Randomised controlled trial and parallel economic evaluation of conventional ventilatory support versus extracorporeal membrane oxygenation for severe adult respiratory failure (CESAR).

OBJECTIVES: To determine the comparative effectiveness and cost-effectiveness of conventional ventilatory support versus extracorporeal membrane oxygenation (ECMO) for severe adult respiratory failure. DESIGN: A multicentre, randomised controlled trial with two arms. SETTING: The ECMO centre at Glenfield Hospital, Leicester, and approved conventional treatment centres and referring hospitals throughout the UK. PARTICIPANTS: Patients aged 18-65 years with severe, but potentially reversible, respiratory failure, defined as a Murray lung injury score > or = 3.0, or uncompensated hypercapnoea with a pH < 7.20 despite optimal conventional treatment. INTERVENTIONS: Participants were randomised to conventional management (CM) or to consideration of ECMO. MAIN OUTCOME MEASURES: The primary outcome measure was death or severe disability at 6 months. Secondary outcomes included a range of hospital indices: duration of ventilation, use of high frequency/oscillation/jet ventilation, use of nitric oxide, prone positioning, use of steroids, length of intensive care unit stay, and length of hospital stay - and (for ECMO patients only) mode (venovenous/veno-arterial), duration of ECMO, blood flow and sweep flow. RESULTS: A total of 180 patients (90 in each arm) were randomised from 68 centres. Three patients in the conventional arm did not give permission to be followed up. Of the 90 patients randomised to the ECMO arm, 68 received that treatment. ECMO was not given to three patients who died prior to transfer, two who died in transit, 16 who improved with conventional treatment given by the ECMO team and one who required amputation and could not therefore be heparinised. Ninety patients entered the CM (control) arm, three patients later withdrew and refused follow-up (meaning that they were alive), leaving 87 patients for whom primary outcome measures were available. CM consisted of any treatment deemed appropriate by the patient's intensivist with the exception of extracorporeal gas exchange. No CM patients received ECMO, although one received a form of experimental extracorporeal arteriovenous carbon dioxide removal support (a clear protocol violation). Fewer patients in the ECMO arm than in the CM arm had died or were severely disabled 6 months after randomisation, [33/90 (36.7%) versus 46/87 (52.9%) respectively]. This equated to one extra survivor for every six patients treated. Only one patient (in the CM arm) was known to be severely disabled at 6 months. Patients allocated to ECMO incurred average total costs of 73,979 pounds compared with 33,435 pounds for those undergoing CM (UK prices, 2005). A lifetime model predicted the cost per quality-adjusted life-year (QALY) of ECMO to be 19,252 pounds (95% confidence interval 7622 pounds to 59,200 pounds) at a discount rate of 3.5%. Lifetime QALYs gained were 10.75 for the ECMO group compared with 7.31 for the conventional group. Costs to patients and their relatives, including out of pocket and time costs, were higher for patients allocated to ECMO. CONCLUSIONS: Compared with CM, transferring adult patients with severe but potentially reversible respiratory failure to a single centre specialising in the treatment of severe respiratory failure for consideration of ECMO significantly increased survival without severe disability. Use of ECMO in this way is likely to be cost-effective when compared with other technologies currently competing for health resources. TRIAL REGISTRATION: Current Controlled Trials ISRCTN47279827.

The INNOVO multicentre randomised controlled trial: neonatal ventilation with inhaled nitric oxide versus ventilatory support without nitric oxide for severe respiratory failure in preterm infants: follow up at 4-5 years.

