Blood-brain barrier permeability of novel [D-arg2]dermorphin (1-4) analogs: transport property is related to the slow onset of antinociceptive activity in the central nervous system

To clarify the pharmacological characteristics of Nalpha-amidino-Tyr-D-Arg-Phe-betaAla-OH (ADAB) and Nalpha-amidino-Tyr-D-Arg-Phe-MebetaAla-OH (ADAMB), mu1-opioid receptor-selective [D-Arg2]dermorphin tetrapeptide analogs, the plasma pharmacokinetics, and the in vivo blood-brain barrier (BBB) transport of these peptides were quantitatively evaluated. The mechanism responsible for the BBB transport of these peptides was also examined. The in vivo BBB permeation influx rates of 125I-ADAB and 125I-ADAMB after an i.v. bolus injection into mice were determined to be 0.0515 +/- 0.0284 microl/(min.g of brain) and 0.0290 +/- 0.0059 microl/(min.g of brain), respectively, both rates being slower than that of 125I-Tyr-D-Arg-Phe-betaAla-OH (125I-TAPA), a [D-Arg2]dermorphin tetrapeptide analog. To elucidate the BBB transport mechanism of ADAB and ADAMB, a conditionally immortalized mouse brain capillary endothelial cell line (TM-BBB4) was used as an in vitro model of the BBB. The internalization of both 125I-ADAB and 125I-ADAMB into cells was concentration-dependent with half-saturation constant (Kd) values of 3.76 +/- 0.83 and 5.68 +/- 1.75 microM, respectively. The acid-resistant binding of both ADAB and ADAMB was significantly inhibited by dansylcadaverine (an endocytosis inhibitor) and poly-l-lysine and protamine (polycations), but it was not inhibited by 2,4-dinitrophenol, or at 4 degrees C. These results suggest that ADAB and ADAMB are transported through the BBB with slower permeation rates than that of TAPA, and this is likely to be a factor in the slow onset of their antinociceptive activity in the central nervous system. The mechanism of the BBB transport of these drugs is considered to be adsorptive-mediated endocytosis.     

Transport of recombinant CD4 through the rat blood-brain barrier in vivo.

One class of potential acquired immunodeficiency syndrome therapeutics are derivatives of recombinant CD4 (rCD4). Therefore, the present investigations use in vivo techniques to measure the rate at which [3H]rCD4 is transported through the blood-brain barrier (BBB). In addition, the binding of labeled rCD4 to isolated human and bovine brain capillaries is measured. These studies show that [3H]CD4 is removed rapidly from the bloodstream with a half-time of 12.6 +/- 0.9 min. The volume of distribution (Vd) of the protein in brain increases with time and reaches a Vd that is 11.1 +/- 1.1-fold greater than the brain Vd of plasma marker, native rat serum albumin. In addition, [3H]rCD4 is extracted rapidly by the kidney and the ratio of rCD4 Vd to native rat serum albumin Vd in the rat kidney reaches 99 +/- 5 at 60 min after i.v. injection. rCD4 is shown to undergo transcytosis through the BBB using an internal carotid artery perfusion/capillary depletion method coupled with gel filtration fast protein liquid chromatography. In conclusion, these studies report the unexpected finding that rCD4 is transportable through the BBB. rCD4 is a cationic protein and the mechanism of rCD4 transport through the BBB may be analogous to the absorptive-mediated transcytosis of other polycationic proteins.     

Evaluation of cationized rat albumin as a potential blood-brain barrier drug transport vector

The present investigations evaluated the effects in rats of repetitive administration of cationized rat albumin over an 8-week period with the future aim of using this modified protein as a vector to transport drugs across the brain capillary endothelial wall, i.e., the blood-brain barrier. Rat albumin was cationized at pH = 7.8 with hexamethylenediamine, and the isoelectric point of the protein was raised from 5.5 to approximately 8. The cationized protein was monomeric based on mobility during sodium dodecylsulfate polyacrylamide gel electrophoresis. After radiolabeling, the cationized rat serum albumin (RSA) was taken up by isolated rat or bovine brain microvessels, whereas radio-labeled native RSA was not taken up by the capillaries in vitro. The brain volume of distribution of the 3H-cationized RSA increased linearly over a 5-hr period after an intravenous injection of the isotope and reached a value of 46 +/- 3 microliter/g (mean +/- S.E.) by 5 hr, whereas the brain volume of distribution of the 125I-native RSA was constant during the 5-hr time period (9.3 +/- 0.7 microliter/g, which is equal to the brain blood volume). The cationized and native RSAs were administered daily (Monday through Friday) at a dose of 1 mg/kg subcutaneously to groups of rats for 4- and 8-week periods. This dosage regimen resulted in no discernible toxicity, based on the findings of normal weight gain, normal tissue histology and normal serum chemistry. Therefore, cationized rat albumin may be used in future studies that use the repetitive administration of cationized rat albumin chimeric peptides for the evaluation of the transport of these substances through the blood-brain barrier in vivo.     

