Jason D. Huber

Peptide drug modifications to enhance bioavailability and blood-brain barrier permeability

Peptides have the potential to be potent pharmaceutical agents for the treatment of many central nervous system derived maladies. Unfortunately peptides are generally water-soluble compounds that will not enter the central nervous system, via passive diffusion, due to the existence of the blood-brain barrier. Peptides can also undergo metabolic deactivation by peptidases, thus further reducing their therapeutic benefits. In targeting peptides to the central nervous system consideration must be focused both on increasing bioavailability and enhancing brain uptake. To date multiple strategies have been examined with this focus. However, each strategy comes with its own complications and considerations. In this review we assess the strengths and weaknesses of many of the methods currently being examined to enhance peptide entry into the central nervous system.

Improved bioavailability to the brain of glycosylated Met-enkephalin analogs

The blood-brain barrier prevents the entry of many potentially therapeutic peptide drugs to the brain. Glycosylation has shown potential as a methodology for improving delivery to the CNS. Previous studies have shown improved bioavailability and improved centrally mediated analgesia of glycosylated opioids. In this study we investigate the effect of glycosylation on the cyclic opioid peptide [D-Cys(2,5),Ser(6),Gly(7)] enkephalin. The peptide was glycosylated on the Ser(6) via an O-linkage with various sugar moieties and alignments. The peptides were then investigated for receptor binding, physiochemical attributes, in situ brain uptake in female Sprague-Dawley rats and antinociception in male ICR mice. Glycosylation resulted in a slight decrease in affinity to the delta-opioid receptor, and mixed effect on binding to the mu-opioid receptor. There was a significant decrease in lipophilicity resulting from glycosylation and a slight reduction in binding to bovine serum albumin. In situ perfusion showed that brain uptake was improved by up to 98% for several of the glycosylated peptides, and the nociceptive profiles of the peptides, in general, followed the rank order of peptide entry to the brain with up to a 39-fold increase in A.U.C.

Protection against hypoxia-induced increase in blood-brain barrier permeability: Role of tight junction proteins and NFκB

Co-culture with glial cells and glia-conditioned media can induce blood-brain barrier properties in microvessel endothelial cells and protect against hypoxia-induced blood-brain barrier breakdown. We examined the effect of two types of glia-conditioned media on brain microvessel endothelial cell permeability and tight junction protein expression, and studied potential mechanisms of action. We found that C6-glioma-conditioned media, but not rat astrocyte-conditioned media, protected against an increase in permeability induced by exposure to 1% oxygen for 24 hours. This hypoxic stress caused an increase in the expression of tight junction proteins claudin-1 and actin, particularly in cells treated with C6-conditioned media. We found that C6-conditioned media has a significantly higher level of both basic fibroblast growth factor and vascular endothelial growth factor. Treatment with C6-conditioned media for 1 or 3 days protects against hypoxia-induced permeability increases, and this protective effect may be mediated by signal transduction pathways terminating at the transcription factor NFκB.

Pain and the blood–brain barrier: obstacles to drug delivery

Delivery of drugs across the blood-brain barrier has been shown to be altered during pathological states involving pain. Pain is a complex phenomenon involving immune and centrally mediated responses, as well as activation of the hypothalamic-pituitary-adrenal axis. Mediators released in response to pain have been shown to affect the structure and function of the blood-brain barrier in vitro and in vivo. These alterations in blood-brain barrier permeability and cytoarchitecture have implications in terms of drug delivery to the central nervous system, since pain and inflammation have the capacity to alter drug uptake and efflux across the blood-brain barrier. An understanding of how blood-brain barrier and central nervous system drug delivery mechanisms are altered during pathological conditions involving pain and/or inflammation is important in designing effective therapeutic regimens to treat disease.

Assessment of Stereoselectivity of Trimethylphenylalanine Analogues of δ-Opioid [D-Pen2,D-Pen5]-Enkephalin