BACKGROUND: Trials of inhaled nitric oxide (iNO) used short term in preterm infants with severe respiratory failure have to date shown no evidence of benefit, and there have been no trials reporting follow-up to 4 years of age. The INNOVO trial recruited 108 infants (55 iNO arm and 53 controls) from 15 neonatal units. By 1 year of age 59% had died, and 84% of the survivors had signs of impairment or disability. OBJECTIVE: This paper reports the long-term clinical effectiveness and costs of adding NO to the ventilator gases of preterm infants with severe respiratory failure. PATIENTS AND METHODS: Children were assessed at age 4-5 years by interview, examination, cognitive and behavioural assessments. The outcome data were divided into seven domains and were described as normal, impaired or disabled (mild, moderate or severe) by the degree of functional loss. RESULTS: 38 of the 43 survivors had follow-up assessments. In the iNO group 62% (34/55) had died or were severely disabled, compared to 70% (37/53) in the no iNO group (RR 0.89, 95% CI 0.67 to 1.16). There was no evidence of difference in the levels of impairment or disability between the two groups in any of the domains studied, or of cost differences, amongst the survivors. CONCLUSION: For this group of babies with severe respiratory failure there was no evidence of difference in the longer-term outcome between those babies allocated to iNO and those who were allocated to no iNO. The challenge is to identify those premature babies who are able to respond to NO with clinically important health improvements. TRIAL REGISTRATION NUMBER: 17821339.

Neonatal ventilation with inhaled nitric oxide vs. ventilatory support without inhaled nitric oxide for infants with severe respiratory failure born at or near term: the INNOVO multicentre randomised controlled trial.

BACKGROUND: Evidence from European centres to support the use of nitric oxide (NO) in mature newborns with evidence of severe respiratory failure is sparse. METHODS: Infants of >33 weeks' gestation, <28 days old, and with severe respiratory failure requiring ventilatory support were randomised to receive or not to receive inhaled NO (iNO). The study was not blinded. RESULTS: Sixty infants were recruited (29 allocated iNO, 31 no iNO) from 15 neonatal units in the UK, Finland, Belgium and the Republic of Ireland. 15/60 recruited babies died, and 8.1% of the survivors (4/45) were classified as severely disabled at 1 year. There was no statistically significant difference between the randomised groups in terms of the primary outcome of death or severe disability by the corrected age of 1 year (relative risk = 0.96 (95% confidence interval = 0.46-2.03); p = 0.86) (Fisher's exact p = 1.00). The costs of NO were outweighed by reduced extra corporeal membrane oxygenation costs in the iNO group. The mean total hospitalisation costs were lower in the iNO group, although the mean difference (1,697 pounds) was not statistically significant (95% confidence interval = -14,472 to 11,478). CONCLUSIONS: The results complement those of previous studies that suggest NO is cost-effective and reduces the need for extra corporeal membrane oxygenation in this group of babies. Overall survival rates compare unfavourably with results of US trials.

Neonatal Ventilation With Inhaled Nitric Oxide Versus Ventilatory Support Without Inhaled Nitric Oxide for Preterm Infants With Severe Respiratory Failure: the INNOVO multicentre randomised controlled trial (ISRCTN 17821339).

BACKGROUND: Although inhaled nitric oxide (iNO) may be a promising treatment for newborn infants with severe respiratory failure, the results from 3 previous small trials were inconclusive. METHODS: Infants of <34 weeks' gestation, <28 days old, and with severe respiratory failure requiring ventilatory support were randomized to receive or not receive iNO. The study was not blinded. FINDINGS: Recruited were 108 infants (55 allocated to receive iNO and 53 not allocated to receive iNO) from 15 neonatal units in the United Kingdom and Republic of Ireland. Fifty-nine percent (64 of 108) died, and 84% of the survivors (37 of 44) had signs of some impairment or disability, 9 (20%) of them classified as severely disabled. There was no evidence of an effect of iNO on the primary outcomes: death or severe disability at 1 year corrected age (relative risk [RR]: 0.99; 95% confidence interval [CI]: 0.76 to 1.29); death or supplemental oxygen on expected date of delivery (RR: 0.84; 95% CI: 0.68 to 1.02); or death or supplemental oxygen at 36 weeks' postmenstrual age (RR: 0.98; 95% CI: 0.87 to 1.12). There was a trend for infants allocated to the iNO group to spend more time on the ventilator (log rank: 3.6), on supplemental oxygen (log rank: 1.4), and in hospital (log rank: 3.5) than those allocated to receive no iNO. This pattern predominantly reflected the infants who died. Mean total costs at 1 year corrected age were significantly higher in the iNO group, partly because of the costs of the gas but mainly because of the difference in initial hospitalization costs. INTERPRETATION: Evidence of prolongation of intensive care and increased costs of such care, without clear beneficial effects, implies that iNO cannot be recommended for preterm infants with severe hypoxic respiratory failure.

