Neurodevelopmental outcome of hypoglycaemia in healthy, large for gestational age, term newborns.

AIMS: To evaluate the effects of transient hypoglycaemia on the first day of life in 75 healthy term large for gestational age (LGA) infants, born to non-diabetic mothers, on their neurodevelopmental outcome at the age of 4 years. METHODS: Screening for hypoglycaemia was performed 1, 3, and 5 hours after birth, and continued if blood glucose levels were low. Treatment with intravenous glucose for hypoglycaemia was started if hypoglycaemia was severe or symptomatic. Patients' development and behaviour was examined at the age of 4 years by the Denver Developmental Scale, a non-verbal intelligence test, and the Child Behaviour Check List. RESULTS: There were no significant differences between children with neonatal normoglycaemia (n = 15) and hypoglycaemia (plasma glucose <2.2 mmol/l 1 hour after birth, or <2.5 mmol/l subsequently; n = 60) in Denver developmental scale scores and child behaviour checklist scores. Although total IQ did not differ between hypoglycaemic and normoglycaemic children, one subscale (reasoning) did (mean difference 9.3, 95% CI 1.3 to 17.2). The correlation between reasoning IQ and neonatal blood glucose levels was weak and not statistically significant. When other definitions for hypoglycaemia were applied, the difference in reasoning IQ was not found. There were no differences in any of the test scores between hypoglycaemic children who had and who had not been treated with intravenous glucose. CONCLUSION: Transient mild hypoglycaemia in healthy, term LGA newborns does not appear to be harmful to psychomotor development at the age of 4 years.

Soluble CD4-PE40 is cytotoxic for a transfected mammalian cell line stably expressing the envelope protein of human immunodeficiency virus (HIV-1), and cytotoxicity is variably inhibited by the sera of HIV-1-infected patients.

Sera were obtained from 50 individuals infected with human immunodeficiency virus type 1 or from HIV-1-uninfected individuals before or after vaccination with recombinant gp160. These sera were evaluated for activity antagonistic to the cell-killing activity of the chimeric Pseudomonas exotoxin hybrid protein, sCD4-PE40. For these studies, Chinese hamster ovary (CHO) cells were transfected with a chimeric plasmid encoding the tat, rev, and envelope genes of HIV-1 and a cell line was selected for stable expression of the envelope glycoproteins at the cell surface (CHO-env). Cytotoxicity of sCD4-PE40 for CHO-env in the presence or absence of added human serum was quantitated spectrophometrically following enzymatic reduction of a tetrazolium bromide within the mitochondria of viable cells (MTT assay). Several HIV+ sera inhibited the cytotoxic activity of sCD4-PE40; the antagonist had properties consistent with those of immunoglobulins in that it was heat stable, absorbed by protein A, and reversible by increasing the concentration of sCD4-PE40. Of 15 HIV+ sera which strongly reacted with gp120, 11 (73%) also potently inhibited sCD4-PE40 cytotoxicity, and cytotoxicity was inhibited by sera from some HIV- individuals after, but not before, immunization with gp160. These data suggested a role for antibody to gp120 in the antagonistic activity. However, not all sera with antibody to gp120 antagonized sCD4-PE40 cytotoxicity and high levels of antagonist activity were frequently (40%) found in HIV+ sera lacking immunoblot-detectable antibody to gp120, or antibody to either CD4 or PE40. Grouping of the HIV+ sera according to the patients' absolute number of CD4+ cells revealed that the degree of inhibition of sCD4-PE40 cytotoxicity approached a Gaussian distribution, suggesting that persons with CD4+ cell counts between 200 and 700/mm3 may be more likely to possess significant levels of serum antagonist. This data have implications for the clinical development of sCD4-PE40 or other sCD4-based therapeutics in the management of HIV-1 infection.

An NMR study of the covalent and noncovalent interactions of CC-1065 and DNA.

