Autoradiographic localization of peripheral benzodiazepine binding sites in the cat brain with [H]PK 11195

“Peripheral type” benzodiazepine binding sites were labelled in cat brain membranes by using [³H]PK 11195. This ligand binds to the “peripheral type” binding sites in a reversible, specific and saturable manner. Cat brain binding sites density (6̃ pmol/mg prot.) was higher than in the rat. Pharmacological specificity was demonstrated by the potency order of displacing agents: PK 11195 > RO5-4864 > dipyridamole > diazepam > clonazepam. A similar characterization was performed in slide mounted brain sections where [³H]PK 11195 also labelled the “peripheral type” benzodiazepine binding sites. The high percentage of specific binding (80%) at 1 nM of [³H[PK 11195 made possible the autoradiographic studies on binding sites distribution. These sites were heterogeneously distributed in the grey matter and absent in white matter. Visual, auditory and other specific sensory relay stations were highly labelled. The blood pressure regulating nuclei, the vestibulo-cerebellar and the extrapyramidal motor system also presented a very high binding density. As previously described in the rat brain, choroid plexus was also strongly labelled by [³H]PK 11195 in the cat.     

Labelling of peripheral-type benzodiazepine binding sites in human brain with [3H]PK 11195: anatomical and subcellular distribution

The peripheral-type benzodiazepine binding site, erstwhile characterized in the rodent and feline brain, has now been characterized in post-mortem human brain using [3H]PK 11195. The kinetics and pharmacological properties of the binding of this ligand are similar to peripheral-type benzodiazepine binding sites elsewhere. The potency of RO5-4864 for this site in human brain is close to that seen in ruminant and carnivore tissues but considerably lower than in rodent tissues. The regional distribution of these binding sites would suggest a neuronal rather than a glial localization. [3H]PK 11195 bound in a similar fashion to slide-mounted sections of human brain, thus allowing quantitative studies of the regional distribution of peripheral-type benzodiazepine binding sites to be made. The binding sites were distributed heterogeneously, but were restricted to the grey matter. Highest densities of binding sites were found in forebrain structures. The localization was not limited to any functional system, nor did it resemble any previously described transmitter system. The similarities between peripheral-type benzodiazepine binding sites in human and in feline brain in terms of their pharmacological characteristics and their regional and subcellular distribution suggest that the cat, rather than the rat, may be the better model for studying a possible role for this site in human cerebral function.     

Peripheral-type benzodiazepine receptors in human glioblastomas: pharmacologic characterization and photoaffinity labeling of ligand recognition site

Peripheral-type benzodiazepine receptors (PBR), unlike central-type benzodiazepine receptors, are found in low concentrations in normal brain. Because PBR have been described in neoplastic cells of neuroglial origin, they have been suggested for imaging human glial tumors and for directing cytotoxic therapy at these tumors. Little information exists, however, on the presence or pharmacology of PBR in human glial tumors. Using radioligand binding techniques, we have demonstrated that 6 out of 6 glioblastoma (GBM) specimens had high concentrations of PBR [( 3H]PK 11195 binding sites) which were significantly greater than in 5 normal human frontal cortex samples. The pharmacologic specificity of these sites differed significantly from that of PBR in human and rat kidney specimens. Saturation binding experiments revealed a small number of high affinity sites and a substantial number of sites of intermediate affinity. Under in vitro binding conditions the more numerous lower affinity site is the major contributor to specific binding measurements. The ligand recognition site of the PBR in human GBM tissue was photoaffinity labeled using [3H]PK 14105, a nitrophenyl analogue of PK 11195. Subsequent SDS-polyacrylamide gel electrophoresis revealed specific incorporation of label into a 17,300 molecular weight component. There was no specific incorporation into normal human frontal cortex, but a component of very similar molecular weight was demonstrated in human kidney. We conclude that human glioblastomas consistently express PBR sites that are present in greater density than in normal human brain. Imaging of human glial tumors with analogues of PK 11195 thus appears feasible. Further molecular characterization of the photoaffinity-labeled PBR may also provide new information on the biology of these tumors.     