Antitumour efficacy of VEGFR2 tyrosine kinase inhibitor correlates with expression of VEGF and its receptor VEGFR2 in tumour models.

During the development of indazolylpyrimidines as novel and potent inhibitors of vascular endothelial growth factor (VEGF) receptor-2 (VEGFR2) tyrosine kinase, we observed that some human tumour xenografts are more sensitive to VEGFR2 kinase inhibitors than others. A better understanding of the basis for this differential response may help to identify a predictive marker that would greatly aid in the identification of a suitable patient population for treatment. One representative compound from the indazolylpyrimidine series is GW654652 that inhibited all three VEGFRs with similar potency. The inhibition of VEGFR2 kinase by GW654652 was about 150 to >8800 more potent than the inhibition of eight other kinases tested. GW654652 inhibited VEGF- and bFGF-induced proliferation in endothelial cells with an IC(50) of 110 and 1980 nM, respectively, and has good pharmacokinetic profile in mouse and dog. We investigated the association between VEGF and VEGFR2 expression and the antitumour efficacy of GW654652, in various xenograft models. Statistically significant associations were observed between the antitumour efficacy of GW654652 in xenografts and VEGF protein (P=0.005) and VEGFR2 expression (P=0.041). The oral dose of GW654652 producing 50% inhibition of tumour growth (ED(50)) decreased with increasing levels of VEGF (r=-0.94); and, in contrast, the ED(50) increased with the increased expression of VEGFR2 (r=0.82). These results are consistent with the observed inverse correlation between VEGF and VEGFR2 expression in tumours. These findings support the hypothesis that VEGF and VEGFR2 expression by tumours may predict the therapeutic outcome of VEGFR kinase inhibitors.

Tacrolimus versus microemulsified ciclosporin in liver transplantation: the TMC randomised controlled trial.

BACKGROUND: Calcineurin inhibitors are the most commonly used immunosuppressive drugs in liver transplantation, but the optimum initial immunosuppression regimen is not known. The aim of our study was to compare tacrolimus with microemulsified ciclosporin, in a regimen with standardised concomitant immunosuppressive therapy. METHODS: In all liver transplant centres in the UK and Republic of Ireland, 606 patients undergoing a first orthotopic liver transplantation were randomly assigned open-label tacrolimus or microemulsified ciclosporin. Primary outcome was the combined frequency (whichever occurred first) of death, retransplantation, or treatment failure for immunological reasons, analysed by intention to treat. FINDINGS: 96% of patients received the treatment allocated to them. The primary outcome was reached in 62 (21%) of 301 patients in the tacrolimus group versus 99 (32%) of 305 allocated microemulsified ciclosporin (relative risk 0.63 [95% CI 0.48-0.84], p=0.001; time-to-event analysis log-rank test p=0.002): deaths (50 [17%] vs 72 [24%]); retransplantations (11 [4%] vs 31 [10%]) treatment failure for immunological reasons (6 [2%] vs 12 [4%]). The relative risk for the composite outcome was in favour of tacrolimus. The main causes of death in both trial groups were sepsis and multiple organ failure (31 [10%] vs 30 [10%]), and the main cause for retransplantation was hepatic artery thrombosis (6 [2%] vs 17 [6%]). Renal dysfunction and the need for antihypertensive therapy were much the same in both groups. Tacrolimus was more diabetogenic. INTERPRETATION: Clinical outcome at 1 year was better with tacrolimus-based immunosuppression than with microemulsified ciclosporin during the first year after liver transplantation. Tacrolimus should be the first choice of calcineurin inhibitor for patients receiving their first liver graft.

