Permeability of the blood-brain barrier to peptides: An approach to the development of therapeutically useful analogs

Peptides have been shown in both in vivo and in vitro systems to cross the blood-brain barrier (BBB) and so affect function on the side contralateral to their origin. Some peptides cross primarily by transmembrane diffusion, a nonsaturable mechanism largely dependent on the lipid solubility of the peptide. Other peptides are transported by saturable systems across the BBB. These transport systems can be in the CNS to blood direction, as in the cases of Tyr-MIF-1 and methionine enkephalin, in the blood to CNS direction, as in the case of peptide T, or bidirectional, as in the case of LHRH. Other factors that also affect the amount of peptide crossing the BBB include binding in blood, volume of distribution, enzymatic resistance, and half-time disappearance from the blood. An in vitro model of the BBB has been characterized and used to confirm that peptides can cross the BBB. Results with the model agree with those obtained in vivo and have been used to study the permeability of the BBB to peptides, the effect of peptides on BBB integrity, the cellular pathway peptides and proteins use to cross the BBB, and the ability of the BBB to degrade peptides. The in vivo and in vitro methods have been used together to develop halogenated enkephalin analogs that are enzymatically resistant, cross the BBB readily to accumulate in areas of the brain rich in opiate receptors, and are powerful analgesics. This shows how the principles elucidated for peptide passage across the BBB can be used to develop therapeutic peptides and how those peptides can be further tested in complementary in vivo and in vitro systems.     

From MIF-1 to endomorphin: the Tyr-MIF-1 family of peptides

The Tyr-MIF-1 family of small peptides has served a prototypic role in the introduction of several novel concepts into the peptide field of research. MIF-1 (Pro-Leu-Gly-NH₂) was the first hypothalamic peptide shown to act “up” on the brain, not just “down” on the pituitary. In several situations, including clinical depression, MIF-1 exhibits an inverted U-shaped dose–response relationship in which increasing doses can result in decreasing effects. This tripeptide also can antagonize opiate actions, and the first report of such activity also correctly predicted the discovery of other endogenous antiopiate peptides. The tetrapeptide Tyr-MIF-1 (Tyr-Pro-Leu-Gly-NH₂) not only shows antiopiate activity, but also considerable selectivity for the mu-opiate binding site. Tyr-W-MIF-1 (Tyr-Pro-Trp-Gly-NH₂) is an even more selective ligand for the mu receptor, leading to the discovery of two more Tyr-Pro tetrapeptides that have the highest specificity and affinity for this site. These are the endomorphins: endomorphin-1 is Tyr-Pro-Trp-Phe-NH₂ and endomorphin-2 is Tyr-Pro-Phe-Phe-NH₂. Tyr-MIF-1 proved, contrary to the then prevailing dogma, that peptides can be saturably transported across the blood–brain barrier by a quantifiable transport system. Unexpectedly, the Tyr-MIF-1 transporter is shared with Met-enkephalin. In the era in which it was doubtful whether a peripheral peptide could exert CNS effects, the Tyr-MIF-1 family of peptides also explicitly showed that they can exert more than one central action that persists longer than their half-lives in blood. These peptides clearly illustrate that the name of a peptide restricts neither its actions nor its conceptual implications.     

Monoclonal antibodies to leucine enkephalin

Monoclonal antibodies to leucine enkephalin have been produced after fusion of mouse myeloma cells with spleen cells from hyper-immune mice. Hybrid clones 2D1 and SL1 were characterised using radioimmunoassay and an enzyme-linked immunosorbent assay. The antibody 2D1 was of low affinity and showed a maximum sensitivity of 0.1ng. The antibody binds equally well to the sulphated leucine enkephalin and to methionine enkephalin. It does not cross-react with dynorphin, methionine enkephalin-arg-phe or oxidised methionine enkephalin. The hybrid clone SL1 appears to be specific for leucine enkephalin. Preliminary immunocytochemical studies have shown that both antibodies bind specifically to leucine enkephalin in defined areas of the central nervous system.     

