Determinants of benzodiazepine brain uptake: lipophilicity versus binding affinity.

Factors influencing brain uptake of benzodiazepine derivatives were evaluated in adult Sprague Dawley rats (n = 8-10 per drug). Animals received single intraperitoneal doses of alprazolam, triazolam, lorazepam, flunitrazepam, diazepam, midazolam, desmethyldiazepam, or clobazam. Concentrations of each drug (and metabolites) in whole brain and serum 1 h after dosage were determined by gas chromatography. Serum free fraction was measured by equilibrium dialysis. In vitro binding affinity (apparent Ki) of each compound was estimated based on displacement of tritiated flunitrazepam in washed membrane preparations from rat cerebral cortex. Lipid solubility of each benzodiazepine was estimated using the reverse-phase liquid chromatographic (HPLC) retention index at physiologic pH. There was no significant relation between brain:total serum concentration ratio and either HPLC retention (r = 0.18) or binding Ki (r = -0.34). Correction of uptake ratios for free as opposed to total serum concentration yielded a highly significant correlation with HPLC retention (r = 0.78, P less than 0.005). However, even the corrected ratio was not correlated with binding Ki (r = -0.22). Thus a benzodiazepine's capacity to diffuse from systemic blood into brain tissue is much more closely associated with the physicochemical property of lipid solubility than with specific affinity. Unbound rather than total serum or plasma concentration most accurately reflects the quantity of drug available for diffusion.

Opioid peptides in Huntington's disease: alterations in prodynorphin and proenkephalin system.

The concentrations of dynorphin A1-8 and Met-enkephalin-Arg6-Gly7-Leu8 were measured in the basal ganglia of postmortem brains from patients with Huntington's disease (HD) and from control subjects. A significant reduction of dynorphin A1-8 concentration was found in caudate nucleus, putamen, external globus pallidus and substantia nigra of HD brains. Levels of Met-enkephalin-Arg6-Gly7-Leu8 were reduced in HD caudate nucleus, putamen, internal and external globus pallidus. These data indicate that both the prodynorphin and proenkephalin opioid peptide system are affected in the basal ganglia in HD.

Isolation and structure of a C-terminally amidated nonopioid peptide, amidorphin-(8-26), from bovine striatum: a major product of proenkephalin in brain but not in adrenal medulla.

We have isolated and sequenced a C-terminally amidated peptide from bovine striatum. The peptide was purified to homogeneity by adsorption to XAD-2 resins and four different HPLC steps. Amino acid composition analysis and gas-phase sequence analysis revealed identity of this peptide with residues 8-26 of the proenkephalin-derived opioid peptide amidorphin, which we have recently isolated from bovine adrenal medulla. C-terminal amidation of amidorphin-(8-26) from bovine striatum was demonstrated by its stability to carboxypeptidase A digestion and full crossreactivity in a radioimmunoassay that required the C-terminal amide group as part of the recognition site. The nonopioid peptide amidorphin-(8-26), which lacks the N-terminal [Met]enkephalin sequence of amidorphin, is a major product of the opioid peptide precursor proenkephalin in the brain. In the adrenal medulla, however, where amidorphin occurs in remarkably high concentrations, amidorphin-(8-26) could not be detected. This is indicative of differential post-translational processing of proenkephalin in different tissues. In the brain, as opposed to the adrenal medulla, amidorphin is further processed at the typical cleavage signals of two basic residues, giving rise to the nonopioid peptide amidorphin-(8-26) and, possibly, to the opioid peptide [Met]enkephalin. Thus, proenkephalin in the brain might be considered as a precursor in which an opioid peptide is linked with a nonopioid peptide of possibly different biological function.

Novel opioid peptide amidorphin: characterization and distribution of amidorphin-like immunoreactivity in bovine, ovine, and porcine brain, pituitary, and adrenal medulla.

We have recently isolated from bovine adrenal medulla a novel C-terminally amidated opioid peptide, amidorphin, which derives from proenkephalin A. Amidorphin revealed a widespread distribution in bovine, ovine, and porcine tissue. Particularly high concentrations of amidorphin immunoreactivity were detected in adrenal medulla, posterior pituitary, and striatum, similar to the major gene products of proenkephalin A. In the adrenal medulla of each species, authentic amidorphin was the predominant immunoreactive form. Pituitary and brain, however, contained predominantly putative N-terminally shortened fragments of amidorphin of a slightly lower molecular weight and shorter retention times on HPLC. In addition, in ovine adrenal medulla, a putative high-molecular-weight form of amidorphin was detected. These findings are indicative of a tissue-specific processing of the proenkephalin A precursor, leading predominantly to authentic amidorphin in the adrenal medulla and further processing to smaller C-terminal fragments in the brain and pituitary.

