Last Day Of Infusion Research Articles

The Effect of Allopregnanolone on Enzymatic Activity of the DNA Base Excision Repair Pathway in the Sheep Hippocampus and Amygdala under Natural and Stressful Conditions.

The neurosteroid allopregnanolone (AL) has many beneficial functions in the brain. This study tested the hypothesis that AL administered for three days into the third brain ventricle would affect the enzymatic activity of the DNA base excision repair (BER) pathway in the hippocampal CA1 and CA3 fields and the central amygdala in luteal-phase sheep under both natural and stressful conditions. Acute stressful stimuli, including isolation and partial movement restriction, were used on the last day of infusion. The results showed that stressful stimuli increased N-methylpurine DNA glycosylase (MPG), thymine DNA glycosylase (TDG), 8-oxoguanine glycosylase (OGG1), and AP-endonuclease 1 (APE1) mRNA expression, as well as repair activities for 1,N6-ethenoadenine (εA), 3,N4-ethenocytosine (εC), and 8-oxoguanine (8-oxoG) compared to controls. The stimulated events were lower in stressed and AL-treated sheep compared to sheep that were only stressed (except MPG mRNA expression in the CA1 and amygdala, as well as TDG mRNA expression in the CA1). AL alone reduced mRNA expression of all DNA repair enzymes (except TDG in the amygdala) relative to controls and other groups. DNA repair activities varied depending on the tissue—AL alone stimulated the excision of εA in the amygdala, εC in the CA3 and amygdala, and 8-oxoG in all tissues studied compared to controls. However, the excision efficiency of lesioned bases in the AL group was lower than in the stressed and stressed and AL-treated groups, with the exception of εA in the amygdala. In conclusion, the presented modulating effect of AL on the synthesis of BER pathway enzymes and their repair capacity, both under natural and stressful conditions, indicates another functional role of this neurosteroid in brain structures.

Humanized 3F8 Anti-GD2 Monoclonal Antibody Dosing With Granulocyte-Macrophage Colony-Stimulating Factor in Patients With Resistant Neuroblastoma

Chimeric and murine anti-GD2 antibodies are active against neuroblastoma, but the development of neutralizing antibodies can compromise efficacy. To decrease immunogenicity, hu3F8, a humanized anti-GD2 antibody, was constructed. To find the maximum-tolerated dose of hu3F8 with granulocyte-macrophage colony-stimulating factor. This phase 1 clinical trial used a 3 + 3 dose-escalation design in a single referral center (Memorial Sloan Kettering Cancer Center, New York, New York). Participants were enrolled from December 24, 2012, through May 3, 2016, with follow-up and analyses through February 28, 2018. Eligibility criteria included older than 1 year and resistant or recurrent neuroblastoma regardless of the number or kinds of prior treatments. All 57 participants met the eligibility criteria, received treatment according to the protocol, and were included in all analyses. Treatment cycles were monthly, if human antihuman antibody remained negative. Each cycle comprised hu3F8 infused intravenously for 30 minutes on Monday, Wednesday, and Friday as well as granulocyte-macrophage colony-stimulating factor administered subcutaneously daily from 5 days before infusion through the last day of infusion. After cycle 2, hu3F8 was increased to the highest dose level that had been confirmed as safe. Toxicity, pharmacokinetics, immunogenicity, and disease response. Of the 57 participants, 34 (60%) were male and 23 (40%) were female (male-to-female ratio of 1.5), with a median (range) age of 6.8 (2.4-31.3) years at enrollment and a median (range) time of 3.1 (0.6-9.0) years since initial chemotherapy. Participants received a median (range) of 4 (1-15) cycles. Treatment was outpatient with reversible neuropathic pain and without unexpected toxic effects. No maximum-tolerated dose was identified. Dose escalation was associated with increased serum levels and proceeded through dosage of 9.6 mg/kg/cycle (approximately 288 mg/m2), which is more than 2.5 times higher than the standard dosage of 75 mg/m2/cycle or 100 mg/m2/cycle of dinutuximab and m3F8. Human antihuman antibody positivity developed in 5 of 57 patients (9%) after cycle 1, including in 1 of 10 patients (10%) not previously treated with anti-GD2 antibody and in 4 of 47 patients (9%) previously exposed to 1 or 2 anti-GD2 antibodies. Antineuroblastoma activity included major responses associated with higher dosing and prolonged progression-free survival despite a history of relapses. This phase 1 clinical trial found hu3F8 to be associated with modest toxic effects, low immunogenicity, and substantial antineuroblastoma activity; phase 2 trials are in progress. ClinicalTrials.gov identifier: NCT01757626.

