History of the Pharmacological Society of Canada 1956-2008.

The Pharmacological Society of Canada (PSC) formed in 1956 and became a constituent society of the Canadian Federation of Biological Sciences (CFBS) in 1958. Over subsequent decades, it met annually with CFBS, matured as a society, and established an identity as the voice of pharmacology in Canada. During the 1980s, it sought a larger stage and bid for, and then hosted, the XIIth International Congress of Pharmacology in Montreal in 1994. The society then participated in several joint meetings with other national pharmacology societies. In 2008, the PSC merged with the Canadian Society for Clinical Pharmacology to form the Canadian Society for Pharmacology and Therapeutics. The following article is a history of the PSC from its formation in the mid-1950s to amalgamation in 2008.

Riboflavin and pyrroloquinoline quinone generate carbon monoxide in the presence of tissue microsomes or recombinant human cytochrome P-450 oxidoreductase: implications for possible roles in gasotransmission.

Carbon monoxide (CO), an endogenously produced gasotransmitter, regulates inflammation and vascular tone, suggesting that delivery of CO may be therapeutically useful for pathologies like preeclampsia where CO insufficiency is implicated. Our strategy is to identify chemicals that increase the activity of endogenous CO-producing enzymes, including cytochrome P-450 oxidoreductase (CPR). Realizing that both riboflavin and pyrroloquinoline quinone (PQQ) are relatively nontoxic, even at high doses, and that they share chemical properties with toxic CO activators that we previously identified, our goal was to determine whether riboflavin or PQQ could stimulate CO production. Riboflavin and PQQ were incubated in sealed vessels with rat and human tissue extracts and CO generation was measured with headspace-gas chromatography. Riboflavin and PQQ increased CO production ∼60% in rat spleen microsomes. In rat brain microsomes, riboflavin and PQQ increased respective CO production approximately fourfold and twofold compared to baseline. CO production by human placenta microsomes increased fourfold with riboflavin and fivefold with PQQ. In the presence of recombinant human CPR, CO production was threefold greater with PQQ than with riboflavin. These observations demonstrate for the first time that riboflavin and PQQ facilitate tissue-specific CO production with significant contributions from CPR. We propose a novel biochemical role for these nutrients in gastransmission.

Structural Insights into Azole-based Inhibitors of Heme Oxygenase-1: Development of Selective Compounds for Therapeutic Applications.

The development of isozyme-selective heme oxygenase (HO) inhibitors promises powerful pharmacological tools to elucidate the regulatory characteristics of the HO system. It is already known that HO has cytoprotective properties with a role in several disease states; thus, it is an enticing therapeutic target. Historically, the metalloporphyrins have been used as competitive HO inhibitors based on their structural similarity to the substrate, heme. However, heme's important role in several other proteins (e.g. cytochromes P450, nitric oxide synthase), results in non-selectivity being an unfortunate side effect. Reports that azalanstat and other non-porphyrin molecules inhibited HO led to a multi-faceted effort over a decade ago to develop novel compounds as potent, selective inhibitors of HO. The result was the creation of the first generation of non-porphyrin based, non-competitive inhibitors with selectivity for HO, including a subset with isozyme selectivity for HO-1. Using X-ray crystallography, the structures of several complexes of HO-1 with novel inhibitors have been elucidated and provided insightful information regarding the salient features required for inhibitor binding. This included the structural basis for non-competitive inhibition, flexibility and adaptability of the inhibitor binding pocket, and multiple, potential interaction subsites, all of which can be exploited in future drug-design strategies. Notably, HO-1 inhibitors are of particular interest for the treatment of hyperbilirubinemia and certain types of cancer. Key features based on this initial study have already been used by others to discover additional potential HO-1 inhibitors. Moreover, studies have begun to use selected compounds and determine their effects in some disease models.

Differential inhibition of rat and mouse microsome heme oxygenase by derivatives of imidazole and benzimidazole.

