Selective heterocyclic amidine inhibitors of human inducible nitric oxide synthase.

The potency and selectivity of a series of 5-hetero-2-iminohexahydroazepines were examined as inhibitors of the three human NOS isoforms. The effect of ring substitution of the 5-carbon for a heteroatom is presented. Potencies (IC(50)'s) for these inhibitors are in the low micromolar range for hi-NOS with some examples exhibiting a 500x selectivity versus hec-NOS.

Attention to infants in the first year.

The time spent by 158 infants in contact with their carers at 6, 13, 26 and 52 weeks was reviewed prospectively. Periods of contact in the categories of (1) physical care, (2) holding the crying or sleeping infant, and (3) playing and interacting with the infant were recorded using 24-h log diaries completed by the mother. The mean total carer contact time over a 24-h day did not change significantly in the first year, varying between 6.5 and 73 h. Between 6 and 52 weeks, time spent by the mother in physical care declined significantly from 207 to 143 min and in holding the crying or sleeping infant from 61 to 17 min (P < 0.05 and 0.0001 respectively). There were no significant changes in the amount of time spent in playing and interacting with the infant over the first year by the mother and father, the time being on average 52.7 and 25.0 min respectively. Play and interaction with a non-parental carer increased significantly from 14 to 69 min (P < 0.0001). Relationships between infant size and holding became weaker as the infant became older. Infant gender, socioeconomic status and duration of breast-feeding did not influence infant contact time.

2-Iminopyrrolidines as potent and selective inhibitors of human inducible nitric oxide synthase.

A series of substituted 2-iminopyrrolidines has been prepared and shown to be potent and selective inhibitors of the human inducible nitric oxide synthase (hiNOS) isoform versus the human endothelial nitric oxide synthase (heNOS) and the human neuronal nitric oxide synthase (hnNOS). Simple substitutions at the 3-, 4-, or 5-position afforded more potent analogues than the parent 2-iminopyrrolidine 1. The effect of ring substitutions on both potency and selectivity for the different NOS isoforms is described. Substitution at the 4- and 5-positions of the 2-iminopyrrolidine yielded both potent and selective inhibitors of hiNOS. In particular, (+)-cis-4-methyl-5-pentylpyrrolidin-2-imine, monohydrochloride (20), displayed potent inhibition of hiNOS (IC50 = 0.25 microM) and selectivities of 897 (heNOS IC50/hiNOS IC50) and 13 (hnNOS IC50/hiNOS IC50). Example 20 was shown to be an efficacious inhibitor of NO production in the mouse endotoxin assay. Furthermore, 20 displayed in vivo selectivity, versus heNOS isoform, by not elevating blood pressure at multiples of the effective dose in the mouse.

2-Iminohomopiperidinium salts as selective inhibitors of inducible nitric oxide synthase (iNOS).

An attractive approach to the treatment of inflammatory conditions such as osteo- and rheumatoid arthritis, inflammatory bowel disease, and sepsis is through the selective inhibition of human inducible nitric oxide synthase (hiNOS) since localized excess nitric oxide (NO) release has been implicated in the pathology of these diseases. A series of monosubstituted iminohomopiperidinium salts possessing lipophilic functionality at ring positions 3, 5, 6, and 7 has been synthesized, and series members have demonstrated the ability to inhibit the hiNOS isoform with an IC50 as low as 160 nM (7). Compounds were found that selectively inhibit hiNOS over the human endothelial constitutive enzyme (heNOS) with a heNOS/hiNOS IC50 ratio in excess of 100 and as high as 314 (9). Potencies for inhibition of hiNOS and the human neuronal constitutive enzyme (hnNOS) are comparable. Substitution in the 3 and 7 positions provides compounds that exhibit the greatest degree of selectivity for hiNOS and hnNOS over heNOS. Submicromolar potencies for 6 and 7 in a mouse RAW cell assay demonstrated the ability of these compounds to inhibit iNOS in a cellular environment. These latter compounds were also found to be orally bioavailable and efficacious due to their ability to inhibit the increase in plasma nitrite/nitrate levels in a rat LPS model.

Substituted 2-iminopiperidines as inhibitors of human nitric oxide synthase isoforms.