Abstract P312: A Novel Selective Melanocortin-4 Receptor Agonist Attenuates Bradycardia and Hyperglycemia in Diabetic Rats

We previously demonstrated that functional brain melanocortin 3/4 receptors (MC3/4R) are required for leptin to exert its chronic cardiovascular and antidiabetic actions. To determine if chronic treatment with a selective MC4R agonist reduces blood glucose and prevents bradycardia in diabetic rats, we used a novel compound with high affinity to MC4R. Male 12-week-old Sprague-Dawley rats (n=5/group) were instrumented with telemetry probes for determination of mean arterial pressure (MAP) and heart rate (HR) 24-hrs/day and an intracerebroventricular (ICV) cannula was placed in the brain lateral ventricle for continuous infusion of the selective MC4R agonist PL6214 (2.5 μg/hr) or MTII (a non-selective MC3/4R agonist, 10 ng/hr) via osmotic minipump. Induction of diabetes caused hyperphagia (20±1 to 32±2 g), hyperglycemia (89±3 to 494±44 mg/dl) and bradycardia (-47 bpm). Chronic infusion of PL6214 for 11 days transiently reduced food intake which returned to diabetic values by day 6 after starting the infusions, whereas chronic infusion of MTII caused a reduction in food intake lasting only 3-4 days. PL6214 reduced blood glucose by 63% on day 2 and 16% by day 11 of infusion, and prevented further bradycardia induced by diabetes (-35±14 bpm on the last day of infusion). Chronic MTII infusion reduced blood glucose by 31% on day 2 and by day 6 glucose levels had already returned to values observed before treatment was started. MTII infusion did not attenuate the bradycardia (-99±13 bpm on the last day of infusion). Diabetes did not alter MAP, while the MC4R agonist increased MAP by 5±1 mmHg compared to control values. These results indicate that selective activation of MC4R agonist attenuates bradycardia and hyperglycemia in type 1 diabetes, and may provide a new strategy for treatment of diabetes. (P20GM104357, NHLBI-PO1HL51971, AHA-SDG5680016 and Palatin Technologies).

Insensitivity of well-conditioned mature sheep to central administration of a leptin receptor antagonist

Ruminants remain productive during the energy insufficiency of late pregnancy or early lactation by evoking metabolic adaptations sparing available energy and nutrients (e.g. higher metabolic efficiency and induction of insulin resistance). A deficit in central leptin signaling triggers these adaptations in rodents but whether it does in ruminants remains unclear. To address this issue, five mature ewes were implanted with intracerebroventricular (ICV) cannula in the third ventricle. They were used in two experiments with an ovine leptin antagonist (OLA) when well-conditioned (average body condition score of 3.7 on a 5 point scale). The first experiment tested the ability of OLA to antagonize leptin under in vivo conditions. Ewes received continuous ICV infusion of artificial cerebrospinal fluid (aCSF), ovine leptin (4 µg/h) or the combination of ovine leptin (4 µg/h) and its mutant version OLA (40 µg/h) for 48 h. Dry matter intake (DMI) was measured every day and blood samples were collected on the last day of infusion. ICV infusion of leptin reduced DMI by 24% (P<0.05), and this effect was completely abolished by OLA co-infusion. A second experiment tested whether a reduction in endogenous leptin signaling in the brain triggers metabolic adaptations. This involved continuous ICV infusions of aCSF or OLA alone (40 µg/h) for 4 consecutive days. The infusion of OLA did not alter voluntary DMI over the treatment period or on any individual day. OLA did not affect plasma variables indicative of insulin action (glucose, non-esterified fatty acids, insulin and the disposition of plasma glucose during an insulin tolerance test) or plasma cortisol, but tended to reduce plasma triiodothyronine and thyroxine (P<0.07). Overall, these data show that a reduction of central leptin signaling has little impact on insulin action in well-conditioned mature sheep. They also raise the possibility that reduced central leptin signaling plays a role in controlling thyroid hormone production.

