Pharmacokinetic analysis of the FAK scaffold inhibitor C4 in dogs.

Inhibition of focal adhesion kinase-vascular endothelial growth factor receptor 3 complex by C4 was previously shown to reduce tumor growth alone and synergistically with other chemotherapeutic agents in animal tumor models. Single and multiple dose IV and oral dosing studies were performed in dogs to determine C4 pharmacokinetics. C4 was administered to 4 dogs at 1.25 or 2.50 mg/kg IV, or 7.50 mg/kg oral gavage. Single- (IV and oral) and multiple- (IV) dose pharmacokinetic samples were collected on days 1 and 3 at pre-dose and 0.5, 1, 2, 4, 8, 24, 120, 144, and 168 h post-dose. C4 concentrations were determined using liquid chromatography with tandem mass spectral detection with a limit of quantitation of 2.50 pg/mL. Pharmacokinetics of C4 was characterized by a 3-compartment model with linear distributional and elimination clearances using Phoenix 64 WinNonlin 6.3. Mean C4 plasma concentration-time profiles revealed a triexponential decline following either IV or oral administration, independent of dose with no accumulation. For the 2.5 mg/kg dose, the median half-life was ~21 h. Median C max and area under the curve (AUC0-24) were similar for days 1 and 3. Oral bioavailability for formulations of PBS, TPGS, Maalox(®), and Pepcid(®) was greatest with TPGS (45 %), followed by Maalox(®) (42 %), Pepcid(®) (37 %), and PBS (30 %). The pharmacokinetic study revealed that C4 has linear pharmacokinetics and does not accumulate following multiple-dose administration. Characterization of C4 pharmacokinetics provides a better understanding of the novel targeted agent, which will help facilitate further development of C4.

A novel fluorescent histamine H(1) receptor antagonist demonstrates the advantage of using fluorescence correlation spectroscopy to study the binding of lipophilic ligands.

BACKGROUND AND PURPOSE: Fluorescent ligands facilitate the study of ligand-receptor interactions at the level of single cells and individual receptors. Here, we describe a novel fluorescent histamine H(1) receptor antagonist (mepyramine-BODIPY630-650) and use it to monitor the membrane diffusion of the histamine H(1) receptor. EXPERIMENTAL APPROACH: The human histamine H(1) receptor fused to yellow fluorescent protein (YFP) was transiently expressed in CHO-K1 cells. The time course of binding of mepyramine-BODIPY630-650 to the H(1) receptor was determined by confocal microscopy. Additionally, fluorescence correlation spectroscopy (FCS) was used to characterize the diffusion coefficient of the H(1) receptor in cell membranes both directly (YFP fluorescence) and in its antagonist-bound state (with mepyramine-BODIPY630-650). KEY RESULTS: Mepyramine-BODIPY630-650 was a high-affinity antagonist at the histamine H(1) receptor. Specific membrane binding, in addition to significant intracellular uptake of the fluorescent ligand, was detected by confocal microscopy. However, FCS was able to quantify the receptor-specific binding in the membrane, as well as the diffusion coefficient of the antagonist-H(1) receptor-YFP complexes, which was significantly slower than when determined directly using YFP. FCS also detected specific binding of mepyramine-BODIPY630-650 to the endogenous H(1) receptor in HeLa cells. CONCLUSIONS AND IMPLICATIONS: Mepyramine-BODIPY630-650 is a useful tool for localizing the H(1) receptor using confocal microscopy. However, its use in conjunction with FCS allows quantification of ligand binding at the membrane, as well as determining receptor diffusion in the absence of significant bleaching effects. Finally, these methods can be successfully extended to endogenously expressed untagged receptors in HeLa cells.

Pyrilamine in the horse: detection and pharmacokinetics of pyrilamine and its major urinary metabolite O-desmethylpyrilamine.