Metalloporphyrin heme oxygenase (HO) inhibitors have made an important contribution to elucidating the role of HO in physiological processes. Nevertheless, their off-target effects have drawn substantial criticism, which prompted us to develop non-porphyrin, azole-based inhibitors of HO. These second-generation HO inhibitors were evaluated using spleen and brain microsomes from rats as native sources of HO-1 and HO-2, respectively. Recently, the use of azole-based inhibitors of HO has been extended to other mammalian species and, as a consequence, it will be important to characterize the inhibitors in these species. The goal of this study was to compare the inhibitory profile of imidazole- and benzimidazole-based inhibitors of HO in a breast-cancer-implanted mouse to that of an untreated rat. For spleen and brain microsomes from both species, HO protein expression was determined by Western blotting and concentration-response curves for imidazole- and benzimidazole-derivative inhibition of HO activity were determined using a headspace gas-chromatographic assay. It was found that the effects on HO activity by imidazole and benzimidazole derivatives were different between the 2 species and were not explained by differences in HO expression. Thus, the HO inhibitory profile should be determined for azole derivatives before they are used in mammalian species other than rats.

Drug-enhanced carbon monoxide production from heme by cytochrome P450 reductase.

Carbon monoxide (CO) formed endogenously is considered to be cytoprotective, and the vast majority of CO formation is attributed to the degradation of heme by heme oxygenases-1 and -2 (HO-1, HO-2). Previously, we observed that brain microsomes containing HO-2 produced many-fold more CO in the presence of menadione and its congeners; herein we explored these observations further. We determined the effects of various drugs on CO production of rat brain microsomes and recombinant human cytochrome P450 reductase (CPR); CO was measured by gas chromatography with reductive detection. Brain microsomes of Sprague-Dawley rats or recombinant human cytochrome P450 reductase (CPR) were incubated with NADPH and various drugs in closed vials in phosphate buffer at pH 7.4 and 37°C. After 15 minutes, the reaction was stopped by cooling in dry ice, and the headspace gas was analyzed for CO production using gas chromatography with reductive (mercuric oxide) detection. We observed drug-enhanced CO production in the presence of both microsomes and recombinant CPR alone; the presence of HO was not required. A range of structurally diverse drugs were capable of amplifying this CO formation; these molecules had structures consistent with redox cycling capability. The addition of catalase to a reaction mixture, that contained activating drugs, inhibited the production of CO. Drug-enhanced CO formation can be catalyzed by CPR. The mechanism of CPR activation was not through classical drug-receptor mediation. Redox cycling may be involved in the drug-induced amplification of CO production by CPR through the production of reactive oxygen species.

Cysteine-independent activation/inhibition of heme oxygenase-2.

Reactive thiols of cysteine (cys) residues in proteins play a key role in transforming chemical reactivity into a biological response. The heme oxygenase-2 (HO-2) isozyme contains two cys residues that have been implicated in binding of heme and also the regulation of its activity. In this paper, we address the question of a role for cys residues for the HO-2 inhibitors or activators designed in our laboratory. We tested the activity of full length recombinant human heme oxygenase-2 (FL-hHO-2) and its analog in which cys265 and cys282 were both replaced by alanine to determine the effect on activation by menadione (MD) and inhibition by QC-2350. Similar inhibition by QC-2350 and almost identical activation by MD was observed for both recombinant FL-hHO-2s. Our findings are interpreted to mean that thiols of FL-hHO-2s are not involved in HO-2 activation or inhibition by the compounds that have been designed and identified by us. Activation or inhibition of HO-2 by our compounds should be attributed to a mechanism other than altering binding affinity of HO-2 for heme through cys265 and cys282.

Structure-Activity Relationships of 1,2-Disubstituted Benzimidazoles: Selective Inhibition of Heme Oxygenase-2 Activity.

Devising ways to up- or down-regulate heme oxygenase activity is attracting much interest as a strategy for the treatment of a variety of disorders. With a view of obtaining compounds that exhibit high potency and selectivity as inhibitors of the heme oxygenase-2 (HO-2) isozyme (constitutive) relative to the heme oxygenase-1 (HO-1) isozyme (inducible), several 1,2-disubstituted 1H-benzimidazoles were designed and synthesized. Specifically, analogues were synthesized in which the C2 substituent was the following: (1H-imidazol-1-yl)methyl, (N-morpholinyl)methyl, cyclopentylmethyl, cyclohexylmethyl, or (norborn-2-yl)methyl. Compounds with the cyclic system in the C2 substituent being a carbocyclic ring, especially cyclohexyl or norborn-2-yl, and the N1 substituent being a ring-substituted benzyl group, especially 4-chlorobenzyl or 4-bromobenzyl, best exhibited the target criteria of high potency and selectivity toward inhibition of HO-2. The new candidates should be useful pharmacological tools and may have therapeutic applications.