PET imaging of human gliomas with ligands for the peripheral benzodiazepine binding site

Human gliomas were imaged in vivo using ligands for the peripheral-type benzodiazepine binding site (or omega 3 binding site) and positron emission tomography (PET). Although gliomas have a high density of the peripheral-type benzodiazepine binding site, PET scans with a selective ligand for this site, [11C] Ro5-4864, failed to demonstrate higher radioactivity levels in human gliomas than in brain. In vitro studies of surgically removed specimens of human glioma demonstrated little binding of Ro5-4864 but high levels of binding of another selective ligand, PK 11195. Scans with [11C]PK 11195 demonstrated increased radioactivity in glioma compared to brain in 8 of 10 patients. Radioactivity in tumor and the ratios of radioactivity in tumor to that in remote gray and in white matter correlated significantly with the specific activity of [11C]PK 11195, suggesting that accumulation represents saturable high-affinity binding. We conclude that the PK 11195 manifests greater binding than Ro5-4864 to the peripheral-type benzodiazepine binding site on human gliomas and that human gliomas can be successfully imaged using [11C]PK 11195 and PET.     

Ontogeny of peripheral-type benzodiazepine receptors in cultured astrocytes and brain from rat

Peripheral-type benzodiazepine receptors (PBRs) in brain are primarily localized within astroglial cells, and the existence of PBR subtypes have been suggested. In the present study the ontogeny of PBRs labeled with [³H]Ro5-4864 and [³H]PK 11195 in cultured astrocytes was compared to their postnatal in-vivo development. The density of [³H]Ro5-4864 binding sites in cultured astrocytes from rat cortex progressively increased from 1- to 3-week-old cultures and did not change further in 5- and 8-week-old cultures. The density of [³H]PK 11195 binding sites progressively increased from 1- to 5-week-old cultures. The density of [³H]PK 11195 binding sites exceeded the density of [³H]Ro5-4864 binding sites by 40–50% in 2-, 5- and 8-week-old cultures. The affinity of the PBR ligands for the receptor sites was increased by 3- to 4-fold from the first to the second week in cultures, and did not change thereafter. A similar developmental pattern of PBRs was observed in rat cortex, except that: first, the difference between the B_max of [³H]PK 11195 and [³H]Ro5-4864 was already apparent in postnatal-week-1 and persisted with maturation; second, the high affinity of the ligands for the receptor sites was apparent from postnatal-week-1 and did not change with maturation. Age-related differences in the ratio between the density of PBRs in astrocytes and rat cortex were also observed. These results lead us to suggest that the development of PBRs in vivo during the first postnatal week is more rapid than the development of the receptors in vitro during the first week in culture. Subsequently, the increased ratio between the density of PBRs in cultured astrocytes and brain with maturation indicates the predominantly astrocytic localization of these receptors. The finding that the density of [³H]PK 11195 binding sites in cultured astrocytes and in rat brain cortex is usually 40–50% greater than the density of [³H]Ro5-4864 binding sites further supports the existence of PBR subtypes.     

Effect of increasing estradiol levels on platelet peripheral benzodiazepine binding sites in women undergoing human menopausal gonadotropin treatment

The effect of increasing serum estradiol levels on platelet [3H]PK 11195 binding was investigated to determine the influence of gonadal steroids on platelet peripheral benzodiazepine binding sites. Serum estradiol and platelet [3H]PK 11195 binding were determined in 10 anovulatory, but otherwise healthy women before and after treatment with human menopausal gonadotropin. Despite a 30-fold increase in posttreatment estradiol, the kinetic parameters of platelet [3H]PK 11195 binding (Kd, Bmax) remained essentially unchanged. These data indicate that peripheral benzodiazepine binding sites on platelets are not influenced by elevated concentrations of estradiol. Thus, it is suggested that variations in circulating levels of this female gonadal steroid are likely not responsible for the modifications in platelet peripheral benzodiazepine binding sites seen in association with various disease states.     