Pyrilamine is an antihistamine used in human and veterinary medicine. As antihistamines produce central nervous system effects in horses, pyrilamine has the potential to affect the performance of racehorses. In the present study, O-desmethylpyrilamine (O-DMP) was observed to be the predominant equine urinary metabolite of pyrilamine. After intravenous (i.v.) administration of pyrilamine (300 mg/horse), serum pyrilamine concentrations declined from about 280 ng/mL at 5 min postdose to about 2.5 ng/mL at 8 h postdose. After oral administration of pyrilamine (300 mg/horse), serum concentrations peaked at about 33 ng/mL at 30 min, falling to <2 ng/mL at 8 h postdose. Pyrilamine was not detected in serum samples at 24 h postdosing by either route. After i.v. injection of pyrilamine (300 mg/horse) O-DMP was recovered at a level of about 20 microg/mL at 2 h postdose thereafter declining to about 2 ng/mL at 168 h postdose. After oral administration, the O-DMP recovery peaked at about 12 microg/mL at 8 h postdose and declined to <2 ng/mL at 168 h postdose. These results show that pyrilamine is poorly bioavailable orally (18%), and can be detected by sensitive enzyme-linked immunosorbent assay tests in urine for up to 1 week after a single administration. Care should be taken as the data suggest that the withdrawal time for pyrilamine after repeated oral administrations is likely to be at least 1 week or longer.

Pyrilamine and O-desmethylpyrilamine detection in equine serum and urine.

Pyrilamine (mepyramine) is an H1-receptor antagonist used in human and veterinary medicine. It has the potential to produce central nervous system effects in horses and therefore may have some impact on an outcome of a horse race. A single oral dose of pyrilamine (300 mg/horse) was given to three animals. Serum samples were collected before drug administration and at 0.25, 0.5, 1, 2, 4, 6, 24, 48, 72, 96, 120, and 144 h, and 7, 8, 9, 10, 11, 12, and 13 days post-administration. Urine samples were collected at 0-1, 1-2, 2-4, 4-6, 24, 48, 72, 96, 120, and 144 h, and 7, 8, 9, 10, 11, 12, 13 days post-administration. Urine and serum samples were initially screened by the pyrilamine enzyme-linked immunosorbent assay (ELISA) kit with subsequent confirmation and quantitation utilizing a newly developed and validated gas chromatography-mass spectrometry (GC-MS) method for pyrilamine and its major metabolite O-desmethylpyrilamine with chlorpromazine as an internal standard. Prior to the basic extraction, urine specimens were hydrolyzed using beta-glucuronidase. The urine extracts as well as the serum samples were then subjected to solid-phase extraction on Bond Elut LRC-PRS columns. Pyrilamine was not found in any of the urine samples but it was present in serum in low concentrations (4-123 ng/mL) up to 6 h after drug administration. The limit of detection and limit of quantitation for the GC-MS method for pyrilamine in serum were 1.5 and 3.1 ng/mL, respectively, and for O-desmethylpyrilamine in urine were 5 and 6.2 ng/mL, respectively. Pyrilamine concentration in serum peaked at 15 min, 30 min, and 1 h in horse #1, #2, and #3, respectively. Urine specimens were screened positive for pyrilamine and its metabolites using ELISA for extended periods of time (4 days in one horse and 9 days in two other animals). Using GC-MS, O-desmethylpyrilamine was detected in urine for 11 days in horse #1, 4 days in horse #2, and 9 days in horse #3. While pyrilamine was eliminated from the bloodstream rather quickly, the metabolite level remained in the urine for days after administration. When evaluating laboratory results, regulators must take into account that a urine sample positive for O-desmethylpyrilamine does not necessarily indicate that the drug remains active in the horse's system, possibly affecting the outcome from the race.

Synthesis and pharmacological activity of fluorescent histamine H1 receptor antagonists related to mepyramine.

Fluorescently labeled histamine H(1) receptor antagonists were synthesized starting from N-demethylmepyramine by introduction of omega-aminoalkyl chains (2-8 methylene groups in length) followed by derivatization of the terminal NH(2) group with various fluorophores (fluorescein, naphthofluorescein, rhodamine, tetramethylrhodamine, BODIPY, dansyl, and nitrobenzoxadiazole (NBD)). On the isolated guinea pig ileum and in a Ca(2+) assay on U373MG human glioblastoma cells the highest H(1) antagonistic activities were found in 5- and 6-carboxyfluorescein labeled compounds with hexa- and octamethylene spacers and in an analogous NBD-aminohexanoyl derivative (pA(2) or pK(B) values in the range: 8.3-9.0; compared to 9.3-9.4 for mepyramine).

GBR compounds and mepyramines as cocaine abuse therapeutics: chemometric studies on selectivity using grid independent descriptors (GRIND).

