Terrence J. Gillespie

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.

The effect of halogenation on blood–brain barrier permeability of a novel peptide drug☆

The utility of a drug depends on its ability to reach appropriate receptors at the target tissue and remain metabolically stable to produce the desired effect. To improve central nervous system entry of the opioid analgesic [D-Pen2, L-Pen5, Phe6] Enkephalin (DPLPE-Phe), our research group synthesized analogs that had chloro, bromo, fluoro, and iodo halogens on the para positions of the phenylalanine-4 residue. This study reports on investigation of the effect of halogenation on stability, lipophilicity, and in vitro blood-brain barrier permeability of a novel enkephalin analog DPLPE-Phe. The stability of each halogenated DPLPE-Phe analog as well as the amidated and nonamidated parent peptide was tested in plasma and brain. All peptides tested had a half-time disappearance >300 min except for DPLPE-Phe-NH2, which was found to have a half-life of 30 min in plasma. Octanol/saline distribution studies indicated addition of halogens to DPLPE-Phe-OH significantly increased lipophilicity except for p-[F-Phe4]DPLPE-Phe-OH. p-[Cl-Phe4]DPLPE-Phe-OH exhibited the most pronounced increase in lipophilicity. Para-bromo and para-chloro halogen additions significantly enhanced in vitro blood-brain barrier permeability, providing evidence for improved delivery to the central nervous system.

Peptide fragments derived from the β-chain of hemoglobin (hemorphins) are centrally active in vivo

A novel tetrapeptide (hemorphin-4) and pentapeptide (hemorphin-5), derived from the beta-chain of hemoglobin, were synthesized by solid-phase methodology, purified and the amino acid sequences confirmed. The central (ICV) effects of hemorphin-4 and -5 were studied in two models of phasic and tonic nociception, the mouse warm water tail-flick assay and hindpaw formalin assay, respectively. Additionally, two physiological endpoints, central modulation of bladder motility and central effects on intestinal propulsion, were studied in rats and mice, respectively. In the tail-flick assay, both peptides (40-100 nmoles) produced a dose-related naloxone-reversible antinociceptive effect when tested 10 min after peptide administration, with the tetrapeptide being slightly more potent than the pentapeptide. No effect was noted for either peptide using the tonic nociception assay, except at a dose of 150 nmoles for hemorphin-5. Inhibition of gastrointestinal propulsion was also not affected by either peptide. However, both peptides (10-40 nmoles) inhibited micturition contractions in a dose-related and naloxone-reversible fashion, with the tetrapeptide being twice as potent as the pentapeptide. These findings provide evidence that hemorphin-4 and -5 exert naloxone-reversible opioid actions in vivo and, therefore, may be physiologically important blood-borne peptides.

A specific enzyme assay for aminopeptidase M in rat brain

A specific enzyme assay for aminopeptidase M (APM) activity on rat brain membranes has been developed through selective use of enzyme inhibitors. Amastatin was the most potent inhibitor (amastatin > actinonin > MDL73347 > bestatin) for purified porcine kidney APM, giving 98% inhibition at a 6 microM concentration, while actinonin, yielded only 57% inhibition at this concentration. Puromycin (10 microM) was used to inhibit puromycin-sensitive aminopeptidase activity in the rat brain membrane preparation. Puromycin (10 microM) had only a slight effect on the Km of porcine kidney APM, and had negligible effect on APM velocity at the high substrate concentration (2 mM) used in the APM assay. The assay produced a linear accumulation of product for increasing amount of rat brain membranes used, and for increasing incubation time. The Km of APM on rat brain membranes for L-Leucine-p-nitroanilide (0.383 mM) was similar to the Km of purified porcine kidney APM (0.558 mM). APM-activity, involved in the metabolism of several biologically important neuropeptides in different brain regions, can be specifically measured with this enzyme assay.

Specificity of Neurotensin Metabolism by Regional Rat Brain Slices

Abstract: Regional differences in neurotensin metabolism and the peptidases involved were studied using intact, viable rat brain microslices and specific peptidase inhibitors. Regional brain slices (2 mm X 230 μm) prepared from nucleus accumbens, caudate‐putamen, and hippocampus were incubated for 2 h in the absence and presence of phosphoramidon, captopril, N‐[1(R,S)‐carboxy‐3‐phenylpropyl]‐Ala‐Ala‐Phe‐p‐ aminobenzoate, and o‐Phenanthroline, which are inhibitors of neutral endopeptidase 24.11, angiotensin‐converting enzyme, metalloendopeptidase 24.15, and nonspecific metallopeptidases, respectively. Neurotensin‐degrading proteolytic activity varied by brain region. Significantly less (35.0 ± 1.6%) neurotensin was lost from hippocampus than from caudateputamen (45.4 ± 1.0%) or nucleus accumbens (47.8 ± 1.1%) in the absence of inhibitors. Peptidases responsible for neurotensin metabolism on brain slices were found to be predominantly metallopeptidases. Metalloendopeptidase 24.15 is of major importance in neurotensin metabolism in each brain region studied. The relative contribution of specific peptidases to neurotensin metabolism also varied by brain region; angiotensin‐converting enzyme and neutral endopeptidase 24.11 activities were markedly elevated in the caudate‐putamen as compared with the nucleus accumbens or hippocampus. Interregional variation in the activity of specific peptidases leads to altered neurotensin fragment formation. The brain microslice technique makes feasible regional peptide metabolism studies in the CNS, which are impractical with synaptosomes, and provides evidence for regional specificity of neurotensin degradation.