Iodinated PK 11195 as an ex vivo marker of neuronal injury in the lesioned rat brain

In order to study the potentials of indirect and direct detection of neuronal damages in humans by single photon emission computerized tomography (SPECT), we compared ex vivo cerebral biodistribution of [125I]PK 11195 with that of [125I]TISCH in a rat model of unilateral striatal excitotoxic lesion. Experiments on in vitro binding with [3H]PK 11195 as a ligand for peripheral type benzodiazepine binding sites (PTBBS) and [3H]SCH 23390 as a ligand for dopamine D1 receptors were also performed to validate our experimental model. We observed a very high increase in the PTBBS and a dramatic decrease in the D1 receptors on the lesioned striatum compared to the intact side. Moreover, we showed that [125I]PK 11195 bound specifically to PTBBS in lesioned cerebral areas ex vivo 5 days after striatal infusion of quinolinic acid (600 nmoles). Increases of 192%, 168%, and 30% were obtained in the striatum, the cortex, and the hippocampus, respectively, on the lesioned side. These results showed that iodinated PK 11195 bound specifically to PTBBS ex vivo and that this binding was dramatically increased at the sites of brain lesion. This ligand could therefore be suitable to detect brain injuries in humans by SPECT, complementing the information given by ligands which image loss in a specific neuron population.     

Central and peripheral type benzodiazepine ligands displace [3H][3-ME-HIS2]TRH from its binding sites in the brain and the anterior pituitary and antagonize the effect of TRH in the rat duodenum.

The effects of central (clonazepam, an agonist, and FG 7142, an inverse agonist), mixed (diazepam) or peripheral type (Ro 5-4864) benzodiazepine receptor ligands on the action of TRH on the transmurally stimulated rat duodenum and binding of [3H][3-Me-His2] TRH in the rat anterior pituitary, hypothalamus, cortex and brainstem have been studied. TRH dose-dependently inhibited the contractions of transmurally stimulated rate duodenum. Clonazepam (5 x 10(-6) M), diazepam (10(-5) M), Ro 5-4864 (10(-5) M) or FG 7142 (10(-5) M) attenuated the response of TRH in the rat duodenum. The action of these compounds was antagonized neither by the central type benzodiazepine antagonist flumazenil nor by peripheral type antagonist PK 11195 but instead PK 11195 itself counteracted TRH. TRH displaced [3H][3-Me-His2]TRH with Ki-values ranging 0.08 to 0.31 microM. Ki-values for clonazepam diazepam, Ro 5-4864, PK 11195 and FG 7142 ranged 6-117 microM, 3-23 microM, 20-67 microM, 20-40 microM and 260-420 microM, respectively, demonstrating fairly weak affinity to TRH-receptors. In saturation experiments, clonazepam and PK 11195 significantly increased KD but not Bmax of the labelled ligand while Ro 5-4864 increased both KD and Bmax. This indicates that all these compounds competitively inhibit the binding of [3H][3-Me-His2]TRH in the CNS which may also be the mechanism for their antagonism of the effect of TRH in the rat duodenum.     

Increased densities of binding sites for the 'peripheral-type' benzodiazepine receptor ligand [3H]PK 11195 in rat brain following portacaval anastomosis.

Using quantitative receptor radioautography, binding sites for the 'peripheral-type' benzodiazepine receptor ligand [3H]PK 11195 were studied in rats 4 week after end-to-side portacaval anastomosis and in sham-operated controls. Portacaval anastomosis resulted in region-selective increases in density of [3H]PK 11195 binding sites in cerebellum, pons greater than thalamus, cerebral cortex greater than hippocampus greater than striatum. Possible mechanisms implicated in these changes include (i) the action of endogenous ligands for the mitochondrial benzodiazepine receptor such as octadecaneuropeptide and (ii) neurotoxic actions of ammonia. In view of the proposed role of these receptors as modulators of intermediary metabolism and neurosteroid biosynthesis, such changes could contribute to the neurochemical mechanisms responsible for portal-systemic encephalopathy.     