Involving consumers in designing, conducting, and interpreting randomised controlled trials: questionnaire survey.

OBJECTIVE: To assess the extent to which consumers are involved in the work of clinical trial coordinating centres in the United Kingdom and the nature of consumers' involvement in randomised trials coordinated by these centres. DESIGN: National surveys using structured questionnaires with some open ended sections. SETTING: 103 clinical trial coordinating centres in the United Kingdom identified through a database assembled in 1997 by the NHS clinical trials adviser. PARTICIPANTS: Named contacts at 62 coordinating centres and investigators in 60 trials that were identified as involving consumers. MAIN OUTCOME MEASURES: Number of coordinating centres and number of trials in which consumers were involved and the nature of consumers' involvement. RESULTS: Of the 62 eligible centres, 23 reported that consumers had already been involved in their work, and most respondents were positive about this involvement. 17 centres planned to involve consumers. 15 centres had no plans to involve consumers, but only four of these considered such involvement irrelevant. Responses from investigators about the 48 individual trials were mostly positive, with respondents commenting that input from consumers had helped refine research questions, improve the quality of patient information, and make the trial more relevant to the needs of patients. CONCLUSIONS: Consumer involvement in the design and conduct of controlled trials seems to be growing and seems to be welcomed by most researchers. Such involvement seems likely to improve the relevance to consumers of the questions addressed and the results obtained in controlled trials.

The Ipswich childbirth study: one year follow up of alternative methods used in perineal repair.

OBJECTIVE: To assess the long term implications of four alternative approaches to postpartum perineal repair. DESIGN: A stratified randomised controlled trial using a 2x2 factorial design. SETTING: Original recruitment at the Maternity Unit at Ipswich Hospital NHS Trust, a district general hospital, between 1992 and 1994. SAMPLE: Seven hundred and ninety three women who had participated in the Ipswich Childbirth Study--a trial among women who had required repair of episiotomy or first or second degree tears--at least one year previously. METHODS: Self-completed postal follow up at least one year after recruitment to trial comparing 1. two-stage repair leaving the skin unsutured with standard three-stage repair, and 2. polyglactin 910 with chromic catgut as suture material for the repair. MAIN OUTCOME MEASURES: Failure to resume pain-free intercourse; persistent perineal pain; perineum feeling different; resuturing; time to resume pain-free intercourse; and dyspareunia. RESULTS: Fewer women allocated two-stage repair reported that the perineum felt different (30% versus 40%; RR 0.75; 95% CI 0.61 to 0.91; 2P < 0.01); otherwise there were no clear differences between the two methods. Women allocated polyglactin 910 were less likely to have dyspareunia (8% versus 13%; RR 0.59, 95% CI 0.39 to 0.91; 2P = 0.02) and less likely to fail to resume pain-free intercourse (8% versus 14%; RR 0.57, 95% CI 0.38 to 0.87; 2P < 0.01). CONCLUSION: Two-stage repair of perineal trauma leaving the skin unsutured appears to reduce the likelihood of the perineum feeling different from before delivery, in addition to less pain and dyspareunia initially; there were no apparent disadvantages. Polyglactin 910 reduces dyspareunia long term, indicating that the short term benefits of this material over chromic catgut persist.

The Ipswich Childbirth Study: 2. A randomised comparison of polyglactin 910 with chromic catgut for postpartum perineal repair.