The binding of the antitumor drug CC-1065 has been studied with nuclear magnetic resonance (NMR) spectroscopy. This study involves two parts, the elucidation of the covalent binding site of the drug to DNA and a detailed investigation of the noncovalent interactions of CC-1065 with a DNA fragment through analysis of 2D NOE (NOESY) experiments. A CC-1065-DNA adduct was prepared, and an adenine adduct was released upon heating. NMR (1H and 13C) analysis of the adduct shows that the drug binds to N3 of adenine by reaction of its cyclopropyl group. The reaction pathway and product formed were determined by analysis of the 13C DEPT spectra. An octamer duplex, d(CGATTAGC.GCTAATCG), was synthesized and used in the interaction study of CC-1065 and the oligomer. The duplex and the drug-octamer complex were both analyzed by 2D spectroscopy (COSY, NOESY). The relative intensity of the NOEs observed between the drug (CC-1065) and the octamer duplex shows conclusively that the drug is located in the minor groove, covalently attached to N3 of adenine 6 and positioned from the 3'----5' end in relation to strand A [d(CGATTA6GC)]. A mechanism for drug binding and stabilization can be inferred from the NOE data and model-building studies.

Protection from carcinogen-induced murine bladder carcinoma by interferons and an oral interferon-inducing pyrimidinone, bropirimine.

Interferons (IFNs) have established activities as antivirals and inhibitors of viral and transplantable tumors. To establish whether IFNs or their inducers can affect induction of carcinogenesis in vivo, the bladder-specific carcinogen N-[4-(5-nitro-2-furyl)-2-thiazolyl]formamide (FANFT) was administered in the diet at 0.11 or 0.13% (w/w) to female C3H/He mice beginning at 7 weeks of age. Mice treated with the IFN-inducing bropirimine [2-amino-5-bromo-6-phenyl-4(3H)-pyrimidinone] i.p. twice a week for 14 weeks starting on day 30 of start of FANFT feeding developed fewer transitional cell carcinomas (TCC) than mice treated with the vehicle. Bropirimine (200 mg/kg twice a week) orally resulted in even greater effectiveness: 6 of 43 bladders with TCC for bropirimine-treated mice versus 24 of 39 for control glycine buffer-treated mice (P less than 0.01, x2 test). Mice treated i.p. daily on days 29 through 210 with 5,000 units of beta interferon (specific activity, 2.0 x 10(8) units/mg) had 0 of 15 TCC while control mice had 7 of 13 TCC (P less than 0.001). Bladders of untreated mice were also significantly heavier than those of beta interferon- or bropirimine-treated mice. This dose of IFN treatment was confirmed as effective in a second experiment, in which mice were treated daily on days 30-223 with 5,000 units alpha/beta interferon (specific activity, 1.2 x 10(7) units/mg). This resulted in 4 of 25 bladders with TCC versus 24 of 39 for control mice (P less than 0.001). A higher dose of IFN (50,000 units alpha/beta interferon daily) was toxic; 24 of 30 mice died within 2 months. IFN and an IFN inducer, bropirimine, inhibited development and progression of FANFT-induced bladder TCC in vivo and thus may have roles as chemopreventive modalities.

Stereoelectronic factors influencing the biological activity and DNA interaction of synthetic antitumor agents modeled on CC-1065.

The synthesis, physicochemical properties, and biological activities of a series of novel spiro cyclopropyl compounds, modeled on the potent antitumor antibiotic CC-1065 (1), are described. Many of these synthetic analogues are significantly more effective than 1 against murine tumors. In particular, compound 27 exhibits high activity and potency. Structure-activity analysis supports a molecular mechanism for biological action involving hydrophobic interaction of the drug with DNA and acid-catalyzed alkylation of DNA.

Antitumor activity of pyrimidinones, a class of small-molecule biological response modifiers.