Systemic injection of kainic acid: gliosis in olfactory and limbic brain regions quantified with [3H]PK 11195 binding autoradiography

Neurodegenerative diseases may result from excessive stimulation of excitatory amino acid receptors by endogenous ligands. Because neuronal degeneration is associated with glial proliferation and hypertrophy, the degenerative changes throughout rat brain following the systemic administration of kainic acid (12 mg/kg) were mapped with quantitative autoradiography of [3H]PK 11195. This radioligand binds to a mitochondrial benzodiazepine binding site (MBBS) on microglia and astrocytes. Analysis of eight horizontal and four coronal brain levels revealed up to 16-fold increases in [3H]PK 11195 binding from 1 to 5 weeks but not 1 day after kainate injection. Increases in [3H]PK 11195 binding were predominantly in ventral limbic brain regions and olfactory projections to neocortical areas, with the olfactory cortex greater than subiculum/CA1 greater than anterior olfactory nucleus, medial thalamic nucleus, and piriform cortex greater than cingulate cortex and rostral hippocampus greater than dentate gyrus, septum, and amygdala greater than entorhinal cortex and temporal cortex. Little or no enhancement of [3H]PK 11195 binding was observed in numerous regions including the caudate-putamen, substantia nigra, nucleus accumbens, olfactory tubercle, cerebellum, thalamic nuclei, choroid plexus, medulla, parietal or occipital cortex, or pons. A 2-fold greater extent of neurodegeneration was obtained in ventral portions of the olfactory bulb, entorhinal cortex, temporal cortex, and dentate gyrus compared with the dorsal portions of these structures. The pattern of increase in [3H]PK 11195 binding closely matched the patterns of neuronal degeneration reported following parenteral kainate injection. These findings strengthen the notion that quantitative autoradiography of [3H]PK 11195 is a valuable tool to quantify the extent of neuronal degeneration. Furthermore, the quantitative changes in [3H]PK 11195 binding in different limbic structures parallel their relative variation in neuropathology observed in Alzheimer's disease but not Huntington's chorea. These findings are in agreement with the idea that excessive stimulation of excitatory amino receptors may contribute to the etiology of Alzheimer's disease.     

Characterization of benzodiazepine receptors in the bovine pineal gland: evidence for the presence of an atypical binding site

Bovine and rat pineal benzodiazepine receptors were characterized using ligands with high affinities for either 'central-type' (CBR) or 'peripheral-type' (PBR) benzodiazepine receptors. The characteristics (Bmax = 83 +/- 10 fmol/mg protein, Kd = 3.88 +/- 0.46 nM) of benzodiazepine receptors in bovine pineal membranes measured with [3H]flunitrazepam (using flunitrazepam to define non-specific binding) were consistent with previously reported values. However, if non-specific binding was defined using Ro 15-1788 (a selective CBR ligand), the Bmax and Kd of [3H]flunitrazepam decreased 51 and 58%, respectively. In addition, when using PK 11195 to determine non-specific binding, the Bmax of [3H]flunitrazepam binding to bovine pineal decreased further (approximately 80%, Kd decreased approximately 39%). Together, these observations strongly suggested the presence of PBR in the bovine pineal. Bovine pineal PBR characterized with [3H]PK 11195 revealed a high density (relative to CBR) of high affinity binding sites (Kd = 1.08 +/- 0.30, Bmax = 776 +/- 33.0 fmol/mg protein). In contrast, when [3H]Ro 5-4864 (1-20 nM) was used to define PBR, no binding was detectable. These observations are in sharp contrast to the rat pineal gland, in which both [3H]Ro 5-4864 and [3H]PK 11195 bind to a large number of PBR with high affinity (Kd approximately equal to 1.9 nM, Bmax approximately equal to 26 pmol/mg protein). Bovine pineal PBR were further characterized with compounds structurally related to either Ro 5-4864 or PK 11195.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of the peripheral-type benzodiazepine receptors in cultured astrocytes: Evidence for multiplicity