Differential effect of plasma protein binding of bupivacaine on its in vivo transfer into the brain and salivary gland of rats

The effect of plasma protein binding of bupivacaine on its transfer into brain and salivary gland was studied using bovine serum albumin, human alpha 1-acid glycoprotein (AAG) and human serum. The in vivo brain extraction and salivary gland extraction of [3H] bupivacaine relative to [14C]butanol were determined with an intracarotid injection technique used on rats. The brain extraction varied inversely with the bovine serum albumin (0-7.5%) and AAG (0-3.0 mg/ml) concentrations. The salivary extraction only slightly varied inversely with the AAG concentration, whereas no significant effects of bovine albumin binding on salivary gland uptake were observed. The in vivo percentage of exchangeable drug in brain or salivary gland capillaries was severalfold greater than the in vitro percentage of unbound drug. The percent values of free drug, brain exchangeable drug and salivary exchangeable drug were 12 +/- 1, 81 +/- 7 and 93 +/- 18% for umbilical cord serum, 8.6 +/- 1.1, 73 +/- 6 and 103 +/- 3% for normal human serum, 5.9 +/- 0.5, 60 +/- 4 and 89 +/- 3% for serum of rheumatoid arthritis patients and 5.0 +/- 0.2, 45 +/- 2 and 83 +/- 3% for serum of metastatic cancer patients. These data indicate that bupivacaine is not transported through the brain capillary wall, i.e., the blood-brain barrier, from the bovine albumin-bound pool, but bupivacaine is partially available for transfer from the circulating AAG-bound pool. However, both bovine albumin-bound and AAG-bound bupivacaine are readily available for transport through salivary gland capillaries.(ABSTRACT TRUNCATED AT 250 WORDS)     

Absorptive-mediated endocytosis of a dynorphin-like analgesic peptide, E-2078 into the blood-brain barrier

The binding and internalization of a novel analog of dynorphin-like analgesic basic peptide, [125I]E-2078 (CH3-[125I] Tyr-Gly-Gly-Phe-Leu-Arg-CH3Arg-D-Leu-NHC2H5), by isolated bovine brain capillaries were investigated. High-performance liquid chromatographic analysis showed that no significant metabolism of [125I] E-2078 occurred during incubation with brain capillaries for 30 min at 37 degrees C. The binding of [125I]E-2078 to brain capillaries increased with time and the steady-state cell-to-medium concentration ratio was 58.5 +/- 2.6 microliters/mg of protein. Approximately one-fourth of the [125I]E-2078 binding was resistant to acid wash, and showed significant dependence on temperature and medium osmolarity. The acid sensitive binding of [125I]E-2078, which presumably represents surface binding, was saturable and the Scatchard plot gave a maximal binding capacity Bmax = 147 +/- 29 pmol/mg of protein, and a half-saturation constant (KD) = 4.62 +/- 0.59 microM. Pretreatment of brain capillaries with phenylarsine oxide, an endocytosis inhibitor, completely suppressed the acid resistant binding of [125I]E-2078, but did not influence the surface binding of [125I]E-2078. The acid resistant binding of [125I] E-2078 was inhibited by poly-L-lysine and protamine, but not inhibited by insulin, transferrin, dynorphin (1-8), beta-neoendorphin, naloxone or poly-L-glutamate. Moreover, in vivo brain extraction of [125I]E-2078 in rats was 368 +/- 55% higher than that of [3H] sucrose and was significantly inhibited by 1 mM of unlabeled E-2078. These results demonstrate that E-2078 is internalized by brain capillaries via absorptive-mediated endocytosis, which is a polycation-sensitive pathway.     