This study was undertaken in an attempt to evaluate the structure-activity relationship of pyrimidinones. Of 20 pyrimidinones tested, only those with a monohalogen substitution at the ortho- or meta-position of the phenyl moiety of the 2-amino-5-halo-6-phenyl-4(3H)-pyrimidinone and ABPP showed statistically significant synergism with cyclophosphamide (CY) against P388 leukemia. Therefore, ABMFPP, AIMFPP, and ABPP were selected for detailed therapeutic evaluation. The pyrimidinone alone had small but significant activity against B16 melanoma with slightly more than a 25% increase in lifespan (ILS); however, when used in combination with CY, ABPP or ABMFPP did not yield an effect greater than treatment with CY alone. Only AIMFPP appeared to produce a more or less additive effect with CY. Although none of these pyrimidinones alone had any significant activity against M5076 tumor, the combination with CY (100 mg/kg) produced a range of 102 to 123% ILS and six to nine of 10 mice per group survived greater than 45 days, whereas the treatment with CY alone yielded only a 48% ILS and none survived greater than 45 days. The synergism of the combination therapy was statistically significant (p less than 0.01). The combination used against L1210 leukemia also appeared to be superior to the treatment with CY alone and produced 25 to 50% long-term survivors (greater than 30 days). The significance of these findings is discussed in terms of its clinical implications.

Pyrimidinones. 3. N-substituted 6-phenylpyrimidinones and pyrimidinediones with diuretic/hypotensive and antiinflammatory activity.

In an extensive analysis of the antiviral and interferon-induction structure-activity relationship of 6-arylpyrimidinones we found that modifications at positions 1-4 of the pyrimidine ring resulted in a loss of activity. However, we uncovered interesting hypotensive and antiinflammatory activity with a series of N-substituted analogues, the results of which we report herein.

Inhibitory effects of interferon-inducing pyrimidinones on the growth of transplantable mouse bladder tumors.

Pyrimidinones are low-molecular-weight compounds which are inducers of interferon in several animal species. They have established antiviral, immunomodulatory, and antitumor effects. Four pyrimidinones as well as another potent interferon inducer, polyriboinosinic-polyribocytidylic acid, and beta-interferon were tested for effects on growth of the transplantable mouse bladder tumor (MBT-2). The pyrimidinones 2-amino-5-bromo-6-phenyl-4(3H)pyrimidinone (ABPP) and 2-amino-5-bromo-6-(3-fluorophenyl)-4(3H)pyrimidinone (ABMFPP) significantly inhibited MBT-2 growth in a dose-dependent manner and with equal potency when injected i.p. every 4 days starting 1 day after tumor cell inoculation. Administration of ABPP p.o. was as effective as i.p. injections. Direct intravesical application of ABPP to transplantable tumors growing in the bladder may be more effective in inhibiting MBT-2 growth than the same dose introduced p.o. Although ABPP (100 mg/kg) has an inhibitory effect comparable to 5000 units of beta-interferon, both pyrimidinones even at 500 mg/kg were less inhibitory of tumor growth than 10 mg of polyriboinosinic-polyribocytidylic acid per kg. The pyrimidinones 2-amino-5-bromo-6-(2,5-difluorophenyl)pyrimidine-4(3H)one (ABDFPP) and 2-amino-5-iodo-6-(2,3-difluorophenyl)pyrimidin-4(3H)one (AIDFPP) were also of comparable potency in inhibiting MBT-2 growth and were more effective on mg/kg basis than both ABPP and ABMFPP. Treatment with ABDFPP or AIDFPP also resulted in long-term cures of up to 40% of mice. In this respect these latter two compounds were superior to treatment with 10 mg of polyriboinosinic-polyribocytidylic acid per kg, a treatment which reduced tumor size but had no effects on tumor incidence. The data suggest that tumors of bladder origin may be particularly sensitive to treatment with pyrimidinones.

Chemoprevention of 7,12-dimethylbenz(alpha)anthracene (DMBA) induced rat mammary tumors by 2-amino-5-bromo-6-phenyl-4(3H)-pyrimidinone (ABPP).

We have studied the chemopreventive effectiveness of 2-amino-5-bromo-6-phenyl-4(3H)-pyrimidinone (ABPP) in 7,12-dimethylbenz(alpha)anthracene (DMBA) induced rat mammary tumors. ABPP is a biological response modifier and has various biological activities, including interferon induction, immunomodulation, and antiviral and antineoplastic properties, both in vitro and in vivo. Fifty-day-old female Sprague-Dawley rats were randomized into two groups and all received 20 mg DMBA by gastric gavage. ABPP at 200 mg/kg weight was given to one group of rats three times a week for 4 weeks, beginning 6 h before DMBA. The other group of rats received the vehicle as controls. The rats were observed for 26 weeks, with weekly measurements of tumor development, size, and weight. Interferon titers were determined on all rats 6 h after the first dose of ABPP. After tumor induction, rats treated with ABPP persistently developed a fewer number of tumors on average than those in the control group. It was found that the mean sizes of total tumor mass per rat were consistently lower in the ABPP group starting 12 weeks after the tumor induction. This reduction of tumor size in ABPP-treated rats correlates significantly with the titers of interferon. Thus, our initial study demonstrates that ABPP seems to have chemopreventive potential, even with a brief duration of treatment, in DMBA-induced rat mammary cancers.