In mammalian brain peripheral benzodiazepine (PBZD) receptors are predominantly localized on astroglial cells. Previous studies utilizing whole membrane preparations from brain and peripheral organs of various species have indicated several distinctions between the drug-receptor interactions of the two prototypic PBZD receptor ligands, PK 11195 and Ro5-4864. The present study was undertaken to determine whether putative differences in the binding of PBZD receptor ligands in homogenates of primary astrocyte cultures can be interpreted as the labeling of PBZD receptor subtypes. Equilib-rium competition and saturation binding experiments in homogenate preparations of primary astrocytes from cerebral cortex of new born rats revealed that [³H]PK 11195 labels twice the number of [³H]Ro5-4864 binding sites. Unlabeled Ro5-4864 competes for [³H]PK 11195 binding in a manner suggesting the existence of multiple PK 11195 bind-ing sites. The competition binding experiments, using various benzodiazepines, indicate that one binding component of PK 11195 corresponds to Ro5-4864 binding sites, whereas the second is different. The latter binding site does not correspond to the central BZD receptor but displays the pharmacological properties of the PBZD receptor. Further differences between the binding of PK 11195 and Ro5-4864 in astrocytes were detected in the presence of ethanol which was more effective in inhibiting the binding of the latter. Subcellular distribution studies indicated, however, that the binding of both [³H]PK 11195 and [³H]Ro5-4864 is associated primarily with the mitochondrial fraction of astro-cytes. Taken together, the present study indicates the existence of non-overlapping PBZD binding sites in astrocytes and thus suggests the existence of PBZD receptor subtypes. It appears, however, that there is no distinction in the subcellular localization of the putative subtypes of the PBZD receptor. © 1993 Wiley-Liss, Inc.     

The positron emission tomography ligand DAA1106 binds with high affinity to activated microglia in human neurological disorders

Chronic microglial activation is an important component of many neurological disorders, and imaging activated microglia in vivo will enable the detection and improved treatment of neuroinflammation. 1-(2-chlorphenyl)-N-methyl-N-(1-methylpropyl)-3-isoquinoline-carbox-amide (PK11195), a peripheral benzodiazepine receptor ligand, has been used to image neuroinflammation, but the extent to which PK11195 binding distinguishes activated microglia and reactive astrocytes is unclear. Moreover, PK11195 may lack sufficient sensitivity for detecting mild neuroinflammation. We hypothesized that N-(2,5-dimethoxybenzyl)-N-(4-fluoro-2-phenoxyphenyl) acetamide (DAA1106), a new ligand that binds specifically to peripheral benzodiazepine receptor, binds to activated microglia in human neurological diseases with higher affinity than does PK11195. We therefore compared the pharmacological binding properties of [3H](R)-PK11195 and [3H]DAA1106 in postmortem tissues from patients with cerebral infarcts, amyotrophic lateral sclerosis, Alzheimer disease, frontotemporal dementia, and multiple sclerosis (n=10 each). In all diseases, [3H]DAA1106 showed a higher binding affinity as reflected by lower dissociation constant (KD) values than that of [3H](R)-PK11195. Moreover, specific binding of both ligands correlated with the presence of activated microglia identified by immunohistochemistry in situ. We conclude that 1) ligands that bind peripheral benzodiazepine receptor mainly label activated microglia in human neurological disorders and that 2) DAA1106 may possess binding characteristics superior to those of PK11195, which may be beneficial for in vivo positron emission tomography imaging.     

