Functional involvement of cerebral diazepam binding inhibitor (DBI) in the establishment of drug dependence

Mechanisms for formation of drug dependence and emergence of withdrawal syndrome are not yet fully understood despite of a huge accumulation of experimental and clinical data. Several clinical features of withdrawal syndrome are considered to be common (i.e., anxiety) among patients with drug dependence induced by different drugs of abuse. In this review, we have discussed the possibility of the functional involvement of diazepam binding inhibitor (DBI), an endogenous neuropeptide for benzodiazepine receptors with endogenously anxiogenic potential, in the development of drug dependence and emergence of its withdrawal symptom. The levels of DBI protein and its mRNA significantly increased in the brain derived from mice dependent on alcohol (ethanol), nicotine and morphine, and abrupt cessation of these drugs facilitated further increase in DBI expression. In the cases of nicotine- and morphine-dependent mice, concomitant administration of antagonists for nicotinic acetylcholine and opioid receptors, respectively, abolished the increase in DBI expression. Therefore, these alterations in DBI expression have a close relationship with formation of drug dependence and/or emergence of withdrawal syndrome and are considered to be a common biochemical process in drug dependence induced by different drugs of abuse.     

A brain octadecaneuropeptide generated by tryptic digestion of DBI (diazepam binding inhibitor) functions as a proconflict ligand of benzodiazepine recognition sites

An octadecaneuropeptide (ODN) produced by the tryptic digestion of DBI was purified and sequenced and its activity on the Vogel test determined. In vitro ODN displaces 3H-diazepam from specific brain recognition sites and injected intraventricularly in thirsty rats facilitates the onset of behavioral inhibition elicited by punishment. The alpha-amide derivative of ODN is devoid of either action. Evidence is presented suggesting that DBI sequence includes at least two replicas of ODN or one replica of ODN and a fragment with similar if not identical amino acid sequence but identical biological activity.     

Acute noise stress in rats increases the levels of diazepam binding inhibitor (DBI) in hippocampus and adrenal gland

We investigated the effect of acute noise-induced stress on the concentrations of diazepam binding inhibitor (DBI) and its processing products in brain regions and adrenal glands of rats. DBI levels in hippocampus began to increase at 15 and 30 min and became significantly higher (+100%) at 90 and 120 min after stress; they returned to normal values at 360 min. While basal DBI levels were similar in the left and right hippocampus, the stress-induced increase of DBI levels was significantly higher in the left compared to the right side. A significant increase was also detected in the adrenals; here, the time course of DBI increase paralleled that of previously reported plasma corticosterone in stressed rats, being significantly higher 30 min after stress, and recovering to normal values at 60 and 90 min. After acute noise-induced stress, no significant change of DBI levels was detectable in cerebral cortex, striatum, hypothalamus and cerebellum. The present study reports for the first time the occurrence of a modification of DBI and its processing products (ODN-like immunoreactivity) in an experimental model of stress, and suggests a role for these neuropeptides in emotional responses.     

A natural processing product of rat diazepam binding inhibitor, triakontatetraneuropeptide (diazepam binding inhibitor 17-50) contains an alpha-helix, which allows discrimination between benzodiazepine binding site subtypes