Selective transport of an anti-transferrin receptor antibody through the blood-brain barrier in vivo

The brain capillary endothelium, which makes up the blood-brain barrier (BBB) in vivo, expresses high concentrations of transferrin receptor, and recent studies show that an antitransferrin receptor monoclonal antibody may function as a BBB drug transport vector. The present report examines the pharmacokinetics of clearance of radiolabeled antitransferrin receptor monoclonal antibody from the bloodstream in rats in vivo, and also assesses the extent to which brain selectively extracts the antibody from the blood compared to other peripheral organs such as liver, kidney, myocardium, or lung. [125I]Mouse immunoglobulin G2a control antibody was cleared monoexponentially with a half-time of 9.8 +/- 2.3 h. The clearance of the [3H]OX-26 antitransferrin receptor antibody from blood was biexponential with half-times of 2.2 +/- 0.8 min (61 +/- 10% of clearance) and 3.9 +/- 0.2 h (39 +/- 4% of clearance). The OX-26 antibody was rapidly taken up by liver during the first 60 min after injection, but this uptake reached rapid saturation, and hepatic OX-26 content actually declined subsequent to the first hour after injection. In contrast, brain continuously extracted the OX-26 antibody from the bloodstream, and the brain volume of distribution of OX-26 reached a value 18-fold greater than the volume of distribution of the mouse immunoglobulin G2a at 5 h after injection. There was no specific uptake of the OX-26 by myocardium or lung, and minor uptake by kidney was observed that also reached saturation within the first 60 min after injection.(ABSTRACT TRUNCATED AT 250 WORDS)     

Transport of Steroid Hormones through the Rat Blood-Brain Barrier: PRIMARY ROLE OF ALBUMIN-BOUND HORMONE

These studies were undertaken to investigate (a) the permeability properties of the blood-brain barrier (BBB) to the major gonadal and adrenal steroid hormones, and (b) the role of the binding proteins of plasma (albumin and specific globulins) in the regulation of BBB steroid hormone transport.The permeability of the BBB to [(3)H]-labeled progesterone, testosterone, estradiol, corticosterone, aldosterone, and cortisol, was measured relative to [(14)C]butanol, a freely diffusable reference, in the barbiturate anesthetized rat using a tissue sampling-single injection technique. The isotopes were rapidly injected in a 200-mul bolus of Ringer's solution (0.1 g/dl albumin) via the common carotid artery and the percent extraction of unidirectional influx of hormone was determined after a single pass through brain: progesterone, 83+/-4%; testosterone, 85+/-1%; estradiol, 83+/-3%; corticosterone, 39+/-2%; aldosterone, 3.5+/-0.8%; and cortisol, 1.4+/-0.3%. The selective permeability of the BBB was inversely related to the number of hydrogen bonds each steroid formed in aqueous solution and directly related to the respective 1-octanol/Ringer's partition coefficient.When the bolus injection was 67% human serum, >95% of the labeled steroid was bound as determined by equilibrium dialysis. However, the influx of the steroids through the BBB was inhibited by human serum to a much less extent than would be expected if only the free (dialyzable) hormone was transported; progesterone, estradiol, testosterone, and corticosterone transport was inhibited 18, 47, 70, and 85% respectively, or in proportion to the steroid binding to plasma globulins. Rat serum (67%) only inhibited the transport of these four hormones, 0, 13, 12, and 69%, respectively, reflecting the absence of a sex hormone-binding globulin in rat plasma. However, neonatal rat serum (67%) inhibited progesterone, testosterone, and estradiol transport 0, 0, and 91%, respectively, consistent with the presence of an estradiol-binding protein in neonatal rat serum.The binding of steroid hormone to bovine albumin in vitro (as determined by equilibrium dialysis) was compared to albumin binding in vivo (as determined by the single injection technique). The ratio of apparent dissociation constant in vivo, K(D)(app), to the in vitro K(D) was: >200 for progesterone, >200 for testosterone, 120 for estradiol, and 7.7 for corticosterone. Assuming the steady-state condition, the K(D)(app)/K(D) was found to be proportional to the BBB permeability for each steroid.These data demonstrate (a) the selective permeability properties of the BBB to the major steroid hormones is proportional to the tendency of the steroid to partition in a polar lipid phase and is inversely related to the number of hydrogen bond-forming functional groups on the steroid nucleus; (b) the presence of albumin in serum may bind considerable quantities of steroid hormone, but exerts little inhibitory effects on the transport of steroids into brain, whereas globulin-bound hormone does not appear to be transported into brain to a significant extent. Therefore, the hormone fraction in plasma that is available for transport into brain is not restricted to the free (dialyzable) fraction, but includes the larger albumin-bound moiety.     

