and 18.1% (p.o. dose) were excreted as unknown metabolite(s) in urine within 24 h of dosing. Only 1.24% (i.v. dose) and 2.6% (p.o. dose) of the total doses were found in urine as 3H2O when the FEAU dose was increased to 50 mg/kg. However, a higher percentage of the total dose (59.6% for the i.v. dose and 61.3% for the p.o. dose) was recovered within 24 h as intact FEAU in rat urine, less than 1.4% (i.v. dose) and 2.7% (p.o. dose) of the total dose were found to be 3H2O, and 5.6% (i.v. dose) and 6.7% (p.o. dose) of the total dose were excreted as known metabolite(s). The distribution ratios for total radioactivity in tissue relative to those in plasma were 0.5 to 1.3 in spleen, testes, muscle, and liver during the first hour after a 10-mg/kg dose in rats. Of the total FEAU radioactivity administered, only 1.38% was excreted in bile as unchanged FEAU. No FEAU glucuronide metabolite was detected. Tissue concentrations of 0.15 to 0.6 microM at 6 h after dosing are in the range of the effective antiviral concentration for FEAU. In conclusion, FEAU administered p.o. to mice and rats was well absorbed; FEAU was rapidly distributed into tissues and remained above in vitro antiviral concentrations for more than 6 h; in mice, [3H]FEAU showed metabolism-mediated tritium exchange with water; and in rats, FEAU was less extensively metabolized than in mice and clearance was primarily via renal processes, mainly in the form of unchanged FEAU.

Effects of ketanserin on blood pressure variability in conscious spontaneously hypertensive rats.

Computerized analysis of blood pressure (BP) was used to study for the effects of ketanserin (Ket) on BP and blood pressure variability (BPV). Rats were instrumented chronically and BP was sampled every 4 ms by a computer from 2:00 to 14:00. Then a single dose of Ket (3 mg.kg-1) was given iv. BP and heart period (HP) were recorded for the next 30 min. The results showed that Ket lowered systolic BP (26.7 kPa to 21.1 kPa), diastolic BP (20.5 kPa to 15.8 kPa), and systolic BPV (1.3 kPa to 0.94 kPa). Otherwise, a positive relationship was found between antihypertensive effects of Ket and BPV. These findings may be of importance in antihypertensive treatment.

Comparisons of anti-human immunodeficiency virus activities, cellular transport, and plasma and intracellular pharmacokinetics of 3'-fluoro-3'-deoxythymidine and 3'-azido-3'-deoxythymidine.

3'-Fluoro-3'-deoxythymidine (FLT), a candidate anti-AIDS compound in clinical trials, showed anti-human immunodeficiency virus type 1 (HIV-1) potency (50% effective concentration, 0.0052 microM) slightly better than or equal to that of 3'-azido-3'-deoxythymidine (AZT) in MT4 cells and was threefold more potent in H9 cells. There was no FLT resistance demonstrable in the AZT-resistant HIV-1 strains. Both FLT and AZT showed low cytotoxicity for MT4 cells, with selectivity indices (efficacy/toxicity ratio) of greater than 47,000 and greater than 33,000, respectively. Cellular permeation of FLT and thymidine (dThd) was greater than that of AZT, and FLT and dThd permeated the cell membranes by a carrier-mediated mechanism as well as by simple diffusion, as indicated by the existence of nitrobenzylthioinosine-5'-monophosphate-sensitive and -insensitive components. By contrast, transport of AZT into cells was by simple diffusion. The intracellular level of the triphosphate of FLT (FLTTP) in MT4 cells was two- to threefold higher than that of AZT (AZTTP) after exposure to 1.8 microM each compound for 12 h. The elimination kinetics of FLTTP and AZTTP in HIV-1-infected MT4 cells in fresh medium showed biphasic patterns, with initial half-lives of 1.03 and 1.09 h, respectively. In phytohemagglutinin-stimulated human peripheral blood lymphocytes, the FLTTP level was increased 59-fold compared with that in unstimulated cells at 12 h, was four- to sixfold higher than the level of AZTTP in stimulated cells at 12 h, and remained four- to fivefold higher during a 4-h elimination period in fresh medium and twofold higher at the end of a 12-h elimination period. Two- to eightfold more [3H]AZT than [3H]FLT was incorporated into the host cell DNA, and both [3H]AZT and [3H]FLT remained persistently incorporated for over 24 h. The incorporated [3H]AZT and [3H]FLT were alkali labile, whereas incorporated [3H]dThd was alkali stable. Pharmacokinetics of FLT in plasma of monkeys after intravenous (i.v.) administration showed that the FLT concentration in plasma declined, with a half-life of 1.19 +/- 0.1 h; the steady-state volume of distribution was 0.93 +/- 0.2 liter/kg of body weight, and total clearance was 0.56 +/- 0.15 liter/kg. Oral bioavailability of FLT was excellent and comparable to i.v. bioavailability in terms of areas under the concentration-time curves for three monkeys. Of the total dose, 41 to 61% was excreted in urine as unchanged FLT, and only 3.2 to 7.4% of the total dose was identified as glucuronide-conjugated FLT in urine 48 h after i.v. administration to monkeys. We conclude that FLT exhibits an anti-HIV-1 potency similar to that of AZT but with slightly better selectivity of effects and with higher intracellular active metabolite levels.