Saturable transport of peptides across the blood-brain barrier

Peptides can be transported across the blood-brain barrier by saturable transport systems. One system, characterized with radioactively labeled Tyr-MIF-1 (Tyr-Pro-Leu-Gly-amide), is specific for some of the small peptides with an N-terminal tyrosine, including Tyr-MIF-1, the enkephalins, beta-casomorphin, and dynorphin (1-8). Another separate system transports vasopressin-like peptides. The choroid plexus has at least one system distinguishable from those above that is capable of uptake and possibly transport of opiate-like peptides. The possibility of saturable transport of other peptides has been investigated to a varying degree. Specificity, stereo-specificity, saturability, allosteric regulation, modulation by physiologic and pharmacologic manipulations, and noncompetitive inhibition have been demonstrated to occur in peptide transport systems and suggest a role for them in physiology and disease.     

Effects of triglycerides, obesity, and starvation on ghrelin transport across the blood-brain barrier

Human ghrelin is transported across the blood-brain barrier (BBB) of normal mice. Here, we studied the effects of triglycerides, obesity, and starvation in retired breeder mice maintained on a high fat diet, mice age-matched to the retired breeders but maintained on normal chow, and 8-week-old mice maintained on breeder chow. The rate of ghrelin transport across the BBB was studied by both the intravenous administration method of multiple-time regression analysis and by the brain perfusion method. We found that (1) obese, aged mice lost the ability to transport intravenously administered ghrelin across the BBB, resulting in an inverse relation between body weight and ghrelin BBB permeability; (2) serum triglycerides promoted transport of intravenously administered ghrelin across the BBB, whereas epinephrine had no effect; (3) fasting tended to promote ghrelin transport across the BBB as most readily shown in brain perfusion studies; (4) evidence suggested that a serum factor promoted ghrelin transport in 8-week-old mice. Overall, these results show that serum factors and physiological states influence the rate at which ghrelin is transported across the blood-brain barrier.     

Methionine sulfoxide is transported by high-affinity methionine and glutamine transport systems in Salmonella typhimurium.

Three lines of evidence indicated that methionine sulfoxide is transported by the high-affinity methionine and glutamine transport systems in Salmonella typhimurium. First, methionine-requiring strains (metE) which have mutations affecting both of these transport systems (metP glnP) were unable to use methionine sulfoxide as a source of methionine. These strains could still grow on L-methionine because they possessed a low-affinity system (or systems) which transported L-methionine but not the sulfoxide. A methionine auxotroph with a defect only in the metP system, which was dependent upon the glnP+ system for the transport of methionine sulfoxide, was inhibited by L-glutamine because glutamine inhibited the transport of the sulfoxide by the glnP+ system. Second, a metE metP glnP strain could be transduced at either the metP or glnP genes to restore its ability to grow on methionine sulfoxide. Third, the transport of [14C]methionine sulfoxide was inhibited by methionine and by glutamine in the metP+ glnP+ strain. No transport was detected in the metP glnP double-mutant strain.     

Transport of Biosynthetic Intermediates: Regulation of Homoserine and Threonine Uptake in Escherichia coli

Homoserine is transported by a single system that it shares with alanine, isoleucine, leucine, phenylalanine, threonine, valine and perhaps cysteine, methionine, serine, and tyrosine. We investigated the regulation of this transport system and found that alanine, isoleucine, leucine, methionine, and valine each repress the homoserine-transporting system. From the concentration resulting in 50% repression of this transport system and the maximal amount of repression, we ranked the amino acids according to their effectiveness in repressing homoserine transport (in decreasing order): leucine>methionine>alanine>valine>isoleucine. The exponential rate of decrease in transport capacity after leucine addition equals the exponential growth rate of the culture, and protein synthesis is necessary for the derepression seen when leucine is removed. Threonine, in addition to using the above system, is transported by a second system shared with serine. We present further evidence for this serine-threonine transport system and show that it is not regulated like the homoserine-transporting system.     

Is there a probenecid sensitive transport system for monoamine catabolites at the level of the brain capillary plexus?