Synthetic peptides related to triakontatetraneuropeptide (TTN) [17TQPTDEEMLFIYSHFKQATVGDVNTDRPGLLDLK50; diazepam binding inhibitor (DBI) 17-50], a natural brain processing product of rat DBI, were analyzed for their physicochemical and ligand-receptor interaction characteristics. The ability of TTN and TTN-related fragments to displace [3H]flumazenil (ethyl-8-fluoro-5,6-dihydro-5-methyl-6-oxo-4H-imidazol[1,5a] [1,4]-benzodiazepine-3-carboxylate) or [3H]Ro 5-4864 [7-chloro-1,3-dihydro-1-methyl-5-(p-chlorophenyl)-2H-1, 4-benzodiazepine-2-one] from their respective benzodiazepine (BZ) binding site subtypes was tested in intact cerebellar culture neurons or in homogenates of cultured astrocytes. These studies indicate that the C-terminal region of TTN, which is also present in DBI 22-50, eicosapentaneuropeptide (DBI 26-50), and octadecaneuropeptide (ODN) (DBI 33-50), but not in DBI 19-41, is essential for interaction with the BZ recognition sites. When the C-terminal lysine of ODN is blocked with an NH2 group, the ability of ODN to interact with the binding of [3H]flumazenil is lost. A comparison analysis of the binding data with the secondary structure characteristics of the peptides demonstrated that TTN (DBI 17-50) and DBI 22-50, which have hydrophobic portions and marked tendencies to produce alpha-helicity, specifically displace (apparent Ki, 5-6 microM) [3H] Ro 5-4864 from astroglial cell binding sites. Peptides (ODN, eicosapentaneuropeptide, OND-NH2) with very low tendencies to form alpha-helices and with virtually no hydrophobic structure were not able to displace Ro 5-4864 at concentrations of up to 100 microM. In contrast, ODN was a good displacer of [3H]flumazenil from intact neurons, with an apparent IC50 of 5 microM. These data suggest that the alpha-helical portion of TTN may be important for BZ receptor recognition and BZ receptor subtype discrimination.     

Protein binding of indomethacin in human cerebrospinal fluid

The binding of the non-steroidal anti-inflammatory drug indomethacin to proteins in human cerebrospinal fluid (CSF), drawn during lumbar puncture from 10 patients affected by lumbosciatica, was measured by equilibrium dialysis and spectrofluorimetry. Similar binding studies on human serum albumin solutions (0.5 and 1 g/L) were performed using the same techniques. The mean binding percentage of indomethacin determined by equilibrium dialysis was 40%. The results obtained by both techniques allowed us to conclude that the binding of indomethacin in CSF was essentially due to albumin.     

3H]5-hydroxytryptamine binding sites in postmortem human brain

Binding sites for [3H]5-hydroxytryptamine (5-HT) in postmortem human frontal cortex, hippocampus and amygdala were studied by displacement with 5-HT selective drugs. The results demonstrated the selective labelling of 5-HT1-like sites by [3H]5-HT in the cortex, with little or no labelling of 5-HT2 or 5-HT3 sites. Self-displacement of the binding of [3H]5-HT is consistent with the presence of a single population of sites, indicating that 5-HT is non-selective for the 5-HT1 subtypes. Around 40% of the 5-HT1 sites in the frontal cortex and amygdala were of the 5-HT1A subtype, in contrast to 60% in the hippocampus. The drug RU 24969 consistently displaced with, a high affinity, a greater proportion of [3H]5-HT sites than did 8-OH-DPAT in all three regions of the brain. The nature of these additional sites was not established. A small proportion (less than 10%) of [3H]5-HT sites in the frontal cortex appeared to be of the 5-HT1C subtype, as these sites were displaced with high affinity by mianserin.     

Comparison of drug concentrations in postmortem cerebrospinal fluid and blood specimens

The concentration of drugs and metabolites in cerebrospinal fluid (CSF) and blood were determined in 282 autopsied cases using liquid-liquid extraction techniques and gas chromatographic analyses. All drugs were confirmed in one matrix by gas chromatography-mass spectrometry. CSF/blood ratios were used to compare the two biological fluids. Classes of drugs evaluated in this study included: benzodiazepines, anticonvulsants, sedatives, opioids, antidepressants, anesthetics, and antihistamines. The majority of the drugs tested were readily detected in CSF specimens. The average CSF/blood ratio for most drugs was in the range of 0.05-0.50. Interpretation of these results is difficult because protein binding, half-life, hydrophobic properties, and pKa of a drug, in addition to survival time after drug use, influence the CSF/blood ratio. While CSF specimens do provide a viable alternative testing matrix when blood specimens are not available, they should not be used to estimate blood drug concentrations.     