Serotonin (5‐HT) Transporter Ligands Affect Plasma 5‐HT in Rats

Dual dopamine (DA)/serotonin (5-HT)-releasing agents are promising candidate medications for stimulant addiction and other disorders. However, certain 5-HT transporter (SERT) substrates are associated with development of idiopathic pulmonary arterial hypertension (IPAH) and valvular heart disease (VHD). According to the "5-HT hypothesis," SERT substrates increase the risk for developing IPAH and VHD by increasing plasma 5-HT. To test this hypothesis directly, we determined the effects of acute and chronic fenfluramine, and other SERT ligands, on plasma 5-HT in male rats. For acute treatments, rats received i.v. vehicle or test drug (0.3 and 1.0 mg/kg), and serial blood samples were withdrawn. For chronic treatments, vehicle or test drug was infused via osmotic minipump (3 and 10 mg/kg/d) for 2 weeks. On the last day of infusion, rats received i.v. fenfluramine challenge (1 mg/kg), and serial blood samples were withdrawn. Plasma 5-HT was measured using ex vivo microdialysis in whole-blood samples. Baseline plasma 5-HT was <1.0 nM. Acute injection of fenfluramine or other SERT substrates caused large (up to 24-fold) dose-dependent increases in plasma 5-HT. Chronic fenfluramine at 3 and 10 mg/kg/d produced 1.7- and 3.5-fold increases in baseline plasma 5-HT, while chronic fluoxetine had no effect. Chronic infusions of fenfluramine or fluoxetine diminished the ability of acute fenfluramine to elevate dialysate 5-HT, and both drugs markedly reduced whole-blood 5-HT. Acute fenfluramine increases plasma 5-HT to concentrations that are below the micromolar levels necessary to produce adverse cardiovascular effects. Chronic fenfluramine and fluoxetine have minimal effects on plasma 5-HT, suggesting that the increased risk for IPAH associated with fenfluramine does not depend upon elevations in plasma 5-HT.

Effect of dexamethasone on pulmonary and renal angiotensin-converting enzyme concentration in fetal sheep during late gestation

Objectives The effect of dexamethasone on tissue angiotensin-converting enzyme (ACE) was investigated in fetal sheep. Study design Pulmonary and renal ACE concentrations were measured in 16 sheep fetuses at between 127 and 131 days of gestation (term 145±2 days): 6 were untreated, whereas 10 were chronically catheterized and infused intravenously with either saline solution (0.9%, n = 4) or dexamethasone (45-60 μg · kg −1 · d −1, n = 6) for the previous 2 days. The dexamethasone dose increased plasma dexamethasone to around one fifth of that measured in newborn human infants delivered after maternal dexamethasone treatment. Results Over the period of infusion, arterial blood pressure increased significantly in the dexamethasone (+6.8±1.5 mm Hg, P<.05) but not saline-treated fetuses (+1.6±0.6 mm Hg). At delivery, pulmonary ACE in the dexamethasone-infused fetuses (1.24±0.26 nmoles hippurate · min −1 · mg protein −1) was significantly greater than in the control fetuses (0.50±0.07 nmoles · min −1 · mg protein −1, P<.005); renal ACE was unchanged by dexamethasone treatment. Overall, pulmonary ACE and blood pressure were correlated on the last day of infusion ( r = 0.70, P<.05). Conclusion The rise in pulmonary ACE seen in dexamethasone-treated sheep fetuses may contribute, in part, to the glucocorticoid-induced increase in blood pressure.

