Multiple-time Regression Research Articles

Blood-brain barrier transport kinetics of NOTA-modified proteins: the somatropin case.

Chemical modifications such as PEG, polyamine and radiolabeling on proteins can alter their pharmacokinetic behavior and their blood-brain barrier (BBB) transport characteristics. NOTA, i.e. 1,4,7-triazacyclononane-1,4,7-triacetic acid, is a bifunctional chelating agent that has attracted the interest of the scientific community for its high complexation constant with metals like gallium. Until now, the comparative BBB transport characteristics of NOTA-modified proteins versus unmodified proteins are not yet described. Somatropin (i.e. recombinant human growth hormone), NOTA-conjugated somatropin and gallium-labelled NOTA-conjugated somatropin were investigated for their brain penetration characteristics (multiple time regression and capillary depletion [CD]) in an in vivo mice model to determine the blood-brain transfer properties. The three compounds showed comparable initial brain influx, with Kin=0.38±0.14 µL/(g×min), 0.36±0.16 µL/(g×min) and 0.28±0.18 µL/(g×min), respectively. CD indicated that more than 80% of the influxed compounds reached the brain parenchyma. All three compounds were in vivo stable in serum and brain during the time frame of the experiments. Our results show that modification of NOTA as well as gallium chelation onto proteins, in casu somatropin, does not lead to a significantly changed pharmacokinetic profile at the blood-brain barrier.

Blood-brain barrier transport kinetics of the neuromedin peptides NMU, NMN, NMB and NT

The neuromedin peptides are peripherally and centrally produced, but until now, it is generally believed that they only function as locally acting compounds without any quantitative knowledge about their blood-brain barrier (BBB) passage. Here, we characterize the transport kinetics of four neuromedins (NMU, NMN, NMB and NT) across the BBB, as well as their metabolization profile, and evaluate if they can act as endocrine hormones.Using the in vivo mouse model, multiple time regression (MTR), capillary depletion (CD) and brain efflux studies were performed. Data was fitted using linear (NMU, NT and NMB) or biphasic modeling (NMU and NMN). Three of the four investigated peptides, i.e. NMU, NT and NMN, showed a significant influx into the brain with unidirectional influx rate constants of 1.31 and 0.75 μL/(g × min) for NMU and NT respectively and initial influx constants (K1) of 72.14 and 7.55 μL/(g × min) and net influx constants (K) of 1.28 and 1.36 × 10−16 μL/(g×min) for NMU and NMN respectively. The influx of NMB was negligible. Only NMN and NT showed a significant efflux out of the brain with an efflux constant (kout) of 0.042 min−1 and 0.053 min−1 respectively.Our results indicate that locally produced neuromedin peptides and/or fragments can be transported through the whole body, including passing the BBB, and taken up by different organs/tissues, supporting the idea that the neuromedins could have a much bigger role in the regulation of biological processes than currently assumed.

The blood-brain barrier permeability properties of plant N-alkylamides

Background: N-alkylamides (NAAs), a large group of secondary metabolites occurring in more than 25 plant families, are potential drug candidates as several biological and medical functions are known such as central nervous activities [1]. Objective: Our general goal was to investigate if NAAs with a different structure, e.g. spilanthol and pellitorine, are able to pass the blood-brain barrier (BBB) and if so, to what extent. Methods: A blood to brain (multiple time regression (MTR)), as well as a brain to blood (efflux) transport experiment were conducted in an in vivo mice model to investigate the initial BBB rate kinetics. The mice were anesthetized, after which the NAA dose solution was injected. Blood was obtained at regular time points after injection, and thereafter, the mice were decapitated and the brains collected. Quantification of the NAAs was done using a bio-analytical LC-MS method. Results: The MTR experiment indicated that spilanthol was able to cross the BBB in mice. A rapid but highly significant influx of spilanthol into the brains was observed with an unidirectional influx rate of 217.0 µl/(g×min). The curve reached a plateau-phase after about 10 minutes exposure time and can be explained by efflux of spilanthol out of the brain or distribution or elimination of spilanthol. The efflux transfer constant calculated for spilanthol was 0.1 min-1. This equals a brain half-time disappearance of 6.4 min. The comparative results of on-going BBB studies, including the permeability properties of pellitorine, will be reported as well. Seen the different possible pharmacological targets of these plant NAAs and their pharmaco-ethnological use, our BBB results may trigger further exploration into the medicinal use of these important plant metabolites.