Probenecid inhibits the transport of the small monocarboxylic acids lactate and propionate from blood to brain but does not affect the transport of 5HIAA or HVA. Neither lactate, 5HIAA, HVA, nor probenecid itself inhibits probenecid uptake into brain from blood and neither lactate nor 5HIAA itself inhibits 5HIAA uptake. These results indicate first that probenecid inhibits the lactate carrier but is itself not transported by that carrier and second that 5HIAA and probenecid are independently transported from blood to brain by a low affinity system, probably by diffusion. Preloading animals with both tryptophan and probenecid increased the apparent transport of lactate, probenecid and 5HIAA but not the transport of glucose. This indicates that the transport of 5HIAA, lactate and probenecid from brain to blood involves a common, saturable carrier. These two sets of data indicate that either the brain capillary transport system is asymmetric or that probenecid-inhibited transport of monoamine catabolites from brain occurs at sites other than the capillary transport system of the blood-brain barrier.     

Imino acid transport in human diploid fibroblasts.

These studies describe the transport of proline and hydroxyproline in human diploid fibroblasts. Inhibition and kinetic analysis demonstrate that proline is actively transported by the “A” neutral amino acid carrier. Proline transport is Na⁺ dependent and is particularly sensitive to sulfhydryl inhibitors and ouabain. Hydroxyproline is also actively transported but its transport is mediated by a system different from those described previously for other neutral amino acids. Hydroxyproline transport requires the presence of Na⁺ and is sensitive to sulfhydryl inhibitors and ouabain. There is little inhibition of hydroxyproline transport in the presence of other amino acids with the exception of methionine. The methionine inhibition of hydroxyproline transport is of the non-competitive type. Little cross-reactivity was exhibited by the systems which transport proline and hydroxyproline. These studies indicate that human skin fibroblasts do not possess an iminoglycine transport system as has been described for many other tissues. The iminoglycine transport system has been identified as the genetic transport defect in iminoglycinuria. Consequently, skin fibroblasts are not an appropriate system for use in diagnosis of this disorder.     

Endogenous methionine enkephalin may play an anticonvulsant role in the seizure-susceptible El mouse

After the intracisternal injection of three protease inhibitors which prevent the degradation of methionine enkephalin (amastatin, Des-Pro²-bradykinin, and phosphoramidon) and a mixture of these protease inhibitors, we investigated the effect on convulsive seizures in the seizure-susceptible El mouse. We also measured the cerebral methionine enkephalin content by high-performance liquid chromatography coupled with radioimmunoassay. Protease inhibitors significantly decreased both the incidence of seizures and the seizure score in El mice in a dose-dependent manner. This anticonvulsant effect was reversed by naloxone (2 mg/kg, sc). The cerebral methionine enkephalin content increased significantly after the administration of protease inhibitors in comparison with saline injection. These findings suggest that it was not protease inhibitors but instead increase of endogenous methionine enkephalin that reduced the incidence of seizures and the seizure score in El mice. Together with our previous data, the present findings support our hypothesis that a deficit in anticonvulsant endogenous methionine enkephalin is involved in the pathogenesis of seizures in the El mouse.     

Sodium-iodide symporter mediates iodide secretion in rat gastric mucosa in vitro

In vivo studies on rats have demonstrated that considerable amounts of iodide are transported from the bloodstream into the gastric lumen. The mechanisms for and functional significance of this transport are poorly understood. Active (driven by Na(+)/K(+)-ATPase) iodide transport into thyroid follicular cells is mediated by the sodium-iodide symporter (NIS), which is also abundantly expressed in gastric mucosa. We aimed to further investigate the iodide transport in gastric mucosa and the possible role of NIS in this transport process. Iodide transport in rat gastric mucosa was studied in vitro in an Ussing chamber system using (125)I as a marker. The system allows measurements in both directions over a mucosal specimen. A considerable transport of iodide (from the serosal to the mucosal side) was established across the gastric mucosa, whereas in the opposite direction (mucosa to serosa), iodide transport was negligible. Sodium perchlorate (NaClO(4)), a competitive inhibitor of NIS, and ouabain, an inhibitor of the Na(+)/K(+)-ATPase, both attenuated gastric iodide transport from the serosal to the mucosal side. To investigate a possible neuroendocrine regulation of the iodide transport identified to occur from the serosal to the mucosal side of the stomach, thyroid-stimulating hormone (TSH), thyrotropin-releasing hormone (TRH), vasoactive intestinal peptide (VIP), histamine, or nitric oxide donor S-nitroso-N-acetyl-D,L-penicillamine (SNAP) was added. None of these substances influenced the iodide transport. We conclude that iodide is actively transported into the gastric lumen and that this transport is at least partly mediated by NIS. Additional investigations are needed to understand the regulation and significance of this transport.     

