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Mutations in the human PANK2 gene are implicated in neurodegenerative diseases such as pantothenate kinase-associated neurodegeneration (PKAN) and result in low levels of coenzyme-A (CoA) in the CNS due to impaired production of phosphopantothenic acid (PPA) from vitamin B5. Restoration of central PPA levels by delivery of exogenous PPA is a recent strategy to reactivate CoA biosynthesis in PKAN patients. Fosmetpantotenate is an oral PPA prodrug. We report here the development of a new PANk2-/- knockout model that allows CoA regeneration in brain cells to be evaluated and describe two new series of cyclic phosphate prodrugs of PPA capable of regenerating excellent levels of CoA in this system. A proof-of-concept study in mouse demonstrates the potential of this new class of prodrugs to deliver PPA to the brain following oral administration and confirms incorporation of the prodrug-derived PPA into CoA.

[Image: see text] The HIV-1 nucleocapsid (NC) protein is a small basic DNA and RNA binding protein that is absolutely necessary for viral replication and thus represents a target of great interest to develop new anti-HIV agents. Moreover, the highly conserved sequence offers the opportunity to escape the drug resistance (DR) that emerged following the highly active antiretroviral therapy (HAART) treatment. On the basis of our previous research, nordihydroguaiaretic acid 1 acts as a NC inhibitor showing moderate antiviral activity and suboptimal drug-like properties due to the presence of the catechol moieties. A bioisosteric catechol replacement approach led us to identify the 5-dihydroxypyrimidine-6-carboxamide substructure as a privileged scaffold of a new class of HIV-1 NC inhibitors. Hit validation efforts led to the identification of optimized analogs, as represented by compound 28, showing improved NC inhibition and antiviral activity as well as good ADME and PK properties.

Ciliary neurotrophic factor (CNTF) is a neurotrophic cytokine able to induce appetite reduction, weight loss and antidiabetic effects. However, its susceptibility to neutralizing anti-CNTF antibodies in patients hampered its use for treatment of human obesity and diabetes. In addition, CNTF has a very short plasma half-life, which limits its use as a therapeutic agent. Solutions, directed to prolong its in vivo effects, vary from the implantation of encapsulated secreting cells to identification of more active variants or chemical modification of the protein itself. PEGylation is a widely used modification for shielding proteins from circulating antibodies and for increasing their plasma half-life. Here, we have selected DH-CNTF, a CNTF variant which has a 40-fold higher affinity for the CNTF receptor α accompanied by an increased activity in cellular assays. The PEGylated DH-CNTF retained the biological activity of native protein in vitro and showed a significant improvement of pharmacokinetic parameters. In an acute model of glucose tolerance, the PEG-DH-CNTF was able to reduce the glycemia in diet-induced obese animals, with a performance equaled by a 10-fold higher dose of DH-CNTF. In addition, the PEGylated DH-CNTF analog demonstrated a more potent weight loss effect than the unmodified protein, opening to the use of CNTF as weight reducing agent with treatment regimens that can better meet patient compliance thanks to reduced dosing schedules.

Huntington's disease (HD) is a neurodegenerative disease caused by polyglutamine expansion in the huntingtin protein. For drug candidates targeting HD, the ability to cross the blood-brain barrier (BBB) and reach the site of action in the central nervous system (CNS) is crucial for achieving pharmacological activity. To assess the permeability of selected compounds across the BBB, we utilized a two-dimensional model composed of primary porcine brain endothelial cells and rat astrocytes. Our objective was to use this in vitro model to rank and prioritize compounds for in vivo pharmacokinetic and brain penetration studies. The model was first characterized using a set of validation markers chosen based on their functional importance at the BBB. It was shown to fulfill the major BBB characteristics, including functional tight junctions, high transendothelial electrical resistance, expression, and activity of influx and efflux transporters. The in vitro permeability of 54 structurally diverse known compounds was determined and shown to have a good correlation with the in situ brain perfusion data in rodents. We used this model to investigate the BBB permeability of a series of new HD compounds from different chemical classes, and we found a good correlation with in vivo brain permeation, demonstrating the usefulness of the in vitro model for optimizing CNS drug properties and for guiding the selection of lead compounds in a drug discovery setting. ©