Transport mechanism of a new behaviorally highly potent adrenocorticotropic hormone (ACTH) analog, ebiratide, through the blood-brain barrier.

The binding and internalization of a novel adrenocorticotropic hormone (ACTH) analog having a potent neuromodulating effect, ebiratide (H-Met(O2)-Glu-His-Phe-D-Lys-Phe-NH(CH2)8NH2), by isolated bovine brain capillaries, were examined. Metabolism of [5-125I-His]ebiratide occurred during a 30-min incubation with bovine brain capillaries at 37 degrees C. In the presence of 20 mM EDTA, added to inhibit this metabolism, the medium, after 30 min of incubation, contained 82.3 +/- 0.5% of the unchanged ebiratide. The total binding and acid-resistant binding of [125I]ebiratide increased with time and reached an equilibrium at about 15 min. The total binding and acid-resistant binding at 30 min (as the cell/medium ratios corrected with [14C]sucrose) were 13.07 +/- 0.86 and 5.00 +/- 0.18 microliters/mg of protein, respectively. The acid-resistant binding showed significant dependence on temperature and medium osmolarity. The [125I]ebiratide binding was significantly inhibited by dansylcadaverine, an endocytosis inhibitor. The saturable acid-resistant binding was obtained by the addition of unlabeled ebiratide (100 nM-5 mM), and the maximal internalization capacity (Bmax) at 30 min was 144.2 pmol/mg of protein, with the half-saturation constant (KD) of 62.1 microM. The nonsaturable acid-resistant binding [cell/medium ratio in the presence of the unlabeled compound (1 mM or more)] was 2.2 microliters/mg of protein. The acid-resistant binding was significantly inhibited by human ACTH, poly-L-lysine, protamine and E-2078, a basic peptide, but was not inhibited by poly-L-glutamate, insulin or transferrin. These results demonstrate that ebiratide is transported through the blood-brain barrier via a basic peptide-specific absorptive-mediated endocytosis.     

Cationization of immunoglobulin G results in enhanced organ uptake of the protein after intravenous administration in rats and primate

Cationization of proteins in general enhances the cellular uptake of these macromolecules, and cationized antibodies are known to retain antigen binding properties. Therefore, cationized antibodies may be therapeutic and allow for "intracellular immunization." The present studies test the hypothesis that the tissue uptake of cationized immunoglobulin G (IgG) after intravenous administration may be greatly increased relative to the uptake of native proteins. The pharmacokinetics of cationized immunoglobulin G clearance from blood, and the volume of distribution of the cationized or native protein (albumin, IgG) for 10 organs was measured both in anesthetized rats and in an anesthetized adult Macaca irus cynomologous monkey. Initial studies on brain showed that serum factors inhibited uptake of 125I-cationized IgG, but not 3H-cationized IgG. The blood-brain barrier permeability surface area product for 3H-cationized IgG was 0.57 +/- 0.04 microliters min-1 g-1. The ratio of the volume of distribution of the 3-H-cationized IgG compared to 3H-labeled native albumin ranged from 0.9 (testis) to 15.7 (spleen) in the rat at 3 hr after injection, and a similarly enhanced organ uptake was observed in the primate. In conclusion, these studies demonstrate that cationization of immunoglobulin greatly increases organ uptake of the plasma protein compared to native immunoglobulins, and suggest that cationization of monoclonal antibodies may represent a potential new strategy for enhancing the intracellular delivery of these proteins.     

Pharmacodynamic and pharmacokinetic characterization of poly(ethylene glycol) conjugation to met-enkephalin analog [D-Pen2, D-Pen5]-enkephalin (DPDPE).