Distribution of diazepam,nordiazepam, and oxazepam between brain extraneuronal space,brain tissue,plasma, and cerebrospinal fluid in diazepam and nordiazepam dependent dogs

The compartmental distribution of diazepam (DZ) and nordiazepam (ND) and their metabolites was studied in DZ and ND dependent dogs. The levels of DZ, and ND and their metabolites were determined during the last week of stabilization in the extraneuronal brain space, in brain tissue, in plasma and in CSF. In these studies dependent dogs were anesthetized with pentobarbital and microdialysis probes were inserted bilaterally into the parietal cortex and perfused with artificial cerebrospinal fluid. Microdialysis probes were also used to determine the unbound parent drugs and their metabolites in plasma. The brain-plasma distribution of total ND and oxazepam (OX) is about equal in ND dependent dogs but in DZ dependent dogs total ND and OX are about 2-fold higher in brain than in plasma. The levels of DZ, ND, and OX in the extraneuronal brain space are similar to their unbound levels in plasma. These data suggest that the concentration of free benzodiazepines in plasma is a good approximation of the concentration in the vicinity of the membrane receptors in the dependent dogs.     

Entry of diazepam and its major metabolite into cerebrospinal fluid

Five dogs received a single 1.0 mg/kg dose of diazepam (DZ) IV. Concentrations of DZ and its major metabolite desmethyldiazepam (DMDZ) were simultaneously measured in plasma and cisternal cerebrospinal fluid (CSF) for up to 8 h after the dose by electron-capture gas-liquid chromatography. DZ was rapidly eliminated from plasma (half-life 0.3–1.3 h); DZ disappearance was mirrored by formation of DMDZ, which in turn was eliminated slowly. Both DZ and DMDZ rapidly penetrated CSF and concentrations in CSF declined parallel with those in plasma. Despite rapid uptake, the extent of CSF transfer of DZ and DMDZ was limited by plasma protein binding. Mean CSF: plasma concentration ratios for DZ (range 0.023–0.137) and DMDZ (range 0.047–0.119) were highly correlated with the unbound fraction in plasma (r=0.95 and 0.80, respectively). Thus DZ and DMDZ concentrations in CSF, presumed to reflect concentrations at the site of action, are determined by unbound plasma concentrations. The intensity of pharmacologic action is more likely to correlate with unbound than with total plasma concentrations.     

Chronic administration of methamphetamine increases the mRNA expression of diazepam binding inhibitor in rat brain

Anxiety is one of the common features of withdrawal syndrome caused by abuse-inducing drugs such as methamphetamine (MAP). The neural pathways associated with anxiety are established within the network sustained by diencephalon, cerebral cortex, cerebellum and hippocampus. Diazepam binding inhibitor (DBI), a peptide consisting of 87 amino acids, serves as an inverse agonist for the type A receptor of the gamma-aminobutyric acid (GABAA receptor) with endogenous anxiogenic potential. We examined the effect of chronic administration of MAP on the mRNA expression of DBI and DBI-related proteins, such as alpha 2 subunit of GABAA receptor (GABA-α2), peripheral-type benzodiazepine receptor (PBR), and pituitary adenylate cyclase-activating polypeptide (PACAP) in seven regions (diencephalon, cerebral cortex, cerebellum, striatum, hippocampus, midbrain, and pons-medulla) of the rat brain. The mRNA expression of DBI increased significantly in all areas of the brain, especially diencephalon, after chronic administration of MAP. The mRNA expression of PBR, GABA-α2 and PACAP increased significantly in all areas of the brain, especially cerebral cortex, after chronic administration of MAP. These results suggest that anxiety is associated with the mRNA expression of DBI as well as DBI-related genes.     

Acute administration of methamphetamine decreases the mRNA expression of diazepam binding inhibitor in rat brain

Chronic administration of methamphetamine (MAP) up-regulated the mRNA expression of diazepam binding inhibitor (DBI) in rat brain, possibly leading to anxiety. Acute effects of MAP on anxiety associated with DBI, however, are not clear. In this study, we examined the effects of acute administration of MAP on behavior related to anxiety and the expression level of DBI mRNA and pituitary adenylate cyclase-activating polypeptide (PACAP) mRNA, calibrated with the glyceraldehydes 3-phosphate dehydrogenase mRNA as the internal control in rat brain. The elevated plus-maze test was applied to the analysis of the possible anxiety-related profile of MAP. Acute administration of MAP (5 mg/kg, intraperitoneal administration) significantly increased spent time in the open-space arms at 4 h after the administration compared with a saline-treated group. The expression of DBI mRNA in a large number of regions of rat brain significantly decreased 2, 4, 8 and 16 h after acute administration of MAP. In contrast, the expression of PACAP mRNA in a large number of regions of rat brain significantly increased 4 and 8 h after the administration of MAP. These results suggest that MAP, at this dose, has an anxiolytic effect, based on the reduction of the putative anxiogenic peptides, DBI.     