A novel role for ezrin in breast cancer angio/lymphangiogenesis.

INTRODUCTION: Recent evidence suggests that tumour lymphangiogenesis promotes lymph node metastasis, a major prognostic factor for survival of breast cancer patients. However, signaling mechanisms involved in tumour-induced lymphangiogenesis remain poorly understood. The expression of ezrin, a membrane cytoskeletal crosslinker and Src substrate, correlates with poor outcome in a diversity of cancers including breast. Furthermore, ezrin is essential in experimental invasion and metastasis models of breast cancer. Ezrin acts cooperatively with Src in the regulation of the Src-induced malignant phenotype and metastasis. However, it remains unclear if ezrin plays a role in Src-induced tumour angio/lymphangiogenesis. METHODS: The effects of ezrin knockdown and mutation on angio/lymphangiogenic potential of human MDA-MB-231 and mouse AC2M2 mammary carcinoma cell lines were examined in the presence of constitutively active or wild-type (WT) Src. In vitro assays using primary human lymphatic endothelial cells (hLEC), an ex vivo aortic ring assay, and in vivo tumour engraftment were utilized to assess angio/lymphangiogenic activity of cancer cells. RESULTS: Ezrin-deficient cells expressing activated Src displayed significant reduction in endothelial cell branching in the aortic ring assay in addition to reduced hLEC migration, tube formation, and permeability compared to the controls. Intravital imaging and microvessel density (MVD) analysis of tumour xenografts revealed significant reductions in tumour-induced angio/lymphangiogenesis in ezrin-deficient cells when compared to the WT or activated Src-expressing cells. Moreover, syngeneic tumours derived from ezrin-deficient or Y477F ezrin-expressing (non-phosphorylatable by Src) AC2M2 cells further confirmed the xenograft results. Immunoblotting analysis provided a link between ezrin expression and a key angio/lymphangiogenesis signaling pathway by revealing that ezrin regulates Stat3 activation, VEGF-A/-C and IL-6 expression in breast cancer cell lines. Furthermore, high expression of ezrin in human breast tumours significantly correlated with elevated Src expression and the presence of lymphovascular invasion. CONCLUSIONS: The results describe a novel function for ezrin in the regulation of tumour-induced angio/lymphangiogenesis promoted by Src in breast cancer. The combination of Src/ezrin might prove to be a beneficial prognostic/predictive biomarker for early-stage metastatic breast cancer.

In vitro Activation of heme oxygenase-2 by menadione and its analogs.

BACKGROUND: Previously, we reported that menadione activated rat, native heme oxygenase-2 (HO-2) and human recombinant heme oxygenase-2 selectively; it did not activate spleen, microsomal heme oxygenase-1. The purpose of this study was to explore some structure-activity relationships of this activation and the idea that redox properties may be an important aspect of menadione efficacy. METHODS: Heme oxygenase activity was determined in vitro using rat spleen and brain microsomes as the sources of heme oxygenase-1 and -2, respectively, as well as recombinant, human heme oxygenase-2. RESULTS: Menadione analogs with bulky aliphatic groups at position-3, namely vitamins K1 and K2, were not able to activate HO-2. In contrast, several compounds with similar bulky but less lipophilic moieties at position-2 (and -3) were able to activate HO-2 many fold; these compounds included polar, rigid, furan-containing naphthoquinones, furan-benzoxazine naphthoquinones, 2-(aminophenylphenyl)-3-piperidin-1-yl naphthoquinones. To explore the idea that redox properties might be involved in menadione efficacy, we tested analogs such as 1,4-dimethoxy-2-methylnaphthalene, pentafluoromenadione, monohalogenated naphthoquinones, α-tetralone and 1,4-naphthoquinone. All of these compounds were inactive except for 1,4-naphthoquinone. Menadione activated full-length recombinant human heme oxygenase-2 (FL-hHO-2) as effectively as rat brain enzyme, but it did not activate rat spleen heme oxygenase. CONCLUSIONS: These observations are consistent with the idea that naphthoquinones such as menadione bind to a receptor in HO-2 and activate the enzyme through a mechanism that may involve redox properties.