Poly(ethylene glycol), or PEG, conjugation to proteins and peptides is a growing technology used to enhance efficacy of therapeutics. This investigation assesses pharmacodynamic and pharmacokinetic characteristics of PEG-conjugated [D-Pen2,D-Pen5]-enkephalin (DPDPE), a met-enkephalin analog, in rodent (in vivo, in situ) and bovine (in vitro) systems. PEG-DPDPE showed increased analgesia (i.v.) compared with nonconjugated form (p < 0.01), despite a 172-fold lower binding affinity for the delta-opioid receptor. [125I]PEG-DPDPE had a 36-fold greater hydrophilicity (p < 0.01) and 12% increase in the unbound plasma protein fraction (p < 0.01), compared with [(125)I]DPDPE. [125I]PEG-DPDPE had a 2.5-fold increase in elimination half-life (p < 0.01), 2.7-fold decrease in volume of distribution (p < 0.01), and a 7-fold decrease in plasma clearance rate (p < 0.01) to [125I]DPDPE. Time course distribution showed significant concentration differences (p < 0.01) in plasma, whole blood, liver, gallbladder, gastrointestinal (GI) content, GI tract, kidneys, spleen, urine, and brain (brain, p < 0.05), between the conjugated and nonconjugated forms. Increased brain uptake of [(125)I]PEG-DPDPE corresponded to analgesia data. [125I]PEG-DPDPE in brain was shown to be 58.9% intact, with 41.1% existing as [125I]DPDPE (metabolite), whereas [125I]DPDPE was 25.7% intact in the brain (at 30 min). In vitro P-glycoprotein affinity was shown for [125I]DPDPE (p < 0.01) but not shown for [125I]PEG-DPDPE. In vitro saturable uptake, with 100 microM DPDPE, was shown for [125I]PEG-DPDPE (p < 0.05). In this study, PEG-conjugated DPDPE seems to act as a prodrug, enhancing peripheral pharmacokinetics, while undergoing hydrolysis in the brain and allowing nonconjugated DPDPE to act at the receptor.     

Targeting rat anti-mouse transferrin receptor monoclonal antibodies through blood-brain barrier in mouse

Drug targeting through the brain capillary endothelium, which forms the blood-brain barrier (BBB) in vivo, may be achieved with peptidomimetic monoclonal antibodies that target peptide transcytosis systems on the BBB in vivo. Murine monoclonal antibodies to the rat transferrin receptor, such as the OX26 monoclonal antibody, are targeted through the BBB on the transferrin receptor in the rat. However, the present studies show the OX26 monoclonal antibody is not an effective brain delivery vector in mice. The emergence of transgenic mouse models creates a need for brain drug-targeting vectors for this species. Two rat monoclonal antibodies, 8D3 and RI7-217, to the mouse transferrin receptor were evaluated in the present studies. Both the RI7-217 and the 8D3 antibody had comparable permeability-surface area products at the mouse BBB in vivo. However, owing to a higher plasma area under the concentration curve, the mouse brain uptake of the 8D3 antibody was higher, 3.1 +/- 0.4% of injected dose [(ID)/g] compared with the brain uptake of the RI7 antibody, 1.6 +/- 0.2% ID/g, at 60 min after i.v. injection. Conversely, the mouse brain uptake of the OX26 antibody, which does not recognize the mouse transferrin receptor, was negligible, 0.06 +/- 0.01% ID/g. The RI7-127 antibody was more selective for brain because this antibody was not measureably taken up by liver. The capillary depletion technique demonstrated transcytosis of the RI7-217 antibody through the mouse BBB in vivo. The brain uptake of the 8D3 antibody was saturable, consistent with a receptor-mediated transport process. In conclusion, these studies indicate rat monoclonal antibodies to the mouse transferrin receptor may be used for brain drug-targeting studies in mice such as transgenic mouse models.     

Hepatic bilirubin uptake in the isolated perfused rat liver is not facilitated by albumin binding.