Cocaine is one of the most widely abused drugs in the industrial world. Substantial evidence has accumulated that the dopamine transporter (DAT) is a key target for cocaine regarding its reinforcing effects. This work describes the application of chemometric methods to a data set of 54 N(1)-benzhydryl-oxy-alkyl-N(4)-phenyl-alk(en)yl-piperazines (GBR compounds) and chemically related mepyramines as putative candidates in cocaine abuse therapy. The aim of the study is to gain insight into the structural requirements that determine the affinity of the data set molecules to the DAT and the serotonin transporter (SERT) as well as their inhibitory potency on dopamine uptake. The compounds in the dataset are described using the recently developed GRID independent descriptors (GRIND), which allow one to obtain fast three-dimensional quantitative structure-activity relationship models without the need of aligning and superimposing the structures; the results are interpreted in a convenient pharmacophoric-like fashion. In the first part of the work, the selectivity of the database molecules for DAT binding vs dopamine reuptake inhibition is investigated. In the second part, the selectivity of the compounds for DAT binding vs SERT binding is studied. In both cases, significant models are obtained, which define the structural features responsible for the respective selectivity profiles. Moreover, the information has potential interest for the design of new derivatives with improved selectivity.

Efficacy and safety of mepyramine-theophylline-acetate in the treatment of asthmatic crisis in children.

Mepyramine-theophylline-acetate (MTA), a theophylline derivative combined with an antihistamine, is used to treat patients with asthma. A double-blind, randomized, prospective, parallel-group study was conducted to evaluate the efficacy and safety of MTA in the treatment of asthmatic crisis in children 2 to 6 years of age. Forty patients with mild-to-moderate asthma were admitted to the study. The MTA group received 8 mg/kg per day of MTA by mouth in three divided doses for 7 days. The other group received 50 microL/kg per day of placebo in three divided doses for 7 days. Salbutamol (albuterol) syrup was used as the rescue drug if manifestations of asthma persisted. Both the MTA group and the placebo group had similar demographic characteristics at baseline. Both groups showed improvement of the asthma symptoms (cough, dyspnea, hypoventilation, and wheezing), as evaluated by the investigators at days 3 and 7. Patient diary scores showed earlier improvements in the MTA group than in the placebo group. Both groups showed improvement in peak flow at days 3 and 7 (P = 0.005). The control group used more doses of salbutamol than the MTA group on days 2 through 6 and globally (mean +/- SD, 6.79 +/- 9.11 doses vs 1.29 +/- 2.23 doses). The improvements in the placebo group were thought to be due to salbutamol. Three MTA patients dropped out of the trial, one because the parents felt that the treatment was not effective and two because of gastrointestinal manifestations (epigastric discomfort and vomiting). In the placebo group, two patients dropped out. One patient had epigastric discomfort and the other had to be treated in the emergency department for an exacerbation of the asthma. We conclude that MTA may be a good therapeutic option for the treatment of asthmatic crisis in children 2 to 6 years of age.

[Synthesis and combined H1-/H2 antagonist activity of mepyramine, pheniramine and cyclizine derivatives with cyanoguanidine, urea and nitroethenediamine partial structures]. / Synthese und kombinierte H1-/H2-antagonistische Aktivität von Mepyramin-, Pheniramin- und Cyclizin-Derivaten mit Cyanoguanidin-, Harnstoff- und Nitroethendiamin-Partialstrukturen.

Compounds with combined histamine H1- and H2-receptor antagonist activity were synthesized by connecting H1- and H2-receptor substructures via cyanoguanidine, urea, or nitroethenediamine moieties. Loss of the strongly basic side-chain nitrogen results in a decrease of H1-receptor activity compared to single reference compounds. At the guinea-pig right atrium (H2-receptor model) compounds with mepyramine or cyclizine structure are also less active than the single references tiotidine, ranitidine, or lamtidine. Nevertheless substances with a pheniramine like partial structure proved to be potent histamine H2-receptor antagonists at the atrium model (about 27 times more active than cimetidine).

[Evaluation of the efficacy and safety of mepifylline in oral solution in children with mild and moderate asthmatic crises]. / Valoración de la eficacia y seguridad de la mepifilina en solución oral en niños con crisis de asma leve a moderada.

Efficacy and safety of mepiphylline, a derivative of theophylline, was evaluated in a group of children, aged 6 to 10 years, with mild or moderate acute asthma. A parallel, randomized, double-blind, placebo controlled, prospective study was performed in 40 children. Twenty one of them received mepiphylline in dose of 8 mg/kg/día divided in 3 during 10 days, and 19 (control group) received placebo. Salbutamol aerosols were available in both groups. Clinical and spirometric data were collected before the beginning of the treatment (pre-and-post-nebulized salbutamol), and at the 3rd, 7th and 10th days. Children and parents cooperated with a diary of symptoms, peak-flow measurements and account of salbutamol used. A total relief of symptoms was found in 14 patients in the mepiphylline group and just 8 of the control group, with no significant differences. Neither spirometry nor diary data showed significant differences. Salbutamol was less than 3 days of unnecessary in 13 patients (61.9%) in the mepiphylline group and 8 patients (42.1%) in the control group (p < 0.05; Kolmogorov-Smirnov two samples Test). We conclude that mepiphylline could be a complementary treatment of mild and moderate acute asthma with a good safety in children.