Selective inhibition of heme oxygenase-2 activity by analogs of 1-(4-chlorobenzyl)-2-(pyrrolidin-1-ylmethyl)-1H-benzimidazole (clemizole): Exploration of the effects of substituents at the N-1 position.

Several analogs based on the lead structure of 1-(4-chlorobenzyl)-2-(pyrrolidin-1-ylmethyl)-1H-benzimidazole (clemizole) were synthesized and evaluated as novel inhibitors of heme oxygenase (HO). Many of the compounds were found to be potent and highly selective for the HO-2 isozyme (constitutive), and had substantially less inhibitory activity on the HO-1 isozyme (inducible). The compounds represent the first report of highly potent and selective inhibitors of HO-2 activity, and complement our suite of selective HO-1 inhibitors. The study has revealed many candidates based on the inhibition of heme oxygenases for potentially useful pharmacological and therapeutic applications.

Structural insights into human heme oxygenase-1 inhibition by potent and selective azole-based compounds.

The development of heme oxygenase (HO) inhibitors, especially those that are isozyme-selective, promises powerful pharmacological tools to elucidate the regulatory characteristics of the HO system. It is already known that HO has cytoprotective properties and may play a role in several disease states, making it an enticing therapeutic target. Traditionally, the metalloporphyrins have been used as competitive HO inhibitors owing to their structural similarity with the substrate, heme. However, given heme's important role in several other proteins (e.g. cytochromes P450, nitric oxide synthase), non-selectivity is an unfortunate side-effect. Reports that azalanstat and other non-porphyrin molecules inhibited HO led to a multi-faceted effort to develop novel compounds as potent, selective inhibitors of HO. This resulted in the creation of non-competitive inhibitors with selectivity for HO, including a subset with isozyme selectivity for HO-1. Using X-ray crystallography, the structures of several complexes of HO-1 with novel inhibitors have been elucidated, which provided insightful information regarding the salient features required for inhibitor binding. This included the structural basis for non-competitive inhibition, flexibility and adaptability of the inhibitor binding pocket, and multiple, potential interaction subsites, all of which can be exploited in future drug-design strategies.

Heme oxygenase inhibition by α-(1H-imidazol-1-yl)-ω-phenylalkanes: effect of introduction of heteroatoms in the alkyl linker.

Several α-(1H-imidazol-1-yl)-ω-phenylalkanes were synthesized and evaluated as novel inhibitors of heme oxygenase (HO). These compounds were found to be potent and selective for the stress-induced isozyme HO-1, showing mostly weak activity toward the constitutive isozyme HO-2. The introduction of an oxygen atom in the alkyl linker produced analogues with decreased potency toward HO-1, whereas the presence of a sulfur atom in the linker gave rise to analogues with greater potency toward HO-1 than the carbon-containing analogues. The most potent compounds studied contained a five-atom linker between the imidazolyl and phenyl moieties, whereas the most HO-1-selective compounds contained a four-atom linker between these groups. The compounds with a five-atom linker containing a heteroatom (O or S) were found to be the most potent inhibitors of HO-2; 1-(N-benzylamino)-3-(1H-imidazol-1-yl)propane dihydrochloride, with a nitrogen atom in the linker, was found to be inactive.

Perinatal iron deficiency combined with a high-fat diet causes obesity and cardiovascular dysregulation.