We have designed and synthesized a series of new imidazole-based compounds structurally related to an antiprotozoal agent with nanomolar activity which we identified recently. The new analogues possess micromolar activities against Trypanosoma brucei rhodesiense and Leishmania donovani and nanomolar potency against Plasmodium falciparum. Most of the analogues displayed IC 50 within the low nanomolar range against Trypanosoma cruzi, with very high selectivity toward the parasite. Discussion of structure-activity relationships and in vitro biological data for the new compounds are provided against a number of different protozoa. The mechanism of action for the most potent derivatives (5i, 6a-c, and 8b) was assessed by a target-based assay using recombinant T. cruzi CYP51. Bioavailability and efficacy of selected hits were assessed in a T. cruzi mouse model, where 6a and 6b reduced parasitemia in animals >99% following intraperitoneal administration of 25 mg/kg/day dose for 4 consecutive days.

[Image: see text] The application of class I HDAC inhibitors as cancer therapies is well established, but more recently their development for nononcological indications has increased. We report here on the generation of improved class I selective human HDAC inhibitors based on an ethylketone zinc binding group (ZBG) in place of the hydroxamic acid that features the majority of HDAC inhibitors. We also describe a novel set of HDAC3 isoform selective inhibitors that show stronger potency and selectivity than the most commonly used HDAC3 selective tool compound RGFP966. These compounds are again based on an alternative ZBG with respect to the ortho-anilide that is featured in HDAC3 selective compounds reported to date.

The blood-brain barrier (BBB) is responsible for the homeostasis between the cerebral vasculature and the brain and it has a key role in regulating the influx and efflux of substances, in healthy and diseased states. Stem cell technology offers the opportunity to use human brain-specific cells to establish in vitro BBB models. Here, we describe the establishment of a human BBB model in a two-dimensional monolayer culture, derived from human induced pluripotent stem cells (hiPSCs). This model was characterized by a transendothelial electrical resistance (TEER) higher than 2000 Ω∙cm2 and associated with negligible paracellular transport. The hiPSC-derived BBB model maintained the functionality of major endothelial transporter proteins and receptors. Some proprietary molecules from our central nervous system (CNS) programs were evaluated revealing comparable permeability in the human model and in the model from primary porcine brain endothelial cells (PBECs).

The identification of a new series of growth inhibitors of Trypanosoma brucei rhodesiense, causative agent of Human African Trypanosomiasis (HAT), is described. A selection of compounds from our in-house compound collection was screened in vitro against the parasite leading to the identification of compounds with nanomolar inhibition of T. brucei growth. Preliminary SAR on the hit compound led to the identification of compound 34 that shows low nanomolar parasite growth inhibition (T. brucei EC50 5 nM), is not cytotoxic (HeLa CC50 > 25,000 nM) and is selective over other parasites, such as Trypanosoma cruzi and Plasmodium falciparum (T. cruzi EC50 8120 nM, P. falciparum EC50 3624 nM).

Neuromedin U (NMU) is an endogenous peptide implicated in the regulation of feeding, energy homeostasis, and glycemic control, which is being considered for the therapy of obesity and diabetes. A key liability of NMU as a therapeutic is its very short half-life in vivo. We show here that conjugation of NMU to human serum albumin (HSA) yields a compound with long circulatory half-life, which maintains full potency at both the peripheral and central NMU receptors. Initial attempts to conjugate NMU via the prevalent strategy of reacting a maleimide derivative of the peptide with the free thiol of Cys34 of HSA met with limited success, because the resulting conjugate was unstable in vivo. Use of a haloacetyl derivative of the peptide led instead to the formation of a metabolically stable conjugate. HSA-NMU displayed long-lasting, potent anorectic, and glucose-normalizing activity. When compared side by side with a previously described PEG conjugate, HSA-NMU proved superior on a molar basis. Collectively, our results reinforce the notion that NMU-based therapeutics are promising candidates for the treatment of obesity and diabetes.