Biochemical characterization of histamine H1 receptors in bovine adrenal medulla.

Bovine adrenal medullary membranes display high affinity and saturable binding to [3H]mepyramine, a selective H1 antagonist, with Kd of 1.5 +/- 0.1 nM and Bmax of 694 +/- 12 fmol/mg protein. [3H]Azidobenzpyramine, an azidobenzamide derivative of mepyramine, was synthesized and used to photolabel the high affinity mepyramine binding sites. Following photolysis, a protein component with an approximate molecular weight of 53-58 kDa was shown to be covalently labeled, as judged by gel filtration and SDS/PAGE; labeling being greatly reduced in the presence of excess unlabeled mepyramine. These results indicate that bovine adrenal medulla expresses a large number of H1 receptors, which are pharmacologically and biochemically indistinguishable from the H1 receptor of many other tissues of various species.

Reversible and irreversible labelling of H1- and H2 -receptors using novel [125I] probes.

We have recently designed the first 125I-labelled probes specific for the histamine H1 and H2 receptors. These reversible and irreversible antagonists are among the most potent H1 and H2 ligands and have enabled investigations into the biochemical and pharmacological properties of these two receptors. In various brain animal species, the ligand binding peptide of the H1 and H2 receptors, as determined by photoaffinity labeling, resides within 56-59 kDa peptides. In contrast, in guinea pig heart, the ligand binding domain of the H1 receptor is characterized by a higher molecular weight (68 kDa), suggesting the presence of an isoform of this protein, clearly differentiable by this biochemical property but not by its pharmacology. The reversible 125I-probes allowed us to extend the pharmacology of these receptors in several biological preparations and in human brain, and to establish their interaction with G-proteins. A detailed mapping of H1 and, for the first time, of H2 receptors, has been achieved in guinea pig brain, establishing their presence in almost all brain areas. These experiments show that there is no correlation between the density of H2 receptor and the activity of adenylate cyclase sensitive to histamine suggesting a molecular heterogeneity of this receptor.

High-performance liquid chromatography of the antihistamine pyrilamine and its N-oxide using electrochemical detection.

The electrochemical behavior of the over-the-counter antihistamine drug pyrilamine and its N-oxide analogue, have been studied by several voltammetric methods. Cyclic voltammograms of pyrilamine maleate in 0.1 M ammonium acetate at pH 7.0 indicated a quasi-reversible electrode process by observing a wave at + 0.85 V and + 1.30 V in the initial anodic sweep followed by a wave at - 1.30 V versus Ag/AgCl. Differential pulse and hydrodynamic voltammetry of pyrilamine and the N-oxide were examined to determine oxidation potentials for use in high-performance liquid chromatography with electrochemical detection (HPLC-ED). Differentiation between pyrilamine and its N-oxide was achieved in HPLC-ED analyses at a detection potential of + 0.7 V and + 0.9 V versus Ag/AgCl with tandem ultraviolet detection at 254 nm. Utility of the HPLC-ED method was demonstrated by the analysis of pyrilamine and the N-oxide in microbial biotransformation samples.

Three histamine receptors (H1, H2 and H3) visualized in the brain of human and non-human primates.

The distribution of histamine H1, H2 and H3 receptors in postmortem human and rhesus monkey brain was examined using receptor autoradiography. [125I]Iodobolpyramine, [125I]iodoaminopotentine and [3H](R) alpha-methylhistamine were used as ligands to label H1, H2 and H3 receptors respectively. The 3 receptor subtypes were identified in the human and monkey brains. Each receptor presented comparable distribution in the two primate brains. H1 and H2 receptors were particularly enriched in the caudate and putamen and observed in other brain areas such as the neocortex and hippocampus. H3-receptors were found to predominate in the basal ganglia where the highest densities were localized in the two segments of the globus pallidus. They were also observed in the hippocampus and cortical areas. The distribution of these 3 histamine receptors in the primate brain suggests the involvement of histaminergic mechanism in the functions of many brain areas. In particular, H2 and H3 receptors could play a role in the regulation of the basal ganglia functions in primates.