A series of analogues of 2-iminopiperidine have been prepared and shown to be potent inhibitors of the human nitric oxide synthase (NOS) isoforms. Methyl substitutions on the 4-position (3) or 4- and 6-positions (8) afforded the most potent analogues. These compounds exhibited IC50 values of 0.1 and 0.08 microM, respectively, for hiNOS inhibition. Substitution with cyclohexylmethyl at the 6-position (13) afforded an inhibitor that showed the best selectivity for hiNOS versus heNOS (heNOS IC50/hiNOS IC50 = 64). Following oral administration, inhibitors were found to decrease serum nitrite/nitrate levels in an in vivo rat endotoxin assay. This series of 2-iminopiperidines were prepared via the described synthetic methodologies. The effect of ring substitutions on potency and selectivity for this class of cyclic amidines as NOS inhibitors is described.

Growth in weight, recumbent length, and head circumference for preterm low-birthweight infants during the first three years of life using gestation-adjusted ages.

Data from 867 preterm low-birthweight participants in the Infant Health and Development Program (IHDP) were used to develop reference data for growth status at an age and for increments from term to 36 month gestation-adjusted age (GAA). Weight, length and head circumference were recorded at 4 month intervals in the first year and at 6 month intervals in the second and third years. Selected percentiles for values at an age (status values) and increments for age intervals are presented in graphs separately for VLBW infants (< or = 1500 g at birth) and for LBW infants (1501-2500 g at birth). Percentiles of weight increments are presented beginning shortly before term for 1 month intervals to 6 month GAA, for 2 month intervals to 12 month GAA, and for 3-month intervals to 36 month GAA. Percentiles for length and head circumference increments are presented from term to 6 months for 2-month intervals, and to 36 month GAA for 3 month intervals. Among LBW infants, boys, had larger status and increment values than girls (P < 0.05), but there were no significant sex-associated differences in VLBW infants for status or increments. The mean status values and increments in weight and head circumference of the LBW infants were larger than those of VLBW infants, but the differences in length were not significant.

Synthesis and evaluation of two positron-labeled nitric oxide synthase inhibitors, S-[11C]methylisothiourea and S-(2-[18F]fluoroethyl)isothiourea, as potential positron emission tomography tracers.

In an effort to develop a tracer for probing inducible nitric oxide synthase (iNOS) levels in vivo utilizing positron emission tomography, we have synthesized and evaluated two positron-emitting iNOS selective inhibitors: S-[11C]methylisothiourea (1b) and S-(2-[18F]fluoroethyl)-isothiourea (3b). Prior to fluorine-18 labeling, the nonradioactive fluoro derivative S-(2-fluoroethyl)isothiourea (3a) was prepared and determined to have a 9-fold higher selectivity for iNOS compared to endothelial NOS (eNOS). Radiochemical synthesis of both compounds, in high radiochemical purity and at high specific activity, was accomplished by the S-alkylation reaction of labeled precursors (11CH3I or 18FCH2CH2OTf) with thiourea. An in vitro model, J774 macrophage cell line, was used to assess the uptake of radiolabeled iNOS inhibitor in response to iNOS induction at the cellular level. Increased cell uptake of these two labeled compounds at stimulated iNOS levels, as well as blocking under controlled in vitro conditions, was observed. Lipophilicity (log P o/w), stability, and tissue biodistribution data of both compounds are reported. Serum stability studies indicate that 3b metabolized much more rapidly compared to the relatively stable 1b in vitro and in vivo. Based on in vitro cell uptake data, both tracers were further evaluated in lipopolysaccharide (LPS)-pretreated rats. LPS has been reported to induce iNOS protein expression in the liver, lung, heart, and kidney and other tissues. The uptake for LPS-pretreated rats (6 h post-treatment) was significantly increased in the liver, kidney, and heart for 3b at 10 min and in the liver and lung for 1b at 30 min. The results suggest that this first generation of radiolabeled inhibitors may be useful for assessing induction of iNOS in vivo with PET.

Inhibitors of human nitric oxide synthase isoforms with the carbamidine moiety as a common structural element.

Identification of potent and selective inhibitors of inducible nitric oxide synthase (NOS) is of great interest because of their therapeutic potential for treatment of diseases mediated by excess production of nitric oxide. We present here a comparison of potency and selectivity for amino acid and nonamino acid based compounds as inhibitors of human inducible, human endothelial constitutive and human neuronal constitutive NOS isoforms. In addition, a novel series of substituted amidines has been identified as NOS inhibitors. 2-Methylthioacetamidine and 2-thienylcarbamidine were the most potent of the series examined with IC50 values of 3.9 and 2.9 microM for human neuronal constitutive NOS. Cyclopropylcarbamidine and 2-thienylcarbamidine were the most potent inhibitors for human inducible NOS with IC50 values of 5.2 and 6.5 microM, respectively. These substituted amidines represent a new class of NOS inhibitors and provide a foundation for potential therapeutic agents.