Bilirubin uptake by the liver is a rapid process of high specificity that has kinetic characteristics which suggest carrier-mediation. In the circulation, bilirubin is readily bound to albumin, from which it is extracted by the liver. Although several studies suggested that it is the small, unbound fraction of bilirubin which interacts with hepatocytes and is removed from the circulation, recent experiments have been interpreted as suggesting that binding to albumin facilitates ligand uptake. A liver cell surface receptor for albumin has been postulated. The present study was designed to examine directly whether albumin facilitates the hepatic uptake of bilirubin and whether uptake of bilirubin depends on binding to albumin. Rat liver was perfused with a protein-free fluorocarbon medium, and single-pass uptake of 1, 10, or 200 nmol of [3H]bilirubin was determined after injection as an equimolar complex with 125I-albumin, with 125I-ligandin, or free with only a [14C]sucrose reference. Uptake of 10 nmol of [3H]bilirubin was 67.5 +/- 3.7% of the dose when injected with 125I-albumin, 67.4 +/- 6.5% when injected with 125I-ligandin, and 74.9 +/- 2.4% when injected with [14C]sucrose (P greater than 0.1). At 200 nmol, uptake fell to 46.4 +/- 3.1% (125I-albumin) and 63.3 +/- 3.4% [( 14C]sucrose) of injected [3H]bilirubin (P less than 0.01), which suggests saturation of the uptake mechanism. When influx was quantitated by the model of Goresky, similar results were obtained. When [3H]bilirubin was injected simultaneously with equimolar 125I-albumin and a [14C]sucrose reference, there was no delay in 125I-albumin transit as compared with that of [14C]sucrose. This suggested that the off-rate of albumin from a putative hepatocyte receptor would have to be very rapid, which is unusual for high affinity receptor-ligand interaction. There was no evidence for facilitation of bilirubin uptake by binding to albumin or for interaction of albumin with a liver cell surface receptor. These results suggest that the hepatic bilirubin uptake mechanism is one of high affinity which can extract bilirubin from circulating carriers such as albumin, ligandin, or fluorocarbon.     

A simple ligand-binding assay for thyroxine-binding globulin on reusable Sephadex columns.

A method for the assay of thyroxine-binding globulin on reusable Sephadex G-25 (fine) columns is described. It depends upon elution by diluted iodothyronine-free serum of protein-bound [125 I]thyroxine from the columns under conditions where binding to thyroxine-binding prealbumin and albumin are abolished. It is simple, rapid and precise and permits determinations in large numbers of samples. Values (mg/l; mean +/- S.D.) were: normals 31.6 +/- 5.4, hyperthyroid 28.3 +/- 4.8, hypothyroid 40.6 +/- 7.5, oral contraceptives 40.1 +/- 6.8, pregnant 50.3 +/- 5.4, cirrhotics 20.7 +/- 4.3. Concentrations were reduced in serum heated at 56 degrees C, while the uptake of [125 I]triiodothyronine was increased. There was a significant negative correlation between thyroxine-binding globulin concentration and triiodothyronine uptake in the heated serum samples and in euthyroid subjects.     

The role and fate of rabbit and human transcobalamin II in the plasma transport of vitamin B12 in the rabbit.

Previous studies have shown that plasma transcobalamin II (TCII) facilitates the cellular uptake of [57Co] vitamin B12 (B12) by a variety of tissues, but the lack of an intrinsic label on the protein moiety of the TCII-B12 complex has made it impossible to determine the role and fate of TCII during this process. We have labeled homogensous rabbit and human TCII with 125I-labeled N-succinimidyl-3-(4-hydroxyphenyl) propionate and have performed in vivo experiments in rabbits. When 125I-labeled rabbit TCII-[57Co] B12 and 131I-labeled bovine albumin were simultaneously injected intravenously, we observed that 125Iand 57Co were cleared from plasma at a faster rate (t1/2 = 1 1/2 h) than 131I and that 125I and 57Co were present in excess of 131I in the kidney, liver, spleen, heart, lung, and small intestine 1/2 h after injection. Later, 57Co remained in excess of 131I, but the ratio of 125I to 131I decreased progressively in all of these plasma and were rapidly excreted in the urine. After 1 h following injection, 57Co was present in excess of 125I in the plasma...     

Mechanism of tubular uptake on human growth hormone in perfused rat kidneys

The mechanism of tubular uptake of labeled human growth hormone ([125I]hGH), a low molecular weight protein (approximate 21,5000 daltons), was studied in isolated perfused rat kidneys. Fractional reabsorption (FR) of [125I]hGH was decreased from 94 to 77% over a period of 80 min as perfusate oncotic pressure was lowered by reducing the albumin concentration from 7.5 to 2.5 g/100ml, whereas greater reductions in fractional sodium (delta 35%) and fluid reabsorption (delta 42%) occurred, indicating that tubular [125I]hGH uptake is likely a specific process not directly dependent upon net fluid and sodium reabsorption. Absolute absorption rates of [125I]hGH filtered loads were inhibited by cytochalasin B, a microfilament disrupter when kidneys were perfused with either albumin concentration. Cytochalasin B inhibited [125I]hGH absorption in both a dose-and time-related manner. The low dose of cytochalasin B (2.5 micrograms/ml) decreased [125I]hGH absorption without significantly altering sodium, fluid or glucose reabsorption. With high doses (5 and 10 micrograms/ml), cytochalasin B affected tubular absorption of [125I]hGH to an extent much greater than sodium, fluid and glucose reabsorption. Inhibition of cytochalasin B on FR[125I]hGH was poorly correlated with the concurrent inhibition of FRNa and FRH2O. Accordingly, tubular reabsorption of [125I]hGH is not directly linked to that of sodium, fluid and glucose. The present studies are consistent with the hypothesis that renal absorption of low molecular-weight proteins is via an endocytotic process involving microfilaments.     

Comparison of in vitro and in vivo models of drug transcytosis through the blood-brain barrier

Drug and solute transport through in vitro and in vivo models of the blood-brain barrier (BBB) were compared to provide a measure of how well the in vitro model predicted BBB permeability found in vivo. The in vitro model employed bovine brain capillary endothelial cells in either primary tissue culture or as a continuous line grown on Transwells and placed in side-by-side diffusion chambers. The in vivo model of BBB transport utilized an internal carotid artery perfusion/capillary depletion method in anesthetized rats. BBB permeability in vivo and in vitro was measured for 15 radiolabeled drugs and for L-[3H]dopa, D-[14C]glucose and [3H]albumin. [3H]- or [14C]sucrose was used in vivo as a blood volume reference. Lipid solubility of each drug was measured based on the 1-octanol/Ringer's partition coefficient. The morphology of the endothelial cell in primary tissue culture was spindle-shaped and the morphology of the endothelial cell in continuous culture was cuboid-shaped. The cuboidal morphology demonstrated a 2-fold greater resistance to solute transport and was used for the majority of the in vitro studies. Drug and solute permeability coefficients (Pe) ranged from 3.9 X 10(-3) to 2.5 X 10(-1) cm/min in vitro and from 1.0 X 10(-5) to 2.1 X 10(-2) cm/min in vivo. The In of the permeability.surface area product in vitro correlated with the In partition coefficient (r = 0.62, P less than .0125) and the In permeability.surface area product in vivo correlated with the In partition coefficient (r = 0.84, P less than .0005).(ABSTRACT TRUNCATED AT 250 WORDS)     

Evaluation of drug penetration into the brain: a double study by in vivo imaging with positron emission tomography and using an in vitro model of the human blood-brain barrier

The blood-brain barrier (BBB) permeabilities of 11 compounds were measured both in vitro with a newly developed coculture-based model of human BBB and in vivo with positron emission tomography (PET). The 11 compounds were fluoropyridinyl derivatives labeled with the positron-emitter fluorine-18, [(18)F]F-A-85380 [2-[(18)F]fluoro-3-[2(S)-2 azetidinylmethoxy]pyridine], and 10 selected N-substituted-azetidinyl and pyrrolidinyl closely related [(18)F]fluoropyridinyl derivatives (including [N'-aromatic/aliphatic]-thioureas, -ureas, and -amides). The in vitro BBB model, a new coculture system of primary human brain endothelial cells and astrocytes, was used to measure the permeability coefficient for each compound. Dynamic PET studies were performed in rats with the same compounds, and a two-compartment model analysis was used to calculate their in vivo permeability coefficients. The 11 derivatives differed in their degree of BBB passage and transport mechanism. The analysis of PET data showed a significant cerebral uptake for six derivatives, for which the in vitro evaluation indicated active influx or free diffusion. Five derivatives displayed low in vivo cerebral uptake, in agreement with the observation of an in vitro active efflux. Overall, there was a remarkable correlation between the in vitro and in vivo permeability coefficients (r = 0.99). This double study proves a close correlationship between the assessment of the BBB passage in vitro and in vivo. The in vitro model of human BBB offers the possibility of subtle discrimination of various BBB permeability degrees and transport mechanisms. Conversely, small animal PET imaging appears suitable to screen directly in vivo brain targeting of drugs or radiopharmaceutical candidates.     

Drug targeting of a peptide radiopharmaceutical through the primate blood-brain barrier in vivo with a monoclonal antibody to the human insulin receptor.

Peptide radiopharmaceuticals are potential imaging agents for brain disorders, should these agents be enabled to undergo transport through the blood-brain barrier (BBB) in vivo. Radiolabeled Abeta1-40 images brain amyloid in tissue sections of Alzheimer's disease autopsy brain, but this peptide radiopharmaceutical cannot be used to image brain amyloid in vivo owing to negligible transport through the BBB. In these studies, 125I-Abeta1-40 was monobiotinylated (bio) and conjugated to a BBB drug delivery and brain targeting system comprised of a complex of the 83-14 monoclonal antibody (mAb) to the human insulin receptor, which is tagged with streptavidin (SA). A marked increase in rhesus monkey brain uptake of the 125I-bio-Abeta1-40 was observed after conjugation to the 8314-SA delivery system at 3 h after intravenous injection. In contrast, no measurable brain uptake of 125I-bio-Abeta1-40 was observed in the absence of a BBB drug delivery system. The peptide radiopharmaceutical was degraded in brain with export of the iodide radioactivity, and by 48 h after intravenous injection, 90% of the radioactivity was cleared from the brain. In conclusion, these studies describe a methodology for BBB drug delivery and brain targeting of peptide radiopharmaceuticals that could be used for imaging amyloid or other brain disorders.     

Uptake and metabolism of thyroid hormones by the rat foetus in early pregnancy

Pregnant rats were injected with trace amounts of [125I]thyroxine [( 125I]T4) or [125I]-tri-iodothyronine [( 125I]T3). Maternal blood, organs and foetuses were removed 1 and 4 h later, homogenized and tri-chloroacetic acid added to precipitate radiolabelled iodothyronines. An hour after injecting [125I]thyroxine, 9-10 day old foetuses contained 21% of the radiolabel found in the same weight of plasma whereas by 13-14 days they contained 0.6%. An hour after injection of [125I]tri-iodothyronine, 9-10 day old foetuses contained 54% of the radioactivity present in the same weight of plasma. When related to tissue protein, the radiolabelled iodothyronine content in 9-10 day old foetuses was comparable with that in maternal brain, heart or ovary. De-iodination of [125I]thyroxine in 9-10 day old foetoplacental units was equal to that in maternal liver but greater than that in brain or heart. It is concluded that substantial amounts of thyroxine and tri-iodothyronine enter the rat foetus in early pregnancy but thyroxine uptake may be minimal in later pregnancy.     

Distribution of radioactivity in the spinal cord after intracerebroventricular and intravenous injection of radiolabeled opioid peptides in mice

The rejection of [beta-125I]endorphin and D-[3H]Ala2-Met-enkephalinamide into the lateral ventricles of mice revealed that radioactivity could be transported rapidly via the cerebrospinal fluid (CSF) down to the lowest part of the spinal cord. The maximal levels of both compounds in thoracic, lumbar and sacral sections of the spinal cord occurred 10 min after administration, which coincided with the peak antinociceptive action of morphine after i.c.v. administration. When [3H]Leu-enkephalin was administered i.c.v., the level of radioactivity in the spinal cord did not increase with time and these levels in spinal cord were considerably lower than those of either [beta125I]endorphin or D-[3H]Ala2-Met-enkephalinamide. This difference in distribution of these compounds, coupled with the observation that [beta-125I] endorphin and D-[3H]Ala2-Met-enkephalinamide were more stable than [3H]Leu-enkephalin in CSF, suggested that radioactivity in the spinal cord of mice treated with [beta-125I]endorphin or D-[3H]Ala2-Met-enkephalinamide was due to unchanged material. Very low levels of radioactivity were found in brain or spinal cord after i.v. administration of either [beta-125I]endorphin or D-[3H]Ala2-Met-enkephalinamide. This latter observation supports the view that endorphins administered i.c.v. enter the spinal cord directly through CSF rather than reentry from the general circulation. The results presented herein support the hypothesis that the antinociceptive action of morphine in mice as measured by the tail-flick procedure can be mediated via the release of endogenous compound(s) from brain into CSF which are transported to lower sections of the spinal cord where they inhibit the tail-flick response.