Distribution of PK11195 binding sites in porcine brain studied by autoradiography in vitro and by positron emission tomography

The cerebral distribution of peripheral-type benzodiazepine binding sites (PBBS) in human brain has been investigated by positron emission tomography (PET) with the specific radioligand [11C]PK11195 in diverse neuropathological conditions. However, little is known about the pattern of PK11195 binding sites in healthy brain. Therefore, we used quantitative autoradiography to measure the saturation binding parameters for [3H]PK11195 in cryostat sections from young Landrace pigs. Specific binding was lowest in the cerebellar white matter (85 fmol mg(-1)) and highest in the caudate nucleus (370 fmol mg(-1)), superior colliculus (400 fmol mg(-1)), and anterior thalamic nucleus (588 fmol mg(-1)). The apparent affinity was in the range of 2-6 nM in vitro, predicting high specific binding in PET studies of living brain. However, the distribution volume (V(d), ml g(-1)) of high specific activity [11C]PK11195 was nearly homogeneous (3 ml g(-1)) throughout brain of healthy Landrace pigs, and was nearly identical in studies with lower specific activity, suggesting that factors in vivo disfavor the detection of PBBS in Landrace pigs with this radioligand. In young, adult G?ttingen minipig brain, the magnitude of V(d) for [11C]PK11195 was in the range 5-10 ml g(-1), and had a heterogeneous distribution resembling the in vitro findings in Landrace pigs. There was a trend toward globally increased V(d) in a group of minipigs with acute MPTP-induced parkinsonism, but no increase in V(d) was evident in the same pigs rescanned at 2 weeks after grafting of fetal mesencephalon to the partially denervated striatum. Thus, [11C]PK11195 binding was not highly sensitive to constituitively expressed PBBS in brain of young Landrace pigs, and did not clearly demonstrate the expected microglial activation in the MPTP/xenograft model of minipigs.     

Localization of the Endogenous Benzodiazepine Receptor Ligand Octadecaneuropeptide and Peripheral Type Benzodiazepine Receptors in the Rat Pituitary

An association of octadecaneuropeptide, an endogenous ligand at the benzodiazepine receptor, with the peripheral type benzodiazepine receptor has been reported in brain as well as a few peripheral tissues. In order to verify whether or not such an association occurs in the rat pituitary gland, we have proceeded to the immunocytochemical localization of octadecaneuropeptide as well as the autoradiographic localization of peripheral type benzodiazepine receptors in rat pituitary, octadecaneuropeptide immunoreactive material was found in high concentrations in the posterior lobe, whereas only a very few cells were labelled in the intermediate lobe. The anterior lobe did not show any specific staining. Immunoelectron microscopy revealed that in the posterior lobe labelling was restricted to pituicytes. Autoradiographic studies demonstrated a strong and uniform labelling in the posterior lobe after incubation with [³H] PK11195, a ligand selective for peripheral type benzodiazepine receptors. In the intermediate lobe, the autoradiographic reaction was restricted to a narrow band adjacent to the hypophysial cleft. No labelling was detected in the anterior lobe. These results demonstrate a close association between octadecaneuropeptide and peripheral type benzodiazepine receptors in the intermediate and posterior lobes of the rat pituitary gland.     

The high affinity peripheral benzodiazepine receptor ligand DAA1106 binds specifically to microglia in a rat model of traumatic brain injury: implications for PET imaging

Traumatic brain injury (TBI) is a significant cause of mortality, morbidity, and disability. Microglial activation is commonly observed in response to neuronal injury which, when prolonged, is thought to be detrimental to neuronal survival. Activated microglia can be labeled using PK11195, a ligand that binds the peripheral benzodiazepine receptor (PBR), receptors which are increased in activated microglia and sparse in the resting brain. We compared the binding properties of two PBR ligands PK11195 and DAA1106 in rats using the controlled cortical impact (CCI) model of experimental TBI. While both ligands showed relative increases with specific binding in the cortex ipsilateral to injury compared to the contralateral side, [(3)H]DAA1106 showed higher binding affinity compared with [(3)H](R)-PK11195. Combined immunohistochemistry and autoradiography in brain tissues near the injury site showed that [(3)H]DAA1106 binding co-registered with activated microglia more than astrocytes. Further, increased [(3)H]DAA1106-specific binding positively correlated with the degree of microglial activation, and to a lesser degree with reactive astrocytosis. Finally, in vivo administration of each ligand in rats with TBI showed greater retention of [(11)C]DAA1106 compared to [(11)C](R)-PK11195 at the site of the contusion as assessed by ex vivo autoradiography. These results in a rat model of TBI indicate that [(11)C]DAA1106 binds with higher affinity to microglia when compared with PK11195, suggesting that [(11)C]DAA1106 may represent a better ligand than [(11)C](R)-PK11195 for in vivo PET imaging of activated microglia in TBI.     