Oral administration of Tyr-MIF-1 stimulates gastric emptying and gastrointestinal motility in rodents.

The effects of orally administered Tyr-MIF-1, an agonist of an endogenous antiopiate system, were examined on gastric emptying in mice and gastrointestinal myoelectric activity in rats. Tyr-MIF-1 (5 mg/kg in mice, 20 mg/kg in rats) accelerated gastric emptying of a methylcellulose test meal, increased the frequency of antral spike bursts, and disrupted intestinal migrating myoelectric complexes. These effects were reproduced by a subcutaneous administration of Tyr-MIF-1 at the same dosage. They were blocked by naloxone (1 mg/kg) but not by the kappa receptor subtype antagonist MR 2266 (1 mg/kg). The GABAA antagonist bicuculline (0.5 mg/kg), but not the GABAB antagonist 2-hydroxysaclofen (4 mg/kg), also antagonized the effects of Tyr-MIF-1. These data demonstrate that oral Tyr-MIF-1 stimulates gastric emptying and gastrointestinal motility through a systemic or central action that involves opioid and GABA systems.     

Role of endogenous opioids in soman (pinacolyl methylphosphonofluoridate)-induced antinociception

The effect of soman poisoning on the levels of methionine enkephalin and beta-endorphin in mice and rats were determined. Soman poisoning produced no significant effect on methionine enkephalin levels in the striatum of rats or mice or beta-endorphin levels in the pituitary gland of mice. In rats beta-endorphin levels were significantly reduced 24 hr post soman poisoning, but returned to control levels by 48 hr. In vitro, the hydrolysis of leucine enkephalin by aminopeptidase was virtually complete by 30 min and found to be the major route of degradation. The release of TYR-GLY-GLY in the presence or absence of puromycin (10 microM) was found to be low (less than or equal to 2.0%). A minor effect on TYR release in the presence of GLY-GLY-PHE-MET (50 microM) was insignificant. Preincubation of mouse striatum homogenates with soman (1 or 10 microM) did not inhibit the hydrolysis of leucine enkephalin. These results suggest that the long term antinociception following soman exposure is not due to either altered concentration of endogenous opioid-like substances or inhibition of the enzymes responsible for their degradation.     

A comparative study of the effect of methionine enkephalin upon the stereospecific binding of an opiate agonist and an antagonist in mice and rats.

The synthetic enkephalins especially methionine enkephalin are more potent in inhibiting the stereospecific binding of ³H-dihydromorphine than that of ³H-naloxone in mouse brain homogenates. Methionine enkephalin is a more potent inhibitor of ³H-dihydromorphine binding in whole mouse brain homogenates than in washed mouse brain membranes. No difference was observed with regard to the inhibitory effect of methionine enkephalin on the binding of ³H-dihydromorphine in whole rat brain homogenates or washed rat brain membranes. The use of different radiolabelled drugs (agonist versus antagonist), different species (mouse versus rat) and/or the variation in the preparation (brain homogenates versus washed membranes) may account for the difference between the IC₅₀ of methionine enkephalin versus ³H-dihydromorphine and versus ³H-naloxone stereospecific binding. The increased inhibitory effect of methionine enkephalin when the supernatant was added to the washed brain membranes supports the hypothesis that methionine enkephalin may be one part of the real endogenous morphine ligand.     