Histamine H1-receptor in heart: unique electrophoretic mobility and autoradiographic localization.

Histamine H1-receptors, visualized in the guinea pig heart by autoradiography using [125I]iodobolpyramine as a specific probe, are abundant in the nodal tissue and cardiac vessels but also occur heterogeneously in the myocardium. Following photoaffinity labeling with [125I]iodoazidophenpyramine and electrophoresis, the ligand binding domain of the heart H1-receptor was shown to be present on a major 68-kDa and a less abundant 54- to 58-kDa protein. The 68-kDa protein displayed a molecular size higher in heart than in all other tissues (56 kDa). This indicates the existence of at least two isoforms of the H1-receptor; the cardiac isoform, however, was pharmacologically indistinguishable from the common isoform studied in cerebellar membranes using available ligands. Its distinct electrophoretic properties suggest that the cardiac isoform may have a unique function.

Characterization of histamine H1 binding sites on human T lymphocytes by means of 125I-iodobolpyramine. Preferential expression of H1 receptors on CD8 T lymphocytes.

The presence of histamine H1 receptors on lymphocytes has been indirectly suggested by the various effects of agonists or antagonists on the functionally distinct T lymphocyte subsets. Recently, a new H1 antagonist, 125I-iodobolpyramine, whose structure is similar to mepyramine, has become available for the detection of H1 receptors in guinea pig brain. When using 125I-iodobolpyramine on human T lymphocytes, the presence of a single highly specific H1 binding site was evidenced. The binding of 125I-iodobolpyramine to human T cells was reversible when using 1000-fold excess of the cold H1 antagonist, d-chlorpheniramine. Binding saturation was achieved at 0.60-0.65 nM of 125I-iodobolpyramine, the binding equilibrium was reached in 20-30 min at 27 degrees C. The dissociation constant was KD = 0.41 +/- 0.07 (mean +/- SE) and the number of receptors per T cell was 3407 +/- 592 (mean +/- SE) as deduced from saturation and kinetic curves. In competition experiments using a panel of H1 ligands, the T cell binding sites detected by 125I-iodobolpyramine showed a pharmacological behavior characteristic of histamine H1 receptors. It was of particular interest that 125I-iodobolpyramine binding displayed clearcut stereoselectivity as assessed by the higher affinity of the d-configuration of chlorpheniramine than the l form. Study of purified CD4 and CD8 T cells showed that twice as much H1 histamine receptors were expressed by CD8 T lymphocytes (6615 +/- 1125) as compared to CD4 T cells (3545 +/- 459). These results underline the need for studying the functional properties of such pharmacologically defined T lymphocyte H1 binding sites.

Photoaffinity labeling and electrophoretic identification of the H1-receptor: comparison of several brain regions and animal species.

The photoaffinity probe [125I]iodoazidophenpyramine was used to label irreversibly the H1-receptor in membranes of several guinea pig brain regions and of the cerebral cortex of the rat, mouse, and pig. Following sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis, two main bands were specifically labeled in all tissues: a 56-kilodalton (kDa) peptide and a 41-47-kDa peptide whose relative importance diminished in the presence of protease inhibitors. This indicates that, in all tissues examined, in spite of evidence for pharmacological heterogeneity, the ligand recognition domain of the H1-receptor resides in a 56-kDa peptide.

A detailed mapping of histamine H1-receptors in guinea-pig central nervous system established by autoradiography with [125I]iodobolpyramine.

[125I]Iodobolpyramine, a potent and selective histamine H1-receptor antagonist derived from mepyramine, was used to generate light microscopic autoradiograms on sections of guinea-pig brain and spinal cord. Histamine H1-receptors were labelled with high sensitivity over a low background as determined using mianserin or other H1-receptor antagonists as competing agents. An atlas of H1-receptors was established using five sagittal sections and 39 frontal sections, the latter serially prepared at 50 micron intervals. Labelled areas were identified by comparison with corresponding, classically stained sections and their density was rated according to an arbitrary scale. Autoradiographic grains were detected in a large variety of gray matter areas whereas they were generally absent from white matter areas. In the cerebral cortex, H1-receptors are present in all areas and layers with a higher density in lamina IV. In the hippocampal formation, H1-receptors display a laminated pattern of distribution and are the most abundant in the dentate gyrus (hilus and molecular layer) and in several areas of the subiculum and commissural complex. In the amygdaloid complex, the highest densities are found in the medial group of nuclei. In the basal forebrain, the striatum is moderately labelled whereas the nucleus accumbens, islands of Calleja and most septal nuclei are highly labelled. In the thalamus, H1-receptors are present in high density, particularly in the anterior, median and lateral groups of nuclei. In the hypothalamus the labelling is highly heterogeneous with high densities in, for example, medial preoptic area, dorsomedial, ventromedial and most posterior nuclei, including the tuberomammillary complex in which histaminergic perikarya and short axons are present.(ABSTRACT TRUNCATED AT 250 WORDS)

Polyclonal and monoclonal antibodies directed against SK & F 94461, a specific H1 histamine receptor ligand.