Topoisomerase II-mediated DNA cleavage activity and irreversibility of cleavable complex formation induced by DNA intercalator with alkylating capability.

A group of chrysophanol and emodin derivatives with DNA-intercalating capability and with or without alkylating potential have been synthesized and shown to have antitumor activity in vitro. The topoisomerase II (Topo II)-mediated DNA cleavage activities induced by representative compounds 3-(2-chloroethylamino) methyl-1,8-dihydroxy-9,10-anthraquinone (SK-31690), 3-bis [(2-chloroethyl)amino]methyl-1,8-dihydroxy-9,10-anthraquinone (SK-31662), and 3-(2-hydroxyethylamino)methy-1,8-dihydroxy-9,10-anthraquinon e (SK-31694), and their cytotoxicities, have been investigated. All three compounds inhibited the kinetoplast DNA decatenation catalyzed by DNA Topo II. These compounds inhibited leukemia cell growth and stimulated, in a dose-dependent manner from 0.5 to 60 microM, the formation of Topo II-DNA cleavable complexes, when 3'-32P-labeled DNA was used. The mapping of Topo II-mediated DNA cleavage sites using HindIII-digested 3'-32P-labeled DNA showed that, at 10 microM, these compounds induced protein-linked DNA breaks that correlated with cytotoxicity, with respect to their maximal efficacy or the reciprocal concentration for the half-maximal effect. The reversibility study showed that the amounts of protein-linked DNA cleavage induced by 4'-(9-acridinylamino)methanesulfon-m-anisidide and VP-16 as well as SK-31694, which lacks alkylating potential, were markedly decreased during 30-sec exposure to 65 degrees or 0.5 M NaCl. In contrast, protein-linked DNA cleavages induced by SK-31662, which has two alkylating functionalities, and by SK-31690, which has one alkylating functionality in its structure, cannot be reversed during the 15-min exposure to 65 degrees or 0.5 M NaCl. These data suggest that Topo II is a major cellular target for cytotoxicity of these compounds. Furthermore, DNA intercalators with alkylating potential interact with Topo II-DNA cleavable complexes in an irreversible manner, with enhanced toxicity.

Synergistic inhibition of human immunodeficiency virus type 1 replication in vitro by two-drug and three-drug combinations of 3'-azido-3'-deoxythymidine, phosphonoformate, and 2',3'-dideoxythymidine.