Pantothenic Acid Transport Through the Blood-Brain Barrier

The unidirectional influx of D-pantothenic acid (PA) across cerebral capillaries, the anatomical locus of the blood-brain barrier, was measured with an in situ rat brain perfusion technique using [3H]D-PA (1.1 Ci/mmol). PA was transported across the blood-brain barrier by a saturable system that could be described by a Michaelis-Menten transport model with a half-saturation concentration and maximal influx rate of 19 microM and 0.21 nmol/g of brain/min, respectively. PA (0.3 microM) transport through the blood-brain barrier was significantly inhibited by probenecid, nonanoic acid, and biotin (all less than or equal to 0.25 mM), but not by penicillin G, pyruvate, beta-hydroxybutyrate, L-leucine (all 1 mM), or poly-L-lysine HBr (1 mg/ml). Probenecid (0.25 mM), nonanoic acid (0.5 mM), and PA (1.0 mM) did not inhibit [3H]L-leucine transport through the blood-brain barrier, whereas 30 microM-L-leucine inhibited [3H]leucine transport to 23% of control values. Thus, PA is transported through the blood-brain barrier by a low-capacity, saturable transport system with a half-saturation concentration approximately 10 times the plasma PA concentration. Although involved in the transfer of PA from blood into brain, this system does not play an important regulatory role in the synthesis of CoA from PA in brain.     

A radiotracer method to study efflux transport of iodide liberated from thyroid hormones via deiodination metabolism in the brain

AimsThyroid hormones (TH) play an important role in the development and functional maintenance of the central nervous system. The purpose of this study was to develop a radiotracer method for studying the in vivo efflux transport of iodide liberated by the TH metabolism in the brain. The rationale of our method is as follows: a radioiodinated compound can enter the brain and rapidly release iodide in situ; the iodide efflux rate can be estimated from the clearance of brain radioactivity after disappearance of the iodinated compound.Main methods6-[¹²⁵I]Iodo-9-pentylpurine ([¹²⁵I]9Pe6IP) was designed to enter the brain and release ¹²⁵I^? by the reaction with glutathione and synthesized from the corresponding bromo derivative in a Br/¹²⁵I exchange reaction. The brain kinetics of radioactivity and radioactive metabolites were investigated after intravenous injection of [¹²⁵I]9Pe6IP into mice. The iodide efflux rate was estimated in mice pretreated with perchlorate, an inhibitor of iodide transport from the brain.Key findingsHigh brain uptake (5.3% injected dose/g) was observed at 1 min, and almost complete conversion of [¹²⁵I]9Pe6IP to ¹²⁵I^? occurred 10 min after injection. The ¹²⁵I^? uptake from the blood was negligible. ¹²⁵I^? was eliminated from the brain along a single-exponential curve with a half-life of 6.0 min. Furthermore, dose-dependent inhibition of ¹²⁵I^? efflux was observed in mice pretreated with perchlorate.SignificanceWe conclude that 9Pe6IP labeled with ¹²⁴I (positron emitter) or ¹²³I (single-photon emitter) may be useful for studying the in vivo efflux transport of iodide in the brain using nuclear medicine imaging devices.     

Alterations within the endogenous opioid system in mice with targeted deletion of the neutral endopeptidase ('enkephalinase') gene

The biological inactivation of enkephalins by neutral endopeptidase (enkephalinase, NEP, EC3.4.24.11) represents a major mechanism for the termination of enkephalinergic signalling in brain. A pharmacological blockade of NEP-activity enhances extracellular enkephalin concentrations and induces opioid-dependent analgesia. Recently, knockout mice lacking the enzyme NEP have been developed [Lu et al., J. Exp. Med. 1995;181:2271-2275]. The present study investigates the functional consequences and biochemical compensatory strategies of a systemic elimination of NEP activity in these knockout mice. Using biochemical and behavioural tests we found that the lack of NEP activity in brain is not compensated by enhanced activities of alternative enkephalin-degrading enzymes. Also no change in enkephalin biosynthesis was detectable by in situ methods quantifying striatal proenkephalin-mRNA levels in NEP-deficient mice compared with wildtype. Only a 21% reduction of mu receptor density in crude brain homogenates of NEP knockout mice was observed, while delta- and kappa-opioid receptor densities were unchanged. This receptor downregulation was also confirmed functionally in the hot-plate paradigm. NEP knockouts developed normally, but showed enhanced aggressive behaviour in the resident-intruder paradigm, and altered locomotor activity as assessed in the photobeam system. Thus, although NEP plays a substantial role in enkephalinergic neurotransmission, the biochemical adaptations within the opioid system of NEP-deficient mice are of only modest nature.     