Quorum Sensing Peptides Selectively Penetrate the Blood-Brain Barrier.

Bacteria communicate with each other by the use of signaling molecules, a process called ‘quorum sensing’. One group of quorum sensing molecules includes the oligopeptides, which are mainly produced by Gram-positive bacteria. Recently, these quorum sensing peptides were found to biologically influence mammalian cells, promoting i.a. metastasis of cancer cells. Moreover, it was found that bacteria can influence different central nervous system related disorders as well, e.g. anxiety, depression and autism. Research currently focuses on the role of bacterial metabolites in this bacteria-brain interaction, with the role of the quorum sensing peptides not yet known. Here, three chemically diverse quorum sensing peptides were investigated for their brain influx (multiple time regression technique) and efflux properties in an in vivo mouse model (ICR-CD-1) to determine blood-brain transfer properties: PhrCACET1 demonstrated comparatively a very high initial influx into the mouse brain (Kin = 20.87 μl/(g×min)), while brain penetrabilities of BIP-2 and PhrANTH2 were found to be low (Kin = 2.68 μl/(g×min)) and very low (Kin = 0.18 μl/(g×min)), respectively. All three quorum sensing peptides were metabolically stable in plasma (in vitro) during the experimental time frame and no significant brain efflux was observed. Initial tissue distribution data showed remarkably high liver accumulation of BIP-2 as well. Our results thus support the potential role of some quorum sensing peptides in different neurological disorders, thereby enlarging our knowledge about the microbiome-brain axis.

Cell-Penetrating Peptides Selectively Cross the Blood-Brain Barrier In Vivo.

Cell-penetrating peptides (CPPs) are a group of peptides, which have the ability to cross cell membrane bilayers. CPPs themselves can exert biological activity and can be formed endogenously. Fragmentary studies demonstrate their ability to enhance transport of different cargoes across the blood-brain barrier (BBB). However, comparative, quantitative data on the BBB permeability of different CPPs are currently lacking. Therefore, the in vivo BBB transport characteristics of five chemically diverse CPPs, i.e. pVEC, SynB3, Tat 47–57, transportan 10 (TP10) and TP10-2, were determined. The results of the multiple time regression (MTR) analysis revealed that CPPs show divergent BBB influx properties: Tat 47–57, SynB3, and especially pVEC showed very high unidirectional influx rates of 4.73 μl/(g × min), 5.63 μl/(g × min) and 6.02 μl/(g × min), respectively, while the transportan analogs showed a negligible to low brain influx. Using capillary depletion, it was found that 80% of the influxed peptides effectively reached the brain parenchyma. Except for pVEC, all peptides showed a significant efflux out of the brain. Co-injection of pVEC with radioiodinated bovine serum albumin (BSA) did not enhance the brain influx of radiodionated BSA, indicating that pVEC does not itself significantly alter the BBB properties. A saturable mechanism could not be demonstrated by co-injecting an excess dose of non-radiolabeled CPP. No significant regional differences in brain influx were observed, with the exception for pVEC, for which the regional variations were only marginal. The observed BBB influx transport properties cannot be correlated with their cell-penetrating ability, and therefore, good CPP properties do not imply efficient brain influx.

N-alkylamides: from plant to brain

Background: Plant N-alkylamides (NAAs) are bio-active compounds with a broad functional spectrum. In order to reach their pharmacodynamic targets, they have to overcome several barriers of the body in the absorption phase. The permeability kinetics of spilanthol (a diene NAA) and pellitorine (a triene NAA) across these barriers (i.e. skin, oral/gut mucosa, blood-brain barrier) were investigated.Methods: The skin and oral mucosa permeability were investigated using human skin and pig mucosa in an ex vivo in vitro Franz diffusion cell set-up. The gut absorption characteristics were examined using the in vitro Caco-2 cell monolayer test system. The initial blood-brain barrier transport kinetics were investigated in an in vivo mice model using multiple time regression and efflux experiments. Quantification of both NAAs was conducted using HPLC-UV and bio-analytical UPLC-MS methods.Results: We demonstrated that spilanthol and pellitorine are able to penetrate the skin after topical administration. It is likely that spilanthol and pellitorine can pass the endothelial gut as they easily pass the Caco-2 cells in the monolayer model. It has been shown that spilanthol also crosses the oral mucosa as well as the blood-brain barrier. Conclusion: It was demonstrated that NAAs pass various physiological barriers i.e. the skin, oral and gut mucosa, and after having reached the systemic circulation, also the blood-brain barrier. As such, NAAs are cosmenutriceuticals which can be active in the brain.Key words: Plant N-alkylamides, pharmacokinetics, mucosa/skin, blood-brain barrier (BBB), cosmenutriceuticals