The effects of 3'-azido-3'-deoxythymidine (AZT), phosphonoformate (PFA), and 2',3'-dideoxythymidine (ddT) and their combination on human immunodeficiency type 1 (HIV-1) replication were studied by measuring the HIV-1 p24 antigen expression and reverse transcriptase (RT) release in HIV-1-infected MT4 cells in vitro. RT activity was also measured in a cell-free system by using poly(rA)-oligo(dT) as the primer-template, and cell growth inhibition was measured in noninfected MT4 cells. The interactions of these two- and three-drug combinations were evaluated by the combination index (CI) method and isobologram techniques. The 50% effective concentrations (EC50s) of AZT, PFA, and ddT were 0.014 to 0.005, 9.4 to 8.8, and 8.4 to 2.5 microM, respectively, for p24 enzyme-linked immunosorbent assays (ELISAs) and 0.005 to 0.0034, 1.43 to 1.37, and 2.87 to 2.83 microM, respectively, for RT activity in vitro; for RT activity in the cell-free system, the EC50s were 0.00019 to 0.00024, 0.012 to 0.02, and 0.00074 to 0.0005 microM, for AZT-5'-triphosphate, PFA, and ddT-5'-triphosphate, respectively. AZT in combination with PFA (1:200) or ddT (1:5) as well as the combination of these three drugs (1:200:5) synergistically inhibited HIV-1 replication and RT activity in the cell-free system over a wide range of drug concentrations, with the CIs ranging from 0.5 to 0.09, in which CIs of less than 1, 1, and greater than 1 indicate synergism, additive effect, and antagonism, respectively. Three- and two-drug combinations of AZT, PFA, and ddT showed similar degrees of synergism against HIV-1 replication in p24 assays and RT release assays, whereas the combination of AZT and ddT was found to be the most selective in terms of its anti-HIV-1 effect versus cytotoxicity. Dose reduction indices calculated from both HIV-1 replication inhibition, as measured by p24 ELISA and by RT activity in the cell-free system, indicated that two- and three-drug combinations at high effect levels and the selected combination ratios allow 2- to 240-fold dose reduction over the single drug alone in terms of their anti-HIV-1 effects. The three-drug combination showed the highest dose reduction index. These finding suggest that increased efficacy and reduced toxicity may be achieved in AIDS therapy by using AZT, PFA, and ddT in two- or three-drug combinations.

Induction of deoxycytidine kinase by 5-azacytidine in an HL-60 cell line resistant to arabinosylcytosine.

Induction of 2'-deoxycytidine kinase (dCK) by 5-azacytidine (5-Aza-C) in a dCK-deficient HL-60 cell line resistant to 1-beta-D-arabinofuranosylcytosine (Ara-C) (HL-60/Ara-C) was examined by measurement of the incorporation of [3H]deoxycytidine ([3H] dCyd) into cellular DNA, the kinetic properties of purified dCK, the cytotoxic potency (IC50 values), and the DNA methylation patterns of 5-Aza-C-treated and untreated cells. Following a 72-hr exposure to 1 microM 5-Aza-C, the incorporation of [3H]dCyd into DNA was increased 6-fold and the total dCK activity was increased 12-fold, with a peak for both on day 6. The onset of dCK induction was dependent on the length of exposure time. The IC50 values for cell growth inhibition by Ara-C on day 3 were 0.08 microM for HL-60 cells, 12.5 microM for HL-60/Ara-C cells, and 0.55 microM for HL-60/Ara-C cells pretreated with 5-Aza-C for 40 hr. The Km and Vmax of dCyd for HL-60 dCK were similar to those for 5-Aza-C-induced HL-60/Ara-C dCK. The restriction enzymes Hpall, which cleaves CCGG sequences but cannot cleave at sites methylated at the internal cytosines (5'-CMeCGG), and Mspl, which cleaves sequences with internal methylated cytosine but cannot cleave at sites methylated at external cytosines (5'-MeCCGG), were used for DNA methylation pattern determination. The newly synthesized DNA of HL-60 wild-type cells was cleaved by Mspl to a greater extent than that of HL-60/Ara-C cells. After exposure to 1 microM 5-Aza-C for 40 hr, methylation patterns of newly synthesized DNA reverted in HL-60/Ara-C cells to a clevage pattern similar to that in HL-60 wild-type cells. When compared with untreated control, DNAs from 5-Aza-C-treated resistant cells were cleaved to a greater extent by Mspl than by Hpall, suggesting that internal cytosine-residue methylation was relatively uninhibited.(ABSTRACT TRUNCATED AT 250 WORDS)

Incorporation and metabolism of 2'-fluoro-5-substituted arabinosyl pyrimidines and their selective inhibition of viral DNA synthesis in herpes simplex virus type 1 (HSV-1)-infected and mock-infected Vero cells.