Anti-interleukin 8 antibody abolishes effects of lipoid nephrosis cytokine.

Supernatant factor from peripheral blood mononuclear cell (PBMC) cultures of idiopathic minimal lesion nephrotic syndrome (IMLNS) patients in relapse induces in vivo albuminuria and increases 35sulfate uptake by glomerular basement membrane (GBM) in rats. The purpose of this study was to evaluate the effect of anti-interleukin 8 (IL8) neutralizing antibody on the effects induced by the supernatant factor. Supernatant factor collected from six cultures of PBMC from IMLNS patients in relapse were combined and aliquotted into two samples. Anti-IL8 neutralizing antibody (750 ng) was added to one. Supernatant factor or supernatant factor and anti-IL8 antibody were infused for 5 days into the left renal artery of Wistar rats using an osmotic pump. On the last day of infusion, rats were injected with 35sulfate (1.0 mCi/200 g) intraperitoneally and killed after 8 h. Glomeruli were isolated and GBM obtained. There was a significant increase in 35sulfate uptake of the infused kidney (169 +/- 52 cpm/mg dry glomerular weight, mean +/- SEM) compared with the uptake seen in the contralateral kidney (116 +/- 41, P < 0.05) when the supernatant factor was infused alone. No significant differences in 35sulfate incorporation were seen between infused kidney (173 +/- 5) and contralateral kidney (190 +/- 49) when supernatant factor and anti-IL8 antibody were administered. A significant increase in albuminuria was seen on the last day of infusion (0.43 +/- 0.11 albumin/ creatinine ratio, mean +/- SEM) compared with the ratio prior to infusion of the supernatant factor alone (0.18 +/- 0.03, P <0.05). No significant differences in urinary albumin/creatinine ratios prior to and on the 5th day of infusion were seen when the supernatant factor was administered with anti-IL8 antibody. Supernatant factor effects were abolished by the addition of anti-IL8 neutralizing antibody, suggesting that the described effects are mediated by IL8.

Effect of interleukin-8 on glomerular sulfated compounds and albuminuria.

To evaluate the effect of interleukin-8 (IL8) on glomerular basement membrane (GBM) sulfated compounds and albuminuria, we infused IL8 in 1% bovine serum albumin (BSA) for 5 days into the left renal artery of Holtzman male rats at the rate of 10 microliters/h using an osmotic pump. Control rats received 1% BSA. A significant increase in urinary albumin/creatinine ratio was seen on the last day of IL8 infusion (0.38 +/- 0.11, mean +/- SEM) when compared with albumin/creatinine ratio prior to infusion (0.19 +/- 0.04, P = 0.04). No significant differences in urinary albumin excretion prior to and after infusion of 1% BSA were observed. On the last day of infusion, rats were injected with 35sulfate (1.0 mCi/200 g body weight) intraperitoneally and killed after 8 h. Glomeruli were isolated and GBM obtained. After 5 days of IL8 administration, there was a significant increase in 35sulfate uptake by GBM of the infused kidney (76 +/- 10 cpm/dry glomerular weight, mean +/- SEM) compared with the uptake seen in the contralateral kidney (53 +/- 9, P = 0.05). The in vivo infusion of IL8 increased the 35sulfate uptake by GBM and augmented the urinary albumin/creatinine ratio, suggesting that IL8 may induce albuminuria by altering the metabolism of the GBM sulfated compounds. This hypothesis needs to be confirmed by studies on glomerular charge selectivity and GBM anionic sites during the course of the infusion. Moreover, the persistence of the effect needs to be evaluated by prolonging the infusion for more than 5 days.