Increase in central and peripheral benzodiazepine receptors following surgery

[3H]Flunitrazepam, [3H]PK 11195, [3H]quinuclidinyl benzilate (QNB) and monoamine oxidase (MAO) A and B activity were measured in male rats 1, 3 and 7 days following laparotomy. The surgery resulted in the up-regulation of central benzodiazepine (BZ) receptors in cerebral cortex and of peripheral BZ binding sites in brain and kidney on the first and third days after operation. This increase was followed by a decrease to normal range 7 days after the surgical procedure. [3H]QNB binding to muscarinic receptors in the cerebral cortex as well as MAO A and B activity in rat cerebral cortex and kidney were not affected by the surgical manipulation. The modulatory effect of surgery on BZ receptors corresponds to stages of the healing process in surgical wounds.     

Chronic diazepam treatment induces an increase in peripheral benzodiazepine binding sites

The effect of chronic (21 days) diazepam treatment (0.5 mg/kg, intraperitoneally) on [3H]PK 11195 binding to the heart and cerebral cortex was studied. A significant increase in the density of peripheral benzodiazepine binding sites in the heart (18%) and cerebral cortex (19%) was observed. Following five days of withdrawal, the maximal binding capacity of these sites returned to normal control values. No alteration in the equilibrium dissociation constant was observed during the treatment and withdrawal period.     

Imaging peripheral benzodiazepine receptors in brain tumors in rats: in vitro binding characteristics

Peripheral benzodiazepine binding constants for transplanted RG-2 gliomas and HD and LK Walker 256 tumors (metastatic breast carcinoma) were determined in Wistar rats using autoradiography. In addition, Kd and Bmax parameters for peripheral benzodiazepine receptors on RG-2 tumors were directly visualized using digital image analysis of autoradiograms. High specific binding of [3H]PK11195, a selective peripheral benzodiazepine ligand, had excellent topographical correlation to areas of histologically verified tumor. Scatchard analysis suggested a single class of peripheral binding sites with similar binding affinities in RG-2 and LK Walker 256 tumors and normal cortex. Bmax was 20-fold greater in glial tumors and 11.6- and 10.6-fold greater in LK and HK Walker 256 tumors, respectively, compared to normal cortex. The location of metastatic tumors, either intracerebrally or subcutaneously, did not effect their Kd or Bmax values. Kd and Bmax values for RG-2 tumors were similar whether determined densitometrically or by direct visualization with image analysis. Binding parameters within normal brain were difficult to visualize by image analysis due to the low level of specific binding. The ability to label specifically intracerebral tumor cells and to characterize the binding parameters shown in this study suggest that peripheral benzodiazepine receptor ligands could be utilized by PET to analyze directly a variety of tumors in humans.     

Central and peripheral benzodiazepine receptors in rat brain and platelets: effects of treatment with diazepam and clobazam.

Tolerance to the effects of benzodiazepines (BZ) may be mediated by changes in benzodiazepine receptors (BZRs). Peripheral BZRs (in brain and platelets) and central BZRs (in brain) were measured in rats following intraperitoneal administration of diazepam and clobazam each for 4 and 12 days. BZRs were measured by binding assays using [3H] PK 11195 (peripheral) and [3H] flunitrazepam (central) as radioligands. Diazepam, but not clobazam, increased peripheral BZR numbers in platelets (both P < 0.005), but not in brain, after 4 and 12 days' treatment compared with appropriate controls. Neither drug altered central BZR affinities or numbers in rat brain. BZ effects on peripheral BZRs in platelets cannot be extrapolated to predict changes in brain receptors, either peripheral or central.