Neuromedin U (NMU) is an endogenous peptide, whose role in the regulation of feeding and energy homeostasis is well documented. Two NMU receptors have been identified: NMUR1, expressed primarily in the periphery, and NMUR2, expressed predominantly in the brain. We recently demonstrated that acute peripheral administration of NMU exerts potent but acute anorectic activity and can improve glucose homeostasis, with both actions mediated by NMUR1. Here, we describe the development of a metabolically stable analog of NMU, based on derivatization of the native peptide with high molecular weight poly(ethylene) glycol (PEG) ('PEGylation'). PEG size, site of attachment, and conjugation chemistry were optimized, to yield an analog which displays robust and long-lasting anorectic activity and significant glucose-lowering activity in vivo. Studies in NMU receptor-deficient mice showed that PEG-NMU displays an expanded pharmacological profile, with the ability to engage NMUR2 in addition to NMUR1. In light of these data, PEGylated derivatives of NMU represent promising candidates for the treatment of obesity and diabetes.

The identification of a new series of P. falciparum growth inhibitors is described. Starting from a series of known human class I HDAC inhibitors a SAR exploration based on growth inhibitory activity in parasite and human cells-based assays led to the identification of compounds with submicromolar inhibition of P. falciparum growth (EC50500 nM) and good selectivity over the activity of human HDAC in cells (up to50-fold). Inhibition of parasital HDACs as the mechanism of action of this new class of selective growth inhibitors is supported by hyperacetylation studies.

The currently approved treatment for hepatitis C virus infections is a combination of Ribavirin and pegylated Interferon. It leads to a sustained virologic response in approximately only half of the patients treated. For this reason there is an urgent need of new therapeutic agents. 2'-C-Methylcytidine is the first nucleoside inhibitor of the HCV NS5B polymerase that was efficacious in reducing the viral load in patients infected with HCV. The application of a monophosphate prodrug approach based on unprecedented cyclic phosphoramidates is reported. Our SAR studies led to compounds that are efficiently converted to the active triphosphate in human hepatocytes.

A variety of new prodrugs of 2'-methyl cytidine based on acyloxy ethylamino phosphoramidates have been synthesized and tested in vitro and in vivo for their biological activity. Compared with the parent drug a 10- to 20-fold increase in formation of nucleotide triphosphate in rat and human hepatocytes could be achieved.

5-(Trifluoroacetyl)thiophene-2-carboxamides were found to be potent and selective class II HDAC inhibitors. This paper describes their further development and the investigation on the cause for the lack of cell-based activity. A rapid screening assay was set up which enabled the identification of more metabolic stable compounds as potent and selective class II HDAC inhibitors.

Liver microsomal preparations are routinely used to predict drug interactions that can occur in vivo as a result of inhibition of cytochrome P450 (CYP)-mediated metabolism. However, the concentration of free drug (substrate and inhibitor) at its intrahepatic site of action, a variable that cannot be directly measured, may be significantly different from that in microsomal incubation systems. Intact cells more closely reflect the environment to which CYP substrates and inhibitors are exposed in the liver, and it may therefore be desirable to assess the potential of a drug to cause CYP inhibition in isolated hepatocytes. The objective of this study was to compare the inhibitory potencies of a series of CYP2D inhibitors in rat liver microsomes and hepatocytes. For this, we developed an assay suitable for rapid analysis of CYP-mediated drug interactions in both systems, using radiolabelled dextromethorphan, a well-characterized probe substrate for enzymes of the CYP2D family. Dextromethorphan demethylation exhibited saturable kinetics in rat microsomes and hepatocytes, with apparent Km and Vmax values of 2.1 vs. 2.8 microM and 0.74 nM x min(-1) per mg microsomal protein vs. 0.11 nM x min(-1) per mg cellular protein, respectively. Quinine, quinidine, pyrilamine, propafenone, verapamil, ketoconazole and terfenadine inhibited dextromethorphan O-demethylation in rat liver microsomes and hepatocytes with IC50 values in the low micromolar range. Some of these compounds exhibited biphasic inhibition kinetics, indicative of interaction with more than one CYP2D isoform. Even though no important differences in inhibitory potencies were observed between the two systems, most inhibitors, including quinine and quinidine, displayed 2-3-fold lower IC50 in hepatocytes than in microsomes. The cell-associated concentrations of quinine and quinidine were found to be significantly higher than those in the extracellular medium, suggesting that intracellular accumulation may potentiate the effect of these compounds. Studies of CYP inhibition in intact hepatocytes may be warranted for compounds that concentrate in the liver as the result of cellular transport.