OBJECTIVE: To compare polyglactin 910 sutures with chromic catgut sutures for postpartum perineal repair. DESIGN: A stratified randomised controlled trial, using a 2 x 2 factorial design. SETTING: The maternity unit at Ipswich Hospital NHS Trust, a district general hospital, between 1992 and 1994. SAMPLE: 1780 women who had sustained an episiotomy or first or second degree tear following a spontaneous or simple instrumental delivery. METHODS: Policies of repair with polyglactin 910 or chromic catgut were compared. Both groups were assessed by a research midwife completing questionnaires at 24 to 48 hours and at ten days postpartum, and by self-completed questionnaires at three months after birth. MAIN OUTCOME MEASURES: 1. 24 to 48 hours postpartum: perineal pain, healing; 2. ten days postpartum: perineal pain, healing and removal of sutures; 3. three months postpartum: perineal pain, removal of sutures, resuturing, dyspareunia, and failure to resume pain-free intercourse. RESULTS: Completed questionnaires were returned for 99% of women at both 24 to 48 hours and ten days and by 93% of women three months postpartum. The two groups were similar at trial entry. Significantly fewer women allocated to the polyglactin 910 reported pain in the previous 24 hours at both 24 to 48 hours (59% vs 67%; RR 0.89, 95% CI 0.83-0.95; 2P < 0.01), and ten days (24% vs 29%; RR 0.81, 95% CI 0.69-0.95; 2P = 0.01). At three months postpartum there was no clear difference between the groups in terms of perineal pain, dyspareunia or failure to resume pain-free intercourse. More women in the polyglactin 910 group reported that some suture material had been removed (12% vs 7%; RR 1.62, 95% CI 1.19-2.21; 2P < 0.01). Three women in the polyglactin 910 group had required resuturing compared with ten in the chromic catgut group (RR 0.30; 95% CI 0.08-1.09; 2P = 0.1). CONCLUSIONS: Using polyglactin 910 rather than chromic catgut for perineal repair leads to about one fewer women among every 20 having perineal pain and using analgesia ten days postpartum. Its only apparent disadvantage is that more women, again estimated as 1 in 20, report having material removed during healing. Data from this and other trials suggest that for every 100 women repaired with a polyglycolic acid-based material, about one fewer will require resuturing.

The Ipswich Childbirth Study: 1. A randomised evaluation of two stage postpartum perineal repair leaving the skin unsutured.

OBJECTIVE: To evaluate a policy of two stage postpartum perineal repair leaving the skin unsutured. DESIGN: A stratified randomised controlled trial using a 2 x 2 factorial design. SETTING: The maternity unit at Ipswich Hospital NHS Trust, a district general hospital, between 1992 and 1994. SAMPLE: 1780 women requiring surgical repair of episiotomy or first or second degree tear following a spontaneous or simple instrumental delivery. METHODS: A policy of two-stage perineal repair leaving skin unsutured was compared with a policy of three stage repair including skin closure with interrupted or subcuticular sutures. Both groups were assessed by a research midwife, blind to the allocation, completing questionnaires at 24 to 48 hours and 10 days postpartum, and by self-completed questionnaires at three months after birth. MAIN OUTCOME MEASURES: 1. 24 to 48 hours postpartum: perineal pain; healing; 2. 10 days postpartum: perineal pain, healing and removal of sutures; 3. three months postpartum: perineal pain, removal of sutures, resuturing, dyspareunia, and failure to resume pain-free intercourse. RESULTS: Completed questionnaires were returned for 99% of women at both 24 to 48 hours and ten days and by 93% of women three months postpartum. No differences were detected in perineal pain at 24 to 48 hours (62% vs 64%; RR 0.96, 95% CI 0.90-1.03; 2P = 0.3) and 10 days (25% vs 28%; RR 0.90, 95% CI 0.77-1.06; 2P = 0.2). Significantly fewer women allocated to two-stage repair reported tight stitches at ten days (14% vs 18%; RR 0.77, 95% CI 0.62-0.96, 2P = 0.02); similar numbers of repairs were judged to be breaking down (five compared with seven women). At three months postpartum fewer women allocated to the two-stage repair reported perineal pain and more had resumed pain-free intercourse. Amongst women who had resumed intercourse there was a significant difference in dyspareunia (15% vs 19%; RR 0.80, 95% CI 0.65-0.99; 2P = 0.04). Significantly fewer women in the two-stage repair group (7% vs 12%; RR 0.61, 95% CI 0.45-0.83; 2P = < 0.01) reported removal of suture material. Four women in the two-stage repair group had required resuturing, compared with nine allocated to the three-stage repair. CONCLUSIONS: Two-stage repair of perineal trauma leaving the skin unsutured appears to reduce pain and dyspareunia three months postpartum. There are no apparent disadvantages, in particular no evidence of an increased risk of breakdown of the repair and resuturing.