Consumption of a high-fat Western diet (WD) and the resultant obesity is linked to a number of chronic pathologies, including cardiovascular dysregulation. The purpose of the present study was to determine whether perinatal iron deficiency (PID) added to the consumption of a WD would precipitate an obese phenotype with exacerbated metabolic and cardiovascular outcomes in adult offspring. Female Sprague Dawley rats were fed either a control (225 mg/kg Fe) or an iron-restricted diet (3-10 mg/kg Fe) prior to and throughout gestation. At birth, all dams were fed an iron-replete diet. At weaning, offspring were fed a normal diet or WD for up to 21 wk. Hemodynamics and locomotor activity were assessed by radiotelemetry starting at 15 wk of age. Iron restriction during pregnancy caused severe anemia in dams and offspring, resulting in 15% lower birth weights in the offspring. PID offspring fed the WD had greater caloric intake and exhibited reduced locomotor activity compared with their normal diet-fed littermates; no such effects were observed in normal iron control offspring. Despite having a similar effect on serum lipid profiles, consumption of the WD had a greater impact on body weight in the PID group, and this weight gain was due largely to visceral adipose tissue accumulation. A significant correlation between visceral adipose tissue weight and mean arterial pressure was observed in the PID offspring but not in controls. These observations demonstrate that PID predisposes offspring to an enhanced response to WD characterized by increased fat accumulation and cardiovascular dysregulation.

A novel, "double-clamp" binding mode for human heme oxygenase-1 inhibition.

The development of heme oxygenase (HO) inhibitors is critical in dissecting and understanding the HO system and for potential therapeutic applications. We have established a program to design and optimize HO inhibitors using structure-activity relationships in conjunction with X-ray crystallographic analyses. One of our previous complex crystal structures revealed a putative secondary hydrophobic binding pocket which could be exploited for a new design strategy by introducing a functional group that would fit into this potential site. To test this hypothesis and gain further insights into the structural basis of inhibitor binding, we have synthesized and characterized 1-(1H-imidazol-1-yl)-4,4-diphenyl-2-butanone (QC-308). Using a carbon monoxide (CO) formation assay on rat spleen microsomes, the compound was found to be ∼15 times more potent (IC(50) = 0.27±0.07 µM) than its monophenyl analogue, which is already a potent compound in its own right (QC-65; IC(50) = 4.0±1.8 µM). The crystal structure of hHO-1 with QC-308 revealed that the second phenyl group in the western region of the compound is indeed accommodated by a definitive secondary proximal hydrophobic pocket. Thus, the two phenyl moieties are each stabilized by distinct hydrophobic pockets. This "double-clamp" binding offers additional inhibitor stabilization and provides a new route for improvement of human heme oxygenase inhibitors.

Selective activation of heme oxygenase-2 by menadione.

While substantial progress has been made in elucidating the roles of heme oxygenases-1 (HO-1) and -2 (HO-2) in mammals, our understanding of the functions of these enzymes in health and disease is still incomplete. A significant amount of our knowledge has been garnered through the use of nonselective inhibitors of HOs, and our laboratory has recently described more selective inhibitors for HO-1. In addition, our appreciation of HO-1 has benefitted from the availability of tools for increasing its activity through enzyme induction. By comparison, there is a paucity of information about HO-2 activation, with only a few reports appearing in the literature. This communication describes our observations of the up to 30-fold increase in the in-vitro activation of HO-2 by menadione. This activation was due to an increase in Vmax and was selective, in that menadione did not increase HO-1 activity.

Rapid, convenient method for screening imidazole-containing compounds for heme oxygenase inhibition.

INTRODUCTION: Sensitive assays for measuring heme oxygenase activity have been based on the gas-chromatographic detection of carbon monoxide using elaborate, expensive equipment. The present study describes a rapid and convenient method for screening imidazole-containing candidates for inhibitory activity against heme oxygenase using a plate reader, based on the spectroscopic evaluation of heme degradation. METHODS: A PowerWave XS plate reader was used to monitor the absorbance (as a function of time) of heme bound to purified truncated human heme oxygenase-1 (hHO-1) in the individual wells of a standard 96-well plate (with or without the addition of a test compound). The degradation of heme by heme oxygenase-1 was initiated using l-ascorbic acid, and the collected relevant absorbance data were analyzed by three different methods to calculate the percent control activity occurring in wells containing test compounds relative to that occurring in control wells with no test compound present. RESULTS: In the cases of wells containing inhibitory compounds, significant shifts in λ(max) from 404 to near 412 nm were observed as well as a decrease in the rate of heme degradation relative to that of the control. Each of the three methods of data processing (overall percent drop in absorbance over 1.5h, initial rate of reaction determined over the first 5 min, and estimated pseudo first-order reaction rate constant determined over 1.5h) gave similar and reproducible results for percent control activity. The fastest and easiest method of data analysis was determined to be that using initial rates, involving data acquisition for only 5 min once reactions have been initiated using l-ascorbic acid. DISCUSSION: The results of the study demonstrate that this simple assay based on the spectroscopic detection of heme represents a rapid, convenient method to determine the relative inhibitory activity of candidate compounds, and is useful in quickly screening a series or library of compounds for heme oxygenase inhibition.