2-Iminopiperidine and other 2-iminoazaheterocycles as potent inhibitors of human nitric oxide synthase isoforms.

A series of 2-iminoazaheterocycles have been prepared and shown to be potent inhibitors of human nitric oxide synthase (NOS) isoforms. This series includes cyclic amidines ranging from five- to nine-membered rings, of which 2-iminopiperidine and 2-iminohomopiperidine were the most potent inhibitors, with IC50 values of 1.0 and 2.0 microM, respectively, for human inducible nitric oxide synthase. This series of cyclic inhibitors was further expanded to include analogs with heteroatoms in the 3-position of the six-membered ring. This modification was tolerated for sulfur and oxygen, but nitrogen reduced the inhibitory potency. The oral administration of 2-iminopiperidine in lipopolysaccharide (LPS)-treated rats inhibited the LPS-induced increase in plasma nitrite/nitrate levels in a dose-dependent manner, demonstrating its ability to inhibit inducible NOS activity in vivo. These cyclic amidines represent a new class of potent NOS inhibitors and the foundation for potential therapeutic agents.

Pneumococcal clearance function of the intact autotransplanted spleen.

OBJECTIVE: To determine if the venous drainage of the spleen into the portal circulation is essential for its ability to protect against encapsulated bacterial challenge. DESIGN AND INTERVENTIONS: Three groups of dogs were randomly assigned to undergo either sham laparotomy, splenectomy, or splenectomy with autotransplantation of the intact spleen into the pelvis and formation of vascular anastomoses to the iliac vessels. Two weeks post-operatively, the dogs received a sublethal intravenous injection of Streptococcus pneumoniae type 25. OUTCOME MEASURES: Bacterial clearances and inflammatory damage to the liver. Bacterial clearance of the autotransplanted spleen should be no different from that of a sham-operation spleen and significantly different from that of a splenectomized animal. In addition, immunologic function of the autotransplanted spleen should not differ from that of a sham-operation spleen in the degree of inflammatory damage to the liver. RESULTS: No differences in bacterial clearance function were found between the animals that had undergone sham laparotomy or splenic autotransplantation. However, bacterial clearance in the splenectomized animals was significantly impaired. Histologic examination of the liver 2 weeks after the bacterial challenge revealed high-grade inflammatory damage to the livers of splenectomized dogs, intermediate liver damage in dogs that underwent autotransplantation, and essentially no damage in dogs that underwent sham laparotomy. Autotransplanted spleens were essentially nonreactive, lacking actively proliferating germinal centers, whereas splenic tissue from sham-operation animals showed reactivity. CONCLUSION: Although bacterial clearance function is unchanged in autotransplanted spleens, this method still does not fully protect the liver from inflammatory damage.

Fetal and postnatal growth to age 2 years by mother's country of birth.

The objective of this study was to measure the physical growth of fetuses and infants in an inner city health district in the north of England and to compare their growth profiles according to mother's country of birth (British Isles or Indian subcontinent). The study was part of the Central Manchester Child Growth Project, a prospective longitudinal study of fetal and postnatal growth and development in a sample from the geographically-defined Central Manchester Health District. Data were collected from the beginning of the second trimester of pregnancy to the age of 2 years. One-hundred seventy-four singleton infants born at term ( > or = 37 weeks) had serial antenatal cephalometry every 3 weeks from the beginning of the second trimester and had serial head, length and weight measurements at birth and at the ages of 6, 13, 26, 52 and 104 weeks. Infants of Indian-born mothers tended to be lighter at birth than those of locally-born mothers, but the difference was not due to lower accumulation of soft tissue. Body length from 6 to 52 weeks in both groups of infants was similar. The major finding was the reduced head size in infants of Indian-born mothers, the difference being significant among boys, evident from mid-pregnancy and persisting postnatally to age 2 years. Reduced fetal growth is associated with the development of cardiovascular disease in adulthood, mortality from ischaemic heart disease being specifically linked with head size at birth. The reduced head size of boys of Indian-born mothers is of interest because male immigrants from the Indian subcontinent who live in England have an increased incidence of non-insulin dependent diabetes and a substantial excess mortality (standardised mortality ratio 313 at ages 20-29) from ischaemic heart disease.