In vitro stability of some reduced peptide bond pseudopeptide analogues of dynorphin A

Eight analogues of DYN A(1-11)-NH2 incorporating the nonhydrolyzable psi [CH2-NH] peptide bond surrogate were tested for their in vitro enzymatic stability in mouse brain homogenates. Results show that the Leu(5)-Arg6 and to a lesser extent the Arg(7)-Ile8 and Ile(8)-Arg9 peptide bonds are the more susceptible to enzymatic cleavage in the native peptide. (Leu5 psi[CH(2)-NH]Arg6)DYN A(1-11)-NH2 exhibits an almost complete resistance to enzymatic cleavage with a half-life greater than 500 min in brain, compared to 42 min for the standard peptide, DYN A(1-11)-NH2.

Changes in opioid receptor selectivity following processing of peptide E: Effect on gut motility

Peptide E is a mu-selective opioid peptide derived from proenkephalin A which contains [Met5]-enkephalin at the amino end and [Leu5]-enkephalin at the carboxyl end. Peptide E is further processed both centrally and peripherally to a [Leu5]-enkephalin-containing fragment which was investigated to determine if processing leads to alterations in receptor selectivity. Peptide E-(15-25) inhibited electrically stimulated contractions in both the mouse vas deferens, longitudinal muscle, myenteric (IC50 = 459 nmol/L), and guinea pig ileum (IC50 = 2630 nmol/L), indicating a sixfold delta-receptor selectivity. When administered intracerebroventricularly to mice, peptide E-(15-25) also produced potent analgesia which was completely antagonized by naloxone pretreatment, but the peptide had no effect on intestinal transit as measured by the radiochromium geometric center method. This is consistent with earlier findings that intracerebroventricular delta-opioid-selective agents are analgesic but do not inhibit intestinal transit. In vitro radioligand binding assays were performed using male Sprague-Dawley rat whole brain homogenates. The IC50 for peptide E against [3H]naloxone was 1.8 nmol/L compared with the delta-opioid ligand, [3H] [D-Pen2, D-Pen5]-enkephalin of 38.8 nmol/L. The IC50 for peptide E-(15-25) against [3H]naloxone was 497 nmol/L, but for [3H] [D-Pen2, D-Pen5]-enkephalin it was 50.6 nmol/L. Therefore, peptide E loses mu-opioid receptor affinity (1.8-497 nmol/L) after proteolytic processing and the loss of the amino terminal tyrosine but maintains a high delta-opioid affinity (38.8-50.6 nmol/L). These studies demonstrate that enzymatic peptide processing of peptide E to peptide E-(15-25) leads to a shift from mu- to delta-receptor selectivity and a different spectrum of biological effects on gut motility.

Degradation of insulin-like growth factors in small intestine of suckling rats

Insulin-like growth factors (IFGs), IGF-I and IGF-II, present in mammalian milk, play an important role during gastrointestinal tract development. In this study we identified and localized the activities of the common intestinal proteolytic enzymes and investigated their degradation effect on IGFs. Results indicated that the enzymatic activities of chymotrypsin, trypsin, and elastase progressed from the lowest in the duodenum, to the highest in the midjejunum, and declined in the ileum. Chymotrypsin exhibited the greatest IGFs degradation activities in neonatal intestinal lumen followed by elastase. These data furnish a potential strategic design to supplement IGFs into milk formulas.

Ethanol treatment alters β-endorphin metabolism by purified synaptosomal plasma membranes

Ethanol administration has been shown to affect beta-endorphin (beta-E) levels in most brain areas. Chronic ethanol treatment has also lead to changes in the levels of Met- and Leu-enkephalin which may be due to recent finding that enkephalin A activity is significantly altered. To determine if proteolytic enzymes responsible for beta-E metabolism at the pSPM are also altered, we studied the effect of chronic ethanol (7% v/v; 8 days) administration on in vitro central beta-E metabolism in male C57/BL mice. Purified SPM was time-course incubated with beta-E (20 microM) for 30-120 min and subjected to HPLC analyses for determination of beta-endorphin and related fragments. Chronic ethanol significantly reduced the half-life for beta-E at the pSPM (T1/2 = 50/min) versus controls (T1/2 = 100.4 min). Chronic ethanol also caused significant accumulation of the behaviorally active alpha- and gamma-type endorphins formed at the pSPM. These results suggest that chronic ethanol treatment leads to an increase in the activity of peptidases responsible for beta-E metabolism at pSPM leading to an increased formation of both alpha- and gamma-type endorphins which may affect alcohol related behaviors.