Pharmacokinetics, metabolism and tumour disposition of 8-chloroadenosine 3',5'-monophosphate in breast cancer patients and xenograft bearing mice

8-Chloroadenosine 3',5'-monophosphate (8-Cl-cAMP) is undergoing phase I clinical trials as an anticancer drug. However, there is debate as to whether it is a prodrug for its 8-Cl-adenosine metabolite. Pharmacokinetics, metabolism and tumour disposition studies have been performed in 7 breast cancer patients receiving continuous infusion (28 day) 8-Cl-cAMP (0.54 or 1.08 mg/kg/day) and tumour biopsies were obtained before and on the last day of infusion. Parallel studies were performed in nude mice bearing the HT29 human colon cancer xenograft after continuous infusion (7 day) of active drug doses (50 or 100 mg/kg/day). Steady state plasma levels (Css) of 8-Cl-cAMP in patients ranged from 0.15-0.72 microM but 8-Cl-adenosine was not detected in plasma. In contrast, 8-Cl-cAMP was not detectable in 3 tumour biopsies but 8-Cl-adenosine was present in 2 samples at high concentrations (1.33 and 2.02 microM). In mice, Css of 8-Cl-cAMP ranged from 3.2-4.6 microM and 8-Cl adenosine was present in plasma only at the higher dose (100 mg/kg/day, peak concentration of 2.3 microM). In the HT29 xenograft, 8-Cl-cAMP levels were considerably lower than in plasma (0.37-1.22 microM) while 8-Cl-adenosine was present at 5.3-21.0 microM and 8-Cl-AMP was found at 11.3-35.7 microM. The fate of 8-Cl-cAMP in human tumours is characterised by extensive metabolism to products which are not generally observed in plasma. These data raise the possibility that 8-Cl-cAMP is a prodrug for a product of its metabolism in human tumours.

EXHAUSTION OF INSULOGENIC RESERVE IN MATURITY-ONSET DIABETIC PATIENTS DURING PROLONGED AND CONTINUOUS HYPERGLYCEMIC STRESS.

Solutions of 15 per cent glucose were administered by continuous intravenous infusion for five to seven days to six normal subjects, seven tolbutamide-responsive adult diabetics, and six tolbutamide-nonresponsive adult diabetics. Blood glucose concentration, plasma insulin activity, and net assimilation of carbohydrate were determined daily. An “insulogenic index,” relating level of circulating insulin activity to magnitude of glycemic stimulus, enabled comparison of the insulin-secreting capacity of respective groups. In nondiabetic subjects, mean daily blood glucose levels remained normal, and carbohydrate loads averaging 717 gm. per day were wholly retained. In both diabetic groups, increasing blood glucose and glycosuria permitted net glucose uptakes of only 88 per cent in tolbutamide-responders and 56 per cent in tolbutamide-nonresponders. Plasma insulin activity increased significantly in all groups. In both normal subjects and tolbutamide-responsive diabetics, enhanced levels appeared on the first day, were sustained at high titers each day thereafter, and exhibited mean maximal value on the last day of infusion. Conversely, in tolbutamide-nonresponsive diabetics, average maximal insulin activity was achieved on the second day, whereafter it declined progressively and sometimes disappeared completely. Compared to an insulogenic index of 100 per cent to represent the greatest secretory response of normal subjects, the corresponding maximal index was 49 per cent for tolbutamide-responders and 17 per cent for tolbutamide-nonresponders. On the last day of infusion, capacity for sustained insulin release was reflected by insulogenic indexes of 55 per cent, 32 per cent and 0.1 per cent in the respective groups. The data indicate that unremitting hyperglycemia tends to exhaust insulogenic reserve in adult-onset diabetics, and that betacytotropic collapse occurs most rapidly in patients with severest clinical intolerance to carbohydrate. This demonstration strengthens the view that maturity-onset diabetes and growth-onset diabetes represent different stages of the same disease. The findings suggest that the fundamental lesion in hereditary human diabetes is either primary beta cell deficiency or impaired responsiveness of peripheral tissues to the action of insulin—or, quite possibly, a combination of both.