The incorporation and metabolism of 2'-fluoro-5-substituted arabinosyl pyrimidine analogs, and their selective inhibition of viral DNA synthesis in herpes simplex virus type 1 (HSV-1)-infected and mock-infected Vero cells were studied by HPLC and CsCl isopycnic density gradient analysis of isolated DNAs. The amounts of radiolabeled analogs incorporated as parent compound following 10 microM exposure for 4 h were 10-fold higher in HSV-1-infected vs mock-infected cells for 2'-fluoro-5-difluoromethyl-Ara-U (F2FMAU); 4.3-fold higher for 5-ethyl deoxyuridine (EdU); 2.6-fold higher for 2'-fluoro-5-methyl-Ara-U (FMAU) and 1.7-fold higher for dThd. For 2'-fluoro-5-ethyl-Ara-U (FEAU), 3.0 pmole of unchanged moiety was incorporated per 10(6) HSV-1-infected cells but no incorporation was detected in mock-infected cells. HPLC profiles showed that the percentages of radiolabeled analogs incorporated as parent compound in the DNA extracted from HSV-1-infected cells were 31.0% for F2FMAU, 99.6% for EdU, 83.5% for FEAU and 98.3% for FMAU; from mock-infected cells, they were 63.6% for F2FMAU, 96.7% for EdU, 97.3% for FMAU and no incorporation into DNA for FEAU was detected. CsCl density gradient analyses of isolated DNA showed that viral DNA synthesis was inhibited 98% by 10 microM FEAU, 92% by 10 microM F2FMAU, 90% by 2 microM FMAU and 80% by 50 microM EdU, whereas cellular DNA synthesis was inhibited by 53, 44, 61, 66 and 54%, respectively. We conclude that: (a) FEAU incorporation into host-cell DNA was not detectable but FEAU was selectively incorporated into HSV-infected cells; (b) FMAU and FEAU were metabolically stable; however, F2FMAU was extensively metabolized; (c) FEAU and F2FMAU were among the most selective inhibitors of HSV-1 DNA synthesis while allowing cellular DNA synthesis to continue.

Antagonistic interactions of hexamethylene bisacetamide in combination with 1-beta-D-arabinofuranosylcytosine, adriamycin and harringtonine on the growth and differentiation of HL-60 cells in vitro.

Selective killing of cancer cells by cytotoxic agents and the conversion of cancerous cells to normal state by differentiation agents represent two basically different approaches in chemotherapy. In this study, we examined the combination of the cell differentiation inducer, hexamethylene bisacetamide (HMBA), and the cytotoxic agents, 1-beta-D-arabinofuranosylcytosine (Ara-C), adriamycin (Adr) and harringtonine (HT), for cytotoxicity and induction of cell differentiation in HL-60 cells by measuring cell growth inhibition, morphological maturation and nitroblue tetrazolium (NBT) reduction. To determine quantitatively whether the effects produced by these combinations were additive, synergistic or antagonistic, we used a computer program based on the median-effect principle and isobologram equations (Adv. Enz. Reg. 22, 27-55, 1984), After 5-day exposure to each drug alone we found that the ED50s for cell growth inhibition were 0.01 microM for Ara-C, 0.012 microM for Adr, 0.017 microM for HT and 2.53 mM for HMBA. ED50s for differentiation were 0.089 microM (morphology), 0.06 microM (NBT) for Ara-C; 0.12 microM (morphology), 0.09 microM (NBT) for Adr; 0.04 microM (morphology) 0.06 microM (NBT) for HT; and 2.55 mM (morphology), 2.43 mM (NBT) for HMBA, respectively. At dose levels from ED50 to ED95, the combinations of Adr/HMBA and HT/HMBA produced antagonistic cytotoxic and cell differentiation effects. The combination of Ara-C/HMBA produced antagonistic cytotoxic and cell differentiation effects. The combination of Ara-C/HMBA produced antagonistic cytotoxic effects but slight synergistic cell differentiation effects. On the basis of this study, we conclude that the equipotency combinations of the above three pairs of drugs do not synergistically enhance cytotoxicity or cell differentiation effects in vitro at effect levels high enough for the successful treatment of acute leukemia. Other combinations of cell differentiation agents with cytotoxic agents or biological response modifiers remain to be explored.