Distribution and characterization of [3H]mesulergine binding in human brain postmortem

Much interest is currently being directed towards serotonin (5-HT) receptors of type 2C (5-HT_2C) because of their possible involvement in the control of different activities, such as the composition of the cerebrospinal fluid, locomotion, feeding, neuronal excitability and anxiety. The limited information regarding their distribution in the human brain prompted us to investigate, and to characterize the binding of [³H]mesulergine, a HT_2C antagonist, in autopsy samples from 24 subjects.The results showed that the [³H]mesulergine binding represented 95% of the total binding and equilibrium saturation binding experiments resulted in a single straight line, consistent with the presence of one site only. The area with the highest density of [³H]mesulergine binding was the choroid plexus, followed at a significantly lower level by the hippocampus, substantia nigra, basal ganglia, amygdala, hypothalamus and prefrontal cortex. The pharmacological profile of the [³H]mesulergine binding was consistent with that of 5-HT_2C receptors, since the most effective displacers were ritanserin, mesulergine and mianserine, followed by clozapine, ketanserine and m-CPP, while other compounds had a negligible or no effect.These findings, showing a wide distribution of [³H]mesulergine binding sites in the human brain, could provide anatomical bases for the different functions attributable to 5-HT_2C receptors in humans.     

Distribution and characterization of [3H]mesulergine binding in human brain postmortem.

Much interest is currently being directed towards serotonin (5-HT) receptors of type 2C (5-HT2C) because of their possible involvement in the control of different activities, such as the composition of the cerebrospinal fluid, locomotion, feeding, neuronal excitability and anxiety. The limited information regarding their distribution in the human brain prompted us to investigate, and to characterize the binding of [3H]mesulergine, a HT2C antagonist, in autopsy samples from 24 subjects. The results showed that the [3H]mesulergine binding represented 95% of the total binding and equilibrium saturation binding experiments resulted in a single straight line, consistent with the presence of one site only. The area with the highest density of [3H]mesulergine binding was the choroid plexus, followed at a significantly lower level by the hippocampus, substantia nigra, basal ganglia, amygdala, hypothalamus and prefrontal cortex. The pharmacological profile of the [3H]mesulergine binding was consistent with that of 5-HT2C receptors, since the most effective displacers were ritanserin, mesulergine and mianserine, followed by clozapine, ketanserine and m-CPP, while other compounds had a negligible or no effect. These findings, showing a wide distribution of [3H]mesulergine binding sites in the human brain, could provide anatomical bases for the different functions attributable to 5-HT2C receptors in humans.     

A pilot study comparing the purity, functionality and isoform composition of alpha-1-proteinase inhibitor (human) products

OBJECTIVE: Alpha-1-proteinase deficiency predisposes affected individuals to early onset pulmonary emphysema, and is treated with an alpha-1-proteinase inhibitor (A1-PI) from pooled human plasma. The objective of this pilot study was to assess analytical parameters of the three A1-PI products (Aralast, Prolastin, Zemaira) that may impact on clinical efficacy, safety, and convenience. These included: purity of the preparation; nature of impurities; functionality; and isoform composition. METHODS: Purity was evaluated using reverse phase and size exclusion chromatography high performance liquid chromatography (RP-HPLC and SEC-HPLC), capillary zone electrophoresis (CZE), sodium dodecyl sulfate polyacrylamide gel electrophoresis, sodium dodecyl sulfate capillary gel electrophoresis and Western blot analysis. The identity of protein impurities was determined by immunonephelometry; functionality by calculating the ratio of mg active A1-P1 present (by anti-neutrophil elastase activity assay) to the mg antigenic A1-PI (by immunonephelometry); and normality of the A1-PI isoform pattern by isoelectric focusing (IEF). Three samples of Zemaira and one sample each of Aralast and Prolastin were available for analysis. RESULTS: Zemaira had the highest specific activity. Using RP-HPLC analysis Zemaira averaged 99% purity, Aralast 70% and Prolastin less than 62%. Using SEC-HPLC Zemaira was 95.98% monomeric, Prolastin 79.00% and Aralast 63.55%. Prolastin had lower activity/mg antigenic A1-PI than the other two products. A shift in isoforms in Aralast was suggested by the results of CZE, and was confirmed by IEF. CONCLUSIONS: Zemaira demonstrated greater purity compared with Aralast and Prolastin. Prolastin had more inactive A1-PI than Zemaira or Aralast. Isoform ratios appeared to be altered in Aralast. The results from this pilot study warrant further investigation.     