SK & F 94461, an aminopentyl analogue of mepyramine, is a recently described H1 receptor antagonist. At variance with the other available H1 receptor ligands, SK & F 94461 offers the possibility of coupling to a protein carrier to render the molecule immunogenic. SK & F 94461 coupled to succinylated bovine serum albumin was used as an immunogen to raise polyclonal antibodies in rabbits and BALB/c mice. In parallel, spleen cells from immunized mice were used to produce hybridomas by somatic cell fusion. Thus, six different murine monoclonal antibodies sharing anti-SK & F 94461 specificity were selected for further detailed characterization of their binding properties. Pharmacologic studies of competitive inhibition using a set of 11 histaminergic agents allowed analysis of the fine specificity of anti-SK & F 94461 antibodies. Both polyclonal and monoclonal anti-SK & F 94461 antibodies showed very high affinity for the immunizing molecule (i.e., Ka values for monoclonal antibodies 8 and 12 were, respectively, 3 X 10(10) and 1.4 X 10(10) M-1). Both types of antibodies bound with high affinity (IC50 ranging from 10(-10) to 10(-12) M) to mepyramine, which has a chemical structure closely resembling that of SK & F 94461. Moreover, these antibodies displayed clear-cut stereoselectivity inasmuch as they bound the d-configuration of chlorpheniramine with significantly higher affinity than the l-form. Thus, all six monoclonal antibodies showed IC50 values 1 to 6 log units lower for d- than for l-chlorpheniramine. For some monoclonal antibodies, spectroscopic and fluorescence spectra studies showed that their different binding capacities correlated with their optical properties. Similarly, polyclonal anti-SK & F 94461 antibodies showed a 500-fold lower affinity for l- than for d-chlorpheniramine. All these results indicate that the polyclonal and the majority of monoclonal anti-SK & F 94461 antibodies recognized with high affinity structural configurations known to be important for the pharmacologic activity of H1 ligands, namely the presence of the dimethylaminoethyl side chain and, with stereochemical selectivity, the d-configuration of chlorpheniramine. These data extend for the first time to an H1 histamine receptor ligand results reported in other hormone systems.

Characterization of histamine H1-receptor binding peptides in guinea pig brain using [125I]iodoazidophenpyramine, an irreversible specific photoaffinity probe.

Aminophenpyramine--i.e., N-(5-[2-(4-aminophenyl)ethanamidopentyl])-N'-(4-methoxybenzyl)-N-m ethyl-N'- (2-pyridinyl)-1,2-ethanediamine, a derivative of mepyramine (pyrilamine), a typical antagonist of histamine at its H1 receptor--was synthesized and converted into [125I)iodoazidophenpyramine, a potential photoaffinity probe for the H1 receptor. In the dark, reversible binding of this probe to cerebellar membranes occurred with a Kd of 1.2 x 10(-11) M and a Bmax of 240 fmol/mg of protein and was inhibited by various H1-receptor antagonists with the expected potencies. These features establish the compound as one of the most potent H1-receptor antagonists known so far. Upon UV irradiation, 5% of the bound radioactivity was covalently incorporated into cerebellar membrane polypeptides as shown by standard NaDodSO4/PAGE. Two bands of 47 and 56 kDa were consistently labeled, labeling being prevented by various H1-receptor antagonists with the expected potencies and stereoselectivity. In the presence of protease inhibitors, labeling of the 56-kDa peptide increased at the expense of the 47-kDa peptide, suggesting that the latter was produced by hydrolysis of the former under the action of membrane proteases. In the absence of 2-mercaptoethanol, a band of 350-400 kDa appeared, apparently at the expense of the lighter bands, suggesting that the latter might be linked by one or more disulfide bridges to a higher molecular mass complex. We propose that at least part of the ligand binding domain of the histamine H1 receptor resides within a subunit of apparent molecular mass 56,000.