Comparison of buprenorphine and meloxicam for postsurgical analgesia in rats: effects on body weight, locomotor activity, and hemodynamic parameters.

Buprenorphine is administered to humans and animals for postoperative pain management, although its use is associated with complications. Alternative analgesics, including the nonsteroidal antiinflammatory meloxicam, are available, but information on their postoperative effects is limited. The objective of the present study was to compare buprenorphine (0.03 mg/kg SC twice daily for 3 d) with meloxicam (2 mg/kg SC initial dose followed by 1 mg/kg SC once daily for 2 d) by assessing parameters relating to postsurgical recovery in rats that underwent surgical implantation of radiotelemetric transducers. Rats treated after surgery with buprenorphine showed greater reductions in body weight, food consumption, locomotor activity, and nighttime heart rates than did meloxicam-treated rats. Buprenorphine and meloxicam treatments both had stimulatory effects on mean arterial pressure and daytime heart rate measurements, although effects on nighttime mean arterial pressure were greater in the buprenorphine-treated rats. In summary, the lesser physiologic changes associated with meloxicam, as compared with buprenorphine, suggest that meloxicam offers advantages for use as a postoperative analgesic after laparotomy and radiotelemetric transducer implantation in rats.

Heme oxygenase inhibition by 1-aryl-2-(1h-imidazol-1-yl/1h-1,2,4-triazol-1-yl)ethanones and their derivatives.

Previous studies by our research group have been concerned with the design of selective inhibitors of heme oxygenases (HO-1 and HO-2). The majority of these were based on a four-carbon linkage of an azole, usually an imidazole, and an aromatic moiety. In the present study, we designed and synthesized a series of inhibition candidates containing a shorter linkage between these groups, specifically, a series of 1-aryl-2-(1H-imidazol-1-yl/1H-1,2,4-triazol-1-yl)ethanones and their derivatives. As regards HO-1 inhibition, the aromatic moieties yielding best results were found to be halogen-substituted residues such as 3-bromophenyl, 4-bromophenyl, and 3,4-dichlorophenyl, or hydrocarbon residues such as 2-naphthyl, 4-biphenyl, 4-benzylphenyl, and 4-(2-phenethyl)phenyl. Among the imidazole-ketones, five (36-39, and 44) were found to be very potent (IC(50)<5 muM) toward both isozymes. Relative to the imidazole-ketones, the series of corresponding triazole-ketones showed four compounds (54, 55, 61, and 62) having a selectivity index >50 in favor of HO-1. In the case of the azole-dioxolanes, two of them (80 and 85), each possessing a 2-naphthyl moiety, were found to be particularly potent and selective HO-1 inhibitors. Three non-carbonyl analogues (87, 89, and 91) of 1-(4-chlorophenyl)-2-(1H-imidazol-1-yl)ethanone were found to be good inhibitors of HO-1. For the first time in our studies, two azole-based inhibitors (37 and 39) were found to exhibit a modest selectivity index in favor of HO-2. The present study has revealed additional candidates based on inhibition of heme oxygenases for potentially useful pharmacological and therapeutic applications.

Recombinant truncated and microsomal heme oxygenase-1 and -2: differential sensitivity to inhibitors.

Recombinant truncated forms of heme oxygenase-1 and -2 (HO-1 and HO-2) were compared with their crude microsomal counterparts from brain and spleen tissue of adult male rats with respect to their inhibition by azole-based, nonporphyrin HO inhibitors. The drugs tested were an imidazole-alcohol, an imidazole-dioxolane, and a triazole-ketone. Both the recombinant and crude forms of HO-2 were similarly inhibited by the 3 drugs. The crude microsomal spleen form of HO-1 was more susceptible to inhibition than was the truncated recombinant form. This difference is attributed to the extra amino acids in the full-length enzyme. These observations may be relevant in the design of drugs as inhibitors of HO and other membrane proteins.