New Sesquiterpene Lactones from Illicium floridanum

In continuation of our phytochemical investigation of Illicium floridanum Ellis (American star anise, star bush), three new sesquiterpene lactones possessing the anisatin-type carbon skeleton (8,9-seco-prezizaane skeleton), 14-acetoxy-3-oxofloridanolide (1), 13-acetoxy-14-(n-butyryloxy)floridanolide (2), and 3beta-acetoxy-14-n-butyryloxy-10-deoxyfloridanolide (3), were isolated from fruits of this plant. Their structures were elucidated by 1D and 2D NMR measurements. The molecular structure of 1 was obtained by single crystal X-ray diffraction. The 11,3-delta-lactone structure of the compound previously described as debenzoyldunnianin in our previous communication, on grounds of NMR spectral evidence and X-ray crystallographic analysis is revised to a delta-lactone closed between C-11 and C-7 (compound 4). The neurotoxic sesquiterpene lactone anisatin (5) and its isomer 2alpha-hydroxyneoanisatin (3-deoxy-2alpha-hydroxyanisatin, 6) were also isolated and identified by spectroscopic means. The presence of the neurotoxin 5 in relatively high amounts in the fruits and leaves confirms and explains early reports on the toxicity of this plant.

Active versus expectant management of third stage of labour: the Hinchingbrooke randomised controlled trial.

BACKGROUND: This study tested the hypotheses that active management of the third stage of labour lowers the rates of primary postpartum haemorrhage (PPH) and longer-term consequences compared with expectant management, in a setting where both managements are commonly practised, and that this effect is not mediated by maternal posture. BACKGROUND: 1512 women judged to be at low risk of PPH (blood loss >500 mL) were randomly assigned active management of the third stage (prophylactic oxytocic within 2 min of baby's birth, immediate cutting and clamping of the cord, delivery of placenta by controlled cord traction or maternal effort) or expectant management (no prophylactic oxytocic, no cord clamping until pulsation ceased, delivery of placenta by maternal effort). Women were also randomly assigned upright or supine posture. Analyses were by intention to treat. FINDINGS: The rate of PPH was significantly lower with active than with expectant management (51 [6.8%] of 748 vs 126 [16.5%] of 764; relative risk 2.42 [95% CI 1.78-3.30], p<0.0001). Posture had no effect on this risk (upright 92 [12%] of 755 vs supine 85 [11%] of 757). Objective measures of blood loss confirmed the results. There was more vomiting in the active group but no other important differences were detected. INTERPRETATION: Active management of the third stage reduces the risk of PPH, whatever the woman's posture, even when midwives are familiar with both approaches. We recommend that clinical guidelines in hospital settings advocate active management (with oxytocin alone). However, decisions about individual care should take into account the weights placed by pregnant women and their caregivers on blood loss compared with an intervention-free third stage.

Proteoglycan expression by human trabecular meshworks.