Duration and pattern of crying in the first year of life.

In a prospective study of fetal and postnatal growth and development in a group of babies whose mothers were residents of an inner-city health district in the north of England, the total amount of crying of 157 infants was recorded at four periods during the first year of life by means of a 24-hour log. The mean number of crying episodes reduced from 4.4 at six weeks to 1.5 at one year. Early crying predicted later crying. It was not possible to predict which babies would cry a lot except that breast-fed infants tended to cry less. Mothers' perceptions of whether their babies cried a lot correlated with their perception of sleep difficulties. Rapid response to crying was associated with significantly less crying overall.

Potential role of nitric oxide in a model of chronic colitis in rhesus macaques.

BACKGROUND/AIMS: Excess nitric oxide formation, via the inducible NO synthase isoform, has been implicated in the pathogenesis of experimental and clinical inflammatory bowel disease. The aim of this study was to assess the site, enzyme source, and magnitude of NO production in juvenile rhesus macaques with idiopathic colitis. METHODS: NO production was assessed systemically from plasma and urine levels of reactive nitrogen intermediates and locally by the formation of [3H]citrulline from [3H]arginine and reduced nicotinamide adenine dinucleotide phosphate (NADPH) diaphorase histochemistry. Inducible NO synthase gene expression was assessed by reverse-transcription polymerase chain reaction. RESULTS: Plasma and urine levels of reactive nitrogen intermediates were greater in colitic animals than in control monkeys by 13- and 5-fold, respectively. NADPH diaphorase activity in normal animals was confined to the myenteric plexus. In colitis, staining was also apparent in crypt abscesses and superficial epithelial and mucosal bands. Gene expression for inducible NO synthase was only found in colitic specimens. Colonic [3H]citrulline formation was markedly elevated in colitic specimens, and the inducible isoform accounted for 58% of total activity. CONCLUSIONS: It is proposed that excess NO, formed via the inducible form of NO synthase, contributes to the mucosal inflammation and symptoms of this idiopathic colitis model.

Suppression of adjuvant-induced arthritis by selective inhibition of inducible nitric oxide synthase.

Adjuvant-induced arthritis is a model of chronic inflammation that exhibits several pathological changes similar to those occurring in rheumatoid arthritis, an autoimmune disease in humans characterized by chronic inflammation of the joints. We have examined the role of inducible nitric oxide synthase in producing the pathological changes associated with adjuvant-induced arthritis. Plasma nitrite concentrations were maximally elevated 14 days following adjuvant administration compared to untreated control animals. Arthritic changes in the paw were first observed between days 10-12 and were maximally elevated 21 days following adjuvant administration. Inducible nitric oxide synthase immunoreactivity was found localized in the synovial tissue from adjuvant-treated rats, while untreated controls exhibited no inducible nitric oxide synthase staining. Two selective inducible nitric oxide synthase inhibitors, aminoguanidine and N-iminoethyl-L-lysine, suppressed the increase in plasma nitrite levels and joint inflammation associated with adjuvant-induced arthritis in a dose-dependent manner. N-Iminoethyl-L-lysine attenuated the inducible nitric oxide synthase immunoreactivity in adjuvant-treated rats. Blood pressure was not affected by the highest dose of N-iminoethyl-L-lysine administered in the drinking water, indicating a lack of inhibition of constitutive nitric oxide synthase.

L-N6-(1-iminoethyl)lysine: a selective inhibitor of inducible nitric oxide synthase.

L-N6-(1-Iminoethyl)lysine (L-NIL) has been synthesized and is shown to be both a potent and selective inhibitor of mouse inducible nitric oxide synthase (miNOS). L-NIL has an IC50 of 3.3 microM for miNOS compared to an IC50 of 92 microM for rat brain constitutive NOS indicating that L-NIL is 28-fold more selective for inducible NOS. L-N5-(1-Iminoethyl)ornithine (L-NIO), which differs from L-NIL by having one less methylene group, has very similar potency for inducible NOS, but lacks selectivity. DL-N7-(1-Iminoethyl)homolysine was also synthesized and found to be substantially less potent than L-NIL or L-NIO, with intermediate selectivity for inducible NOS. These data suggest that L-NIL may be useful as a selective inhibitor of inducible NOS for determining the role of this enzyme in disease models.