A new approach for chemoselective ligation of peptides to dauno- and doxorubicin through an oxime bond is presented. The method does not require protecting groups on the peptide moiety.

We describe an improved genetic immunization strategy for eliciting a full spectrum of anti-hepatitis C virus (HCV) envelope 2 (E2) glycoprotein responses in mammals through electrical gene transfer (EGT) of plasmid DNA into muscle fibers. Intramuscular injection of a plasmid encoding a cross-reactive hypervariable region 1 (HVR1) peptide mimic fused at the N terminus of the E2 ectodomain, followed by electrical stimulation treatment in the form of high-frequency, low-voltage electric pulses, induced more than 10-fold-higher expression levels in the transfected mouse tissue. As a result of this substantial increment of in vivo antigen production, the humoral response induced in mice, rats, and rabbits ranged from 10- to 30-fold higher than that induced by conventional naked DNA immunization. Consequently, immune sera from EGT-treated mice displayed a broader cross-reactivity against HVR1 variants from natural isolates than sera from injected animals that were not subjected to electrical stimulation. Cellular response against E2 epitopes specific for helper and cytotoxic T cells was significantly improved by EGT. The EGT-mediated enhancement of humoral and cellular immunity is antigen independent, since comparable increases in antibody response against ciliary neurotrophic factor or in specific anti-human immunodeficiency virus type 1 gag CD8+T cells were obtained in rats and mice. Thus, the method described potentially provides a safe, low-cost treatment that may be scaled up to humans and may hold the key for future development of prophylactic or therapeutic vaccines against HCV and other infectious diseases.

Leptin is an adipocyte-secreted hormone that regulates body weight and exerts effects on hematopoiesis, reproduction, and immunity. The leptin receptor (OBR) shares sequence similarity and signaling capabilities with receptors for cytokines of the ciliary neurotrophic factor (CNTF) family. Our previous finding that CNTF and leptin exert similar anti-obesity effects and activate common neuronal signaling pathways, prompted us to investigate whether leptin may share with CNTF the ability to regulate the expression of specific neuronal genes. To this end, we established a cell line, derived from the murine septal cholinergic neuronal cell line SN-56, which stably expresses OBR. In this cell line, termed SN-56/OBR, leptin induces STAT transcription factor activation and STAT-dependent reporter gene expression in a manner similar to that of CNTF. Furthermore, in SN-56/OBR cells both CNTF and leptin produce changes in neurotransmitter and neuropeptide phenotype characteristic of cholinergic neurons, such as an increase in choline acetyltransferase and vasoactive intestinal polypeptide, and a decrease in neuropeptide Y expression. SN-56/OBR cells thus constitute an interesting new model system to investigate leptin action in cells of central nervous system origin. Possible physiological implications of OBR's intrinsic ability to regulate cholinergic phenotypic markers are discussed.

A new approach for chemoselective ligation of peptides to dauno- and doxorubicin through an oxime bond is presented. The method does not require protecting groups on the peptide moiety.

5-Aryl-2-(trifluoroacetyl)thiophenes were identified as a new series of class II HDAC inhibitors (HDACi). Further development of this new series led to compounds such as 6h, a potent inhibitor of HDAC4 and HDAC6 (HDAC4 WT IC