PURPOSE: Proteoglycans may serve important roles in trabecular meshwork structure or function. Detailed molecular characterization and identification of specific trabecular proteoglycan core proteins has been limited. METHODS: Radiolabeled proteoglycans were extracted from cultured human trabecular meshworks and subjected to ion exchange and molecular sieve chromatography. Peaks were subjected to glycosaminoglycan content analysis. Reverse transcription with polymerase chain reaction was used to identify trabecular mRNAs of several common proteoglycan core proteins. Western immunoblots of trabecular extracts were also utilized to identify these proteoglycan core proteins. RESULTS: The proteoglycans elute from ion exchange columns at high salt and molecular sieve column profiles, and they exhibit broad peaks typical of the proteoglycan microheterogeneity seen in other tissues. The four common glycosaminoglycan side-chains were identified on these proteoglycans. Trabecular cells in organ or cell culture contain mRNAs coding for decorin, biglycan, versican, perlecan and a basement membrane glycoprotein, SPARC. Syndecan-1 transcripts were present at very low levels, while aggrecan transcripts were not detectable. Decorin, biglycan, versican and perlecan core proteins were also identified by immunoblots of trabecular cell extracts. CONCLUSIONS: Several common proteoglycans are expressed by trabecular cells in organ explant or cell culture. Their general characteristics are not unlike those found in other tissues. These proteoglycans may serve important functions in the trabecular outflow pathway.

Agouti regulation of intracellular calcium: role of melanocortin receptors.

Several dominant mutations at the murine agouti locus cause a syndrome of marked obesity and insulin resistance. We have recently reported that intracellular free Ca2+ concentration ([Ca2+]i) is elevated in viable yellow mice. Because [Ca2+]i has a key role in the pathogenesis of insulin resistance, obesity, and hypertension, the role of the purified agouti gene product in regulating [Ca2+]i was evaluated in a number of cell types. Purified murine agouti induced slow, sustained increases in [Ca2+]i in A7r5 vascular smooth muscle cells and 3T3-L1 adipocytes in a dose-dependent fashion. In L6 skeletal myocytes, agouti stimulated an increase in [Ca2+]i with an apparent concentration eliciting 50% of the maximal response (EC50) of 62 nM. This response was substantially inhibited by Ca2+ entry blockade with nitrendipine. To determine whether melanocortin receptors play a role in agouti regulation of [Ca2+]i, we examined the effect of melanocortin peptides and agouti in cells stably transfected with human melanocortin receptors. Human embryonic kidney cells (HEK-293 cells) transfected with either the human melanocortin 1 receptor (MC1R) or melanocortin 3 receptor responded to human agouti with slow, sustained increases in [Ca2+]i, whereas nontransfected HEK-293 cells with no melanocortin receptors did not respond to agouti. Dose-response curves in the MC1R line showed that agouti had an EC50 of 18 nM, which is comparable to that for agouti antagonism of (125)I-Nle,D-Phe-alpha-melanocyte-stimulating hormone binding in the same cell line. This direct effect of agouti on stimulating increases in [Ca2+]i suggests a potential mechanism for agouti-induced insulin resistance.

Mutations in the carboxyl terminus of the agouti protein decrease agouti inhibition of ligand binding to the melanocortin receptors.

Several mutations that cause ectopic expression of the agouti gene result in obesity, hyperinsulinemia, and yellow coat color. A candidate pathway for agouti induced obesity and hyperinsulinemia is through altered signaling by melanocortin receptors, as agouti normally regulates coat coloration through antagonism of melanocortin receptor 1. Furthermore, melanocortin peptides mediate functions including steroidogenesis, lipolysis, and thermoregulation. We report apparent inhibition dissociation constants for mouse and human agouti protein inhibition of ligand binding to the melanocortin receptors, to determine which of these receptors might be involved in agouti induced diabetes. The similarity in the apparent K(I) values for agouti inhibition of ligand binding to the brain melanocortin receptors 3 and 4 (mouse: K(I) app = 190 +/- 74 and 54 +/- 18 nM; human: K(I) app = 140 +/- 56 and 70 +/- 18 nM, respectively) suggests that the MC3-R is a potential candidate for a receptor mediating the effects of agouti protein overexpression. Agouti residues important for melanocortin receptor inhibition were identified through the analysis of deletion constructs and site-specific variants. Val83 is important for inhibition of binding to MC1-R (K(I) app for Val83Ala agouti increased 13-fold relative to wild-type protein). Arg85, Pro86, and Pro89 are important for selective inhibition of binding between MC1-R and MC3-R and MC4-R as their apparent K(I) values are essentially unchanged at MC1-R, while they have increased 6-10-fold relative to wild-type protein at MC3-R and MC4-R.