Source of Methyl Groups in Brain and Nerve Tissue in the Rat

Previous studies that demonstrated that mouse brain accumulated significantly more radioactivity from subcutaneously administered 5-methyltetrahydrofolate labelled in the methyl group compared to the label in the folate moiety are open to two interpretations. The methyl group could have been transferred to another compound (probably methionine) prior to its transport into the brain. Alternatively, if plasma 5-methyltetrahydrofolate per se is significantly involved in the provision of methyl groups to brain and nerve tissue it would be expected that the folate moiety would be returned to the plasma to complete the cycle and thus would appear not to have been taken up. In this article, using competition experiments that exploit the differences in the mechanism of transport of methionine and 5-methyltetrahydrofolate into brain and nerve, evidence is presented that in the rat the methyl group of 5-methyltetrahydrofolate is transported after its conversion to methionine.     

Enkephalins are transported by a novel eukaryotic peptide uptake system

We have identified an oligopeptide transporter in the yeast Saccharomyces cerevisiae which mediates the uptake of tetra- and pentapeptides, including the endogenous opioids leucine enkephalin (Tyr-Gly-Gly-Phe-Leu) and methionine enkephalin (Tyr-Gly-Gly-Phe-Met). The transporter is encoded by the gene OPT1. Yeast expressing OPT1 can utilize enkephalins to satisfy amino acid auxotrophic requirements for growth. The transport of radiolabeled leucine enkephalin exhibits saturable kinetics, with a K(m) of 310 microM. Transport activity is optimum at acidic pH and sensitive to reagents which uncouple oxidative phosphorylation, suggesting an energy dependence on the proton gradient. Growth, transport, and chromatographic data indicate that leucine enkephalin is not hydrolyzed in the extracellular medium and as such is translocated intact across the cell membrane. The system is specific for tetra- and pentapeptides and can be inhibited by the opioid receptor antagonists naloxone and naltrexone. To date, this is the first example of a eukaryotic transport system which can use enkephalins as a substrate, opening the possibility that a homologue exists in higher eukaryotes.     

Melanocyte-Stimulating Hormone Release-Inhibiting Factor-1 (MIF-1) Can Be Formed from Tyr-MIF-1 in Brain Mitochondria but Not in Brain Homogenate

Abstract: Two samples of the peptide tyrosine-melanocyte-stimulating hormone release-inhibiting factor-1 (Tyr-MIF-1; Tyr-Pro-Leu-Gly-NH₂) were tritiated on different amino acids (Tyr or Pro) and incubated together at 37°C with fractions of rat brain. The amount of intact tetrapeptide remaining was determined by HPLC. By 3 min, most of the Tyr-MIF-1 was degraded. Because similar amounts of [³H]Pro and [³H]Tyr appeared after incubation of the Tyr-MIF-1 peptides in brain homogenate, even as early as 30 s, examination of only this crude preparation would misleadingly indicate that Tyr-MIF-1 is not a precursor of melanocyte-stimulating hormone release-inhibiting factor-1 (MIF-1; Pro-Leu-Gly-NH₂) in brain tissue. However, incubation of the mitochondrial fractions of brain under the same conditions resulted in more than three times as much [³H]Tyr being formed as [³H]Pro, with accompanying accumulation of MIF-1. Addition of excess MIF-1 to the mitochondrial fraction completely suppressed the formation of MIF-1 and more than doubled the amount of Tyr-MIF-1 remaining intact. When Tyr-MIF-1 tritiated only on the Tyr was added to the mitochondrial fraction, the main peaks of radioactivity appeared only at the positions of Tyr and Tyr-MIF-1, not at the position of Tyr-Pro. The results indicate that Tyr-MIF-1 can serve as a precursor of MIF-1 in brain mitochondria, an effect not evident when crude brain homogenate is used.