Pyrimidinones. 1. 2-Amino-5-halo-6-aryl-4(3H)-pyrimidinones. Interferon-inducing antiviral agents.

Interferon induction and antiviral activity was discovered with 2-amino-5-bromo-6-phenyl-4(3H)-pyrimidinone. An analogue study incorporating a series of 2-amino-5-substituted-6-arylpyrimidinones revealed that the most potent interferon inducers were mono- and difluorophenyl analogues. These same analogues were also potent antiviral agents against Semliki Forest virus and herpes simplex type 1. In addition the monomethoxyphenyl analogues were potent antiviral agents but weak interferon inducers. Relatively modest structural changes led to dramatic changes in bioactivity. There was a relatively poor correlation between levels of circulating interferons induced and systemic antiviral activity.

Benzisoxazolones: antimicrobial and antileukemic activity.

An unusual acid-mediated rearrangement of o-nitrostyrene oxide afforded 1-(hydroxymethyl)-2,1-benzisoxazol-3(1H)-one which exhibited broad-spectrum antimicrobial and cytotoxic activity. A number of analogues were prepared by employing a modified zinc-reduction procedure on o-nitrobenzoate. Several of these analogues exhibited interesting antipseudomonal activity in agar and broth but were ineffective in vivo.

Characterization of an adduct between CC-1065 and a defined oligodeoxynucleotide duplex.

CC-1065 is a potent antitumor antibiotic produced by Streptomyces zelensis. The drug binds covalently through N-3 of adenine and lies within the minor groove of DNA. Previous studies indicated that CC-1065 reacted with adenine in DNA to yield a thermally labile product that could be used to reveal its sequence specificity. These studies also provided insight into a DNA sequence (5'-CGGAGTTAGGGGCG-3') which should bind one molecule of CC-1065 in an unambiguous manner. This sequence, which contains the CC-1065 adenine binding site within the sequence 5'-TTA-3' was chemically synthesized together with the complementary strand. CC-1065 reacted with the oligoduplex to give an adduct that maintained the B-DNA form and had a final CD spectrum similar to those of the CC-1065 complexes formed with calf thymus DNA. The above 14mer was 5' end-labelled with 32P, annealed with its complementary strand, reacted with CC-1065 and heated. Drug-mediated strand breakage was evaluated on a sequencing gel. A single break occurred in the labelled strands to give a fragment that migrated as an 8.5mer; subsequent piperidine treatment produced a fragment that migrated as a 7mer, which is the size expected from the known binding of CC-1065 at adenine in 5'-TTA-3' sequences.

Characteristics of N-formyl-methionyl-leucyl-phenylalanine as an inducer of lysosomal enzyme release from human neutrophils.

N-formyl-methionyl-leucyl-phenylalanine (FMLP) stimulated a time- and concentration-dependent release of granule associated beta-glucuronidase and lysozyme but not cytoplasmic lactate dehydrogenase from human neutrophils. Maximum discharge of lysome activity released is insignificant when cells are not preincubated with cytochalasin B prior to being exposed to FMLP (10(-10)-10(-7) M); although 11.2 +/- 1.3 and 12.4 +/- 1.1% of total activity for beta-glucuronidase and lysozyme, respectively, is secreteer, had no effect on FMLP-elicited lysosomal enzyme extrusion. 8-(N,N-diethylamino)-octyl-3,4,5-trimethoxybenzoate hydrochloride (TMB-8), a purported antagonist of intracidating the role of calcium in the mechanism of lysosomal enzyme release.