Abstract : [D‐Pen2,D‐Pen5]‐Enkephalin (DPDPE) is an enzymatically stable δ‐opioid receptor‐selective peptide, which was modified by the trimethylation of the Phe4 residue to give β‐methyl‐2′,6′‐dimethylphenylalanine (TMP), resulting in four conformations : (2R,3S)‐β‐Phe‐DPDPE, (2R,3R)‐β‐Phe‐DPDPE, (2R,3S)‐β‐Phe‐DPDPE, and (2S,3R)‐β‐Phe‐DPDPE. Synthesis was by solid‐phase techniques using enantiomerically pure amino acids to give the four optically pure diastereoisomer peptides. The potency and selectivity (δ‐ versus μ‐opioid receptor) were evaluated by radioreceptor binding in rat brain, with a μ/δ ratio decrease for all TMP conformations, compared with the parent compound (DPDPE). Octanol/buffer distribution analysis showed enhanced lipophilicity of all TMP forms, with a sixfold enhancement associated with (2S,3S)‐TMP. In situ vascular perfusion in anesthetized rats showed a 1.6‐fold (p < 0.01) increase in the ratio of brain uptake for (2S,3S)‐TMP and a 1.5‐fold (p < 0.01) decrease in uptake for (2R,3R)‐TMP. Saturability of (2S,3S)‐TMP was shown (p < 0.01) against 100 μM unlabeled DPDPE, showing a shared nondiffusionary transport system. P‐glycoprotein affinity was shown in situ for the parent and (2S,3S)‐TMP (p < 0.01). Protein binding capacity of the TMP compounds in rat plasma and in situ mammalian bovine serum albumin‐Ringer showed (2R,3S)‐TMP and (2S,3R)‐TMP with the lowest degree of protein binding (p < 0.01), and (2S,3S)‐TMP and (2R,3R)‐TMP with comparable affinities to DPDPE. Analgesia, via intravenous administration, showed significantly reduced (p < 0.01) end effect and time course for (2R,3R)‐TMP, (2R,3S)‐TMP, and (2S,3R)‐TMP as compared with DPDPE. These results demonstrate that topographical modification in a conformationally restricted peptide can significantly modulate potency and receptor selectivity, binding capacity, enzymatic stability, lipophilicity, P‐glycoprotein affinity, and blood‐brain barrier permeability, resulting in a change of bioavailability, and thereby provides insight for future peptide drug design.

Effect of reduced flow on blood-brain barrier transport systems

Pathological states (i.e. stroke, cardiac arrest) can lead to reduced blood flow to the brain potentially altering blood-brain barrier (BBB) permeability and regulatory transport functions. BBB disruption leads to increased cerebrovascular permeability, an important factor in the development of ischemic brain injury and edema formation. In this study, reduced flow was investigated to determine the effects on cerebral blood flow (CBF), pressure, basal BBB permeability, and transport of insulin and K+ across the BBB. Anesthetized adult female Sprague-Dawley rats were measured at normal flow (3.1 ml min(-1)), half flow (1.5 ml min(-1)), and quarter flow (0.75 ml min(-1)), using bilateral in situ brain perfusion for 20 min followed by capillary depletion analysis. Reduction in perfusion flow rates demonstrated a modest reduction in CBF (1.27-1.56 ml min(-1) g(-1)), a decrease in pressure, and no significant effect on basal BBB permeability indicating that autoregulation remained functional. In contrast, there was a concomittant decrease in BBB transport of both insulin and K+ with reduced flow. At half and quarter flow, insulin transport was significantly reduced (R(Br)%=17.2 and R(Br)%=16.2, respectively) from control (R(Br)%=30.4). Additionally, a significant reduction in [86Rb+] was observed at quarter flow (R(Br)%=2.5) as compared to control (R(Br)%=4.8) suggesting an alteration in ion homeostasis as a result of low flow. This investigation suggests that although autoregulation maintains CBF, BBB transport mechanisms were significantly compromised in states of reduced flow. These flow alterations may have a significant impact on brain homeostasis in pathological states.

Viability of microvascular endothelial cells to direct exposure of formalin, λ-carrageenan, and complete Freund's adjuvant

We investigated three inflammatory agents to establish if these substances elicit a direct effect on the functional and structural integrity of the blood-brain barrier. Cellular cytotoxicity and paracellular permeability were assessed in vitro using primary bovine brain microvascular endothelial cells exposed to formalin, lambda-carrageenan, or complete Freunds adjuvant for 1, 3, or 72 h, respectively. Results showed that only the highest concentration (0.025%) of formalin produced a decrease in cell viability (approximately 34%) and a significant increase in cell permeability to [(14)C]sucrose at 120 min (approximately 137%). Brain perfusion using female Sprague-Dawley rats showed no difference in paracellular permeability to [(14)C]sucrose for any inflammatory agent. Western blot analyses were performed on isolated rat brain microvessels to assess the structural integrity of blood-brain barrier tight junctions. Results indicate that expression of zonula occludens-1, occludin, claudin-1, and actin remain unchanged following intravenous exposure to inflammatory agents. This study confirms that changes seen at the blood-brain barrier following a peripheral inflammation are due to physiological responses to the given inflammatory agent and not to any direct interaction between the inflammatory agent and the brain microvasculature.