Agouti antagonism of melanocortin binding and action in the B16F10 murine melanoma cell line.

Several dominant mutations at the murine agouti locus result in the expression of a number of phenotypic changes, including a predominantly yellow coat color, obesity, and hyperinsulinemia. The mutants exhibit ectopic overexpression of normal agouti protein, suggesting that agouti regulates coat coloration by direct antagonism of the alpha-melanocyte-stimulating hormone receptor. We have tested this hypothesis by examining agouti inhibition of both melanocortin-stimulated cyclic adenosine monophosphate production and the binding of a radioactive melanocortin analog in the murine B16F10 melanoma cell line. Inhibition of melanocortin-induced cyclic nucleotide accumulation did not require preincubation of the cells with agouti and was independent of the agonist used. Furthermore, inhibition of both agonist binding to and activation of melanocortin receptor could be described by a simple competitive model with similar inhibition constants of 1.9 and 0.9 nM, respectively. The mutually exclusive binding of agouti and melanocortin was verified by cross-linking experiments using a radiolabeled alpha-melanocyte-stimulating hormone analog. Competitive inhibition of alpha-melanocyte-stimulating hormone binding can account for the effects of agouti on coat coloration and suggests the possibility that the other phenotypic changes observed on agouti overexpression may be due to direct action of agouti at a novel melanocortin receptor(s).

Plant antitumor agents. 30. Synthesis and structure activity of novel camptothecin analogs.

A large number of camptothecin (CPT) analogs have been prepared in the 20S, 20RS, and 20R configurations with a number of ring A substituents. Topoisomerase I (T-I) inhibition data (IC50) have been obtained by standard procedures. In general, substitution at the 9 or 10 positions with amino, halogeno, or hydroxyl groups in compounds with 20S configuration results in compounds with enhanced T-I inhibition. Compounds in the 20RS configuration were less active in vitro and in vivo and those in the 20R configuration were inactive. Compounds with 10,11-methylenedioxy substitution on ring A displayed a marked increase in potency in the T-I inhibition assay. The activities of some of the analogs as determined in a variety of in vivo assays including the L-1210 mouse leukemia assay were, in general, in accord with T-I inhibition. A number of water-soluble analogs such as 20-glycinate esters, 9-glycinamides, or hydrolyzed lactone salts were prepared and tested in in vitro and in vivo assays. In general, these compounds were less active than CPT both in terms of T-I inhibition and life prolongation in the L-1210 assay. However, certain 20-glycinate esters showed good in vivo activity after iv administration.

Adaptive immune defects and delayed rejection of allogeneic tumor cells in beige mice.

The effect of the beige (bg) mutation on adaptive allogeneic tumor rejection was examined by monitoring tumor cell survival in vivo using [131I]iododeoxyuridine-prelabeled cells. Accelerated elimination of allogeneic tumor cells normally begins 8 days after ip injection and is due to active immune responses. Two independent mutations to beige on two different inbred backgrounds (C57BL/6J bgJ and DBA/2JCo bg8J) were tested, and bg/bg mice showed a 1-day delay in immune elimination of allogeneic cells. This delayed rejection was not due to a defect in clearing label from dead cells, nor to an inability to effect antibody-induced killing in vivo. Both humoral and cell-mediated responses against the allogeneic tumor cells were significantly lower in bg/bg than in +/bg mice.