The fasting polypeptide FGF21 can enter brain from blood

FGF21 recently has been proposed as a missing link in the biology of fasting, raising the question of whether it directly reaches the brain. We used multiple time-regression analysis to quantify the influx rate of this polypeptide across the blood–brain barrier (BBB), size-exclusion chromatography to examine degradation, capillary depletion to differentiate entry into brain parenchyma from retention in the microvasculature, and measurement of efflux rate to determine a possible confounding effect on measurement of entry. FGF21 was 94% intact in serum and 75% in brain 10 min after intravenous bolus delivery. Its influx rate was 0.23 ± 0.12 μl/g-min, nearly four times faster than that of the vascular marker albumin. At 10 min, about 0.5% of the administered FGF21 was present in a gram of brain tissue. Of this, 70% reached the parenchyma of the brain. Co-injection of excess FGF21 failed to inhibit the influx, showing a lack of saturation. Efflux, which occurred at the same rate as the bulk reabsorption of cerebrospinal fluid, also was not saturable. In summary, FGF21 shows significant, non-saturable, unidirectional influx across the BBB.

Permeation of hepatocyte growth factor across the blood–brain barrier

Hepatocyte growth factor (HGF), mainly produced and acting in the periphery, attenuates cerebral ischemia-induced cell death and thus shows therapeutic potential in CNS regeneration. Accordingly, we tested its ability to permeate the blood–brain barrier (BBB). HGF was stable in the circulating blood of adult mice for up to 20 min, as HPLC showed intact 125I-HGF in both serum and brain homogenate. Multiple time regression analysis revealed a rapid blood-to-brain influx rate of 0.38 ± 0.07 μl/g min, faster than might be expected for a protein of this size. Although excess unlabeled HGF failed to inhibit of the influx of 125I-HGF in mice, the use of a higher dose of unlabeled HGF in cellular uptake studies showed the presence of saturable endocytosis. Furthermore, capillary depletion studies showed that about 32% of the HGF present in brain entered the parenchymal compartment in contrast to the 11% entrapped in endothelial cells 10 min after intravenous bolus injection. The amount of HGF that crossed the BBB in intact form was substantial and could be physiologically important in the CNS.

Differential BBB interactions of three ingestive peptides: Obestatin, ghrelin, and adiponectin

Endogenous compounds, including ingestive peptides, can interact with the blood–brain barrier (BBB) in different ways. Here we used in vivo and in vitro techniques to examine the BBB permeation of the newly described satiety peptide obestatin. The fate of obestatin in blood and at the BBB was contrasted with that of adiponectin. By the sensitive multiple time-regression method, obestatin appeared to have an extremely fast influx rate to the brain whereas adiponectin did not cross the BBB. HPLC analysis, however, showed the obestatin result to be spurious, reflecting rapid degradation. Absence of BBB permeation by obestatin and adiponectin was in contrast to the saturable transport of human ghrelin reported previously. As a positive control, ghrelin showed saturable binding and endocytosis in RBE4 cerebral microvessel endothelial cells. By comparison, obestatin lacked specific binding and endocytosis, and the small amount internalized showed rapid intracellular degradation before the radioactivity was released by exocytosis. The differential interactions of obestatin, adiponectin, and ghrelin with the BBB illustrate their distinctive physiological interactions with the CNS.

Entry of CART into brain is rapid but not inhibited by excess CART or leptin.