A pilot study comparing the purity,functionality and isoform composition of alpha-1-proteinase inhibitor (human) products

ABSTRACT

Summary text: Objective: Alpha-1-proteinase deficiency predisposes affected individuals to early onset pulmonary emphysema, and is treated with an alpha-1-proteinase inhibitor (A1-PI) from pooled human plasma. The objective of this pilot study was to assess analytical parameters of the three A1-PI products (Aralast, Prolastin, Zemaira) that may impact on clinical efficacy, safety, and convenience. These included: purity of the preparation; nature of impurities; functionality; and isoform composition.

Methods: Purity was evaluated using reverse phase and size exclusion chromatography high performance liquid chromatography (RP-HPLC and SEC-HPLC), capillary zone electrophoresis (CZE), sodium dodecyl sulfate polyacrylamide gel electrophoresis, sodium dodecyl sulfate capillary gel electrophoresis and Western blot analysis. The identity of protein impurities was determined by immunonephelometry; functionality by calculating the ratio of mg active A1-P1 present (by anti-neutrophil elastase activity assay) to the mg antigenic A1-PI (by immunonephelometry); and normality of the A1-PI isoform pattern by isoelectric focusing (IEF). Three samples of Zemaira and one sample each of Aralast and Prolastin were available for analysis.

Results: Zemaira had the highest specific activity. Using RP-HPLC analysis Zemaira averaged 99% purity, Aralast 70% and Prolastin less than 62%. Using SEC-HPLC Zemaira was 95.98% monomeric, Prolastin 79.00% and Aralast 63.55%. Prolastin had lower activity/mg antigenic A1-PI than the other two products. A shift in isoforms in Aralast was suggested by the results of CZE, and was confirmed by IEF.

Conclusions: Zemaira demonstrated greater purity compared with Aralast and Prolastin. Prolastin had more inactive A1-PI than Zemaira or Aralast. Isoform ratios appeared to be altered in Aralast. The results from this pilot study warrant further investigation.     

A double-blind outpatient study of diazepam (Valium) and placebo

Summary Investigators reporting clinical studies of diazepam tend to be enthusiastic about its effectiveness in controlling anxiety, but two careful double-blind studies on hospitalized patients found diazepam relatively ineffective. This investigation covering an outpatient population of one hundred found that treatment with diazepam was not significantly superior to placebo. Moreover, diazepam treatment was also associated in some patients with the emergence of suicidal thoughts or tendencies and paranoid tendencies in others.This study was undertaken by the Psychopharmacology Research and Treatment Unit. It was supported in part by a grant from the U.S. Public Health Service (MH 05090).     

Alcohol in cerebrospinal fluid (CSF) and alcoholism.

Alcohol levels were measured in 15 cerebrospinal fluid (CSF) samples and 14 blood samples from grade III and IV male alcoholic patients with signs of nervous system involvement, and compared with levels detected in 11 CSF samples and 11 blood samples from abstemious patients or patients with grade I or II alcoholism whose CSF had been found to be normal by routine analysis (controls). Among the alcoholic patients, alcohol levels were lower in the CSF than in blood, whereas the opposite was true for the controls. The possible mechanisms underlying this difference are discussed and the need for further study of this topic is emphasized.