Demonstration and characterization of alpha-human atrial natriuretic factor in human plasma.

This paper describes a highly specific and sensitive radioimmunoassay for alpha-human atrial natriuretic factor (alpha-hANF), the C-terminal 28-amino-acid residue portion of human prepro-ANF in human plasma. A novel extraction and prepurification procedure allowed for detection of levels of immunoreactive-alpha-hANF as low as 0.5 fmol/ml. In normotensive subjects, levels in the range 1-23 fmol/ml (mean = 8.9 fmol/ml) were found. Combined gel permeation and HPLC analysis demonstrated that this ir-alpha-hANF was comprised virtually exclusively of authentic 28-residue alpha-hANF. No evidence for occurrence of larger precursor forms in human plasma was acquired. A heterogenous group of hypertensive patients displayed considerably higher levels (mean = 62.2 fmol/ml), of interest in view of the hypotensive properties of ANF.

Isolation and structure of a novel C-terminally amidated opioid peptide, amidorphin, from bovine adrenal medulla.

Biologically active peptide hormones and neurotransmitters have been shown to be enzymatically liberated from larger, inactive precursor molecules by tissue-specific post-translational processing, particularly at the typical cleavage signals of paired basic residues. Subsequent N-terminal or C-terminal modifications may be of importance in regulating the biological activities of these peptides. C-terminal alpha-amidation is considered to be essential for the biological function of several non-opioid peptides. Here we present the isolation and structure of a novel C-terminally amidated opioid peptide, amidorphin, from bovine adrenal medulla. Amidorphin and the recently isolated octapeptide metorphamide (adrenorphin) are the only endogenous opioid peptides in mammals known to possess a C-terminal amide group. The amino acid sequence of amidorphin corresponds to the sequence 104-129 of bovine proenkephalin A. Very high concentrations of amidorphin were detected in bovine adrenal medulla and in a further endocrinological system, the hypothalamic-neurohypophyseal axis. Amidorphin may therefore be considered to be a major gene product of the opioid peptide precursor proenkephalin A in these endocrine tissues.

Ontogenetic development of the pro-enkephalin B (= pro-dynorphin) opioid peptide system in the rat pituitary.

The postnatal development of several pro-enkephalin-B-derived opioid peptides - dynorphin 1-17, dynorphin 1-8, dynorphin B, alpha-neo-endorphin and beta-neo-endorphin - was examined in rat pituitary lobes. The concentrations of pro-enkephalin-B-derived peptides from the anterior pituitary were between 4- and 12-fold and those from the neurointermediate pituitary between 17- and 122-fold lower in newborn as compared to adult rats. Similarly, the concentrations of vasopressin in the neurointermediate pituitary increased 50-fold between birth and adulthood; those of oxytocin, however, increased more than 540-fold over this period. The molecular weight pattern of dynorphin 1-17, dynorphin 1-8, dynorphin B, alpha- and beta-neo-endorphin-immunoreactive peptides in the anterior and neurointermediate pituitary did not differ between 3-day-old pups and adult rats. In the neurointermediate pituitary, the major immunoreactive components had the same chromatographic properties as synthetic dynorphin 1-17, dynorphin 1-8, dynorphin B, alpha- and beta-neo-endorphin, respectively, on gel filtration and high-performance liquid chromatography (HPLC). This indicates that neonatal rats were already capable of processing the precursor pro-enkephalin B into these various opioid peptides. In newborn rats, however, the amount of dynorphin 1-8 in the neurointermediate pituitary was three times lower than that of its putative intermediate precursor peptide dynorphin 1-17. Similarly, the amount of beta-neo-endorphin was almost four times lower than that of its putative precursor alpha-neo-endorphin. In contrast, in the neurointermediate pituitary of adult rats, dynorphin 1-17 and dynorphin 1-8, in addition to a alpha- and beta-neo-endorphin, occurred in equimolar amounts.(ABSTRACT TRUNCATED AT 250 WORDS)