Hypothalamic growth hormone (GH)-releasing hormone (GHRH) stimulates the synthesis and release of GH from the pituitary gland. barrier. Brain endothelial cells joined by tight junctions form the blood-brain barrier a “barrier” between the general circulation and the CNS. In this study we administered a GHRH antagonist (JV-1-42) and showed that after i.v. injection iodinated JV-1-42 (131I-JV-1-42) enters the brain intact at a rate of 0.8514 μl/g per min with a serum half-life of 12.2 min. A one-site binding hyperbolic model indicated that the maximal percent of i.v. dose taken up per gram of brain was 0.41%. Coinjection of unlabeled JV-1-42 indicated that the transport from blood to brain is not saturable; however transport from brain to blood is saturable and involves P-glycoprotein. Taken together these results demonstrate that i.v.-administered 131I-JV-1-42 readily crosses the blood-brain barrier and accumulates in Rabbit Polyclonal to TSEN54. the brain. This finding indicates that GHRH antagonists could provide a potential treatment for malignant glioblastomas. for 10 min. Aliquots of 100 μl were taken in triplicate from each phase and total radioactivity was measured for each aliquot in a gamma counter. The partition coefficient was calculated from the following formula: Determination of Rate of Clearance of 131I-JV-1-42 from the Serum. Male CD-1 mice (Charles River Breeding Laboratories) weighing 20-25 g were anesthetized with 0.2 ml of i.p. urethane. Next the skin from the neck was removed to expose the jugular vein and carotid artery. Each mouse was given an injection into the jugular vein of 0.2 ml of lactated Ringer’s solution (LR) (Baxter Health Care Deerfield IL) containing 1% BSA (LR-BSA) and 3 × 105 cpm of 131I-JV-1-42. Blood was collected from the carotid artery 2-120 min after i.v. injection the mouse was decapitated and the brain removed and weighed. The whole arterial blood was centrifuged at E-7050 (Golvatinib) 5 0 × for 10 min and the level of radioactivity determined in the serum and brain. To determine rate of clearance of 131I-JV-1-42 from the serum after i.v. injection the log of the injected dose in each milliliter of serum (log %Inj/ml) was plotted against time (min). Determination of Blood-to-Brain Influx Rate. The rate of uptake of 131I-JV-1-42 from the blood to the brain was determined by using multiple time regression analysis (23 24 For the purpose of this analysis a graph was created in which the brain/serum E-7050 (Golvatinib) ratios for time points ranging from 2 to 60 min after i.v. injection E-7050 (Golvatinib) were plotted against their respective exposure times (Expt). The slope of the linear portion of this relation represents the unidirectional influx rate (intercept represents the initial volume of distribution (is time is the level of radioactivity in the serum at time represents the brain/serum ratio at time = 0 was estimated in mice overdosed with urethane. These mice were killed and 15 min later injected with 131I-JV-1-42. Brains were removed from these mice 10 min after injection. The log of the mean E-7050 (Golvatinib) level of radioactivity in each brain was plotted against time (in minutes). Two sets of three mice were used at each of the five time points and the mean of the three mice in each set was used. Efflux curves were calculated with = 10 based on triplicates. To determine whether efflux of 131I-JV-1-42 was saturable 100 ng of unlabeled JV-1-42 per mouse was included in the injection. To determine the role of P-glycoprotein (P-gp) 100 ng of cyclosporin A per mouse or 5 nM verapamil per mouse was included in the injection per mouse to test for ability to inhibit influx. Brain samples were collected 10 min after injection and results are expressed as the mean percent of the intracerebroventricular (i.c.v.) injected dose taken up per gram of brain (%Inj/Brain). Capillary Depletion With and Without Vascular Washout. The capillary depletion method (26) as modified for mice (27) was performed to separate the cerebral capillaries from the parenchymal tissue so the compartmental distribution of 131I-JV-1-42 could be determined. Male CD-1 mice were anesthetized by i.p. injection of 0.2 ml of urethane. Each mouse received an injection of 3 × 105 cpm of 131I-JV-1-42 and 3 × 105 cpm 99mTc-albumin in 0.8 ml of LR-BSA into the jugular vein. Mice were then divided into two groups (washout or no washout). Next the abdomen was opened and arterial blood was collected from the abdominal aorta at.