Synthesis and biological effects of 2'-fluoro-5-ethyl-1-beta-D-arabinofuranosyluracil.

2'-Fluoro-5-ethyl-1-beta-D-arabinofuranosyluracil (FEAU) was synthesized, and its biological activities were compared with those of 2'-fluoro-5-methyl-1-beta-D-arabinofuranosyluracil (FMAU). Earlier studies indicated that both compounds showed potent anti-herpes simplex virus activity, with a 50% effective dose (ED50) of less than 0.25 microM. In the present study the cell growth inhibitory activity of FEAU (ED50, 200 to 2,060 microM) was found to be about 100-fold less than that of FMAU. With an ED50 ranging from 630 to 3,700 microM, FEAU only weakly inhibited thymidine incorporation into DNA, as compared with FMAU with an ED50 of 9 to 28 microM. Following exposure to [2-14C]FEAU (100 microM), 0.48 pmol/10(6) cells per h was incorporated into the DNA of herpes simplex virus type 1-infected Vero cells, whereas no detectable incorporation was found in uninfected Vero cells or L1210 cells. The Ki of FEAU for thymidine kinase purified from human leukemic cells was greater than 150 microM. For herpes simplex virus type 1- and 2-encoded thymidine kinases, the Kis were 0.6 and 0.74 microM, respectively. Both FEAU and FMAU were relatively nontoxic for mice, with a 50% lethal dose of greater than 800 mg/kg per day (four intraperitoneal doses). However, the lethal dose of FEAU for dogs was 100 mg/kg per day (10 intravenous doses), a dose which is 40- to 80-fold greater than the toxic dose of FMAU. These results suggest that FEAU is a worthy candidate for further development as an antiherpetic agent.

Cell differentiation effects of 2'-fluoro-1-beta-D-arabinofuranosyl pyrimidines in HL-60 cells.

A group of 2'-fluoro and 5-substituted arabinosyl pyrimidines and a group of base-substituted pseudoisocytidine analogs were evaluated for their capacity to induce differentiation in the human promyelocytic leukemia cell line, HL-60. These compounds were compared to 1-beta-D-arabinofuranosylcytosine (Ara-C) by monitoring: (1) inhibition of cell growth; (2) morphological maturation; (3) nitroblue tetrazolium (NBT) reduction; (4) expression of a myeloid differentiation antigen, Mo1; and (5) inhibition of colony formation. Exposure of logarithmically growing cells for 5 days to Ara-C, 2'-fluoro-Ara-C (FAC), 2'-fluoro-5-methyl-Ara-C (FMAC) and 2'-fluoro-5-ethyl-Ara-C (FEAC) resulted in cell growth inhibition at ED50 concentrations of 0.007, 0.11, 1.7 and 18 microM, and at cytostatic concentrations of 0.1, 0.5, 5.0 and 50 microM, respectively. These compounds induced granulocytic and monocytic maturation, reduction of NBT, increased expression of Mo1 antigen and a decrease or loss of both cell proliferation and colony formation in semisolid medium. There were few, if any, cell differentiation effects for the uracil nucleosides and pseudoisonucleosides tested. We found that Ara-C was the most cytotoxic of the compounds, and that when comparing absolute numbers of differentiated cells, i.e. percent of positive cells multiplied by the number of viable cells, FAC, FMAC and FEAC were superior to Ara-C inducing differentiation of HL-60 cells.