Cocaine- and amphetamine-regulated transcript (CART) is a new anorectic peptide found in the brain and periphery. It is closely associated with leptin, an anorectic agent saturably transported across the blood-brain barrier (BBB). Using multiple time-regression analysis, we found that CART has a rapid rate of entry into brain from blood. However, there was no self-inhibition with CART, even when perfused in blood-free buffer or in fasted mice, showing a lack of saturation. HPLC showed that at least 58% of the injected CART reached brain tissue in intact form, and capillary depletion with and without washout showed that the CART was not bound to endothelial cells or adherent to vascular components. There was no evidence for an efflux system out of the brain for CART. Thus CART can cross the BBB from blood to brain, but its rapid rate of entry is not inhibited by excess CART or leptin.

Passage of leptin across the blood-testis barrier.

Leptin is a 17-kDa protein, secreted by fat, that controls adiposity and has been proposed to have numerous effects on reproduction in the mouse. To assess whether the effects of leptin on testicular function are direct, we determined whether leptin can cross the murine blood-testis barrier. Multiple time regression analysis showed that a small amount of blood-borne leptin is able to enter the testis but does so by a nonsaturable process. In addition, no significant expression of leptin receptors was found at the Leydig cells or Sertoli cells of the testis. This compares with the presence of a saturable transport system for leptin at the blood-brain barrier and abundant receptors for leptin at the leptomeninges, neurons, and choroid plexus of the central nervous system (CNS). These results support the hypothesis that the effects of leptin on reproductive function are not mediated at the level of the testis but indirectly, probably through the CNS.

Nonsaturable entry of neuropeptide Y into brain.

Neuropeptide Y (NPY) is found and is active both in the periphery and brain, but its crossing of the blood-brain barrier (BBB) in either direction has not been measured. We used multiple time-regression analysis to determine that radioactively labeled NPY injected intravenously entered the brain much faster than albumin, with an influx constant of 2.0 x 10(-4) ml. g. -1. min-1. However, this rate of entry was not significantly changed by injection of 10 microgram/mouse of excess NPY, by leptin, or by food deprivation. HPLC showed that most of the NPY entering the brain was intact, and capillary depletion with and without washout showed that the NPY did not remain bound to endothelial cells or associated with vascular elements. Perfusion in a blood-free solution eliminated binding to serum proteins as an explanation for the lack of saturation. Efflux of labeled NPY from the brain occurred at the same rate as albumin, reflecting the normal rate of reabsorption of cerebrospinal fluid. Thus NPY can readily enter the brain from blood by diffusion across the BBB.

Blood-endothelial cell and blood-brain transport of L-proline, alpha-aminoisobutyric acid, and L-alanine.

We report the application of multiple time regression analysis with the in situ brain perfusion technique to measure the rates of passage between blood and brain for [14C] L-proline, [14C] L-alanine, and [14C] alpha-aminoisobutyric acid (AIB) and their rapidly reversible volumes following perfusion of these amino acids from 10 to 60 seconds. We also report on their mechanism of transport. Proline diffused through the blood-brain barrier with a transfer coefficient (Kin) of 0.55 +/- 0.15 x 10(-4) ml/s/g and had no reversible compartment. AIB had a low Kin of 0.68 +/- 0.14 x 10(-4) ml/s/g and a significant reversible volume of 4.34 +/- 0.51 x 10(-3) ml/g in parietal cortex. L-alanine had the highest transfer coefficient, 3.11 +/- 0.26 x 10(-4) ml/s/g, and a reversible volume of 10.03 +/- 0.93 x 10(-3) ml/g in the same cerebral region. Postwash procedures which remove any radiotracer in the vasculature and capillary depletion were performed for alanine and AIB, as they had significant reversible compartments, to test the possibility of rapid efflux from the endothelial cells. Results obtained from wash and capillary depletion procedures suggest that a rapid efflux could occur from endothelial cells after entry of alanine and AIB. Mechanisms of transport for L-alanine and AIB were investigated using amino acids (5 mM) as substrates and inhibitors of different amino acid transport systems. AIB transport was reduced by plasma and L-leucine and unchanged by sodium-free buffer, confirming its passage by the L1 system. L-alanine uptake was sodium-independent and not reduced by plasma. L-serine, L-cysteine, L-leucine and L-phenylalanine produced similar inhibition (66%) while L-alanine produced a lower inhibition (41%). L-arginine increased alanine uptake in cortex and thalamus. Adding L-serine to L-phenylalanine reduced the uptake only in cortex and hippocampus. These data suggest that L-alanine is transported by another L transport system different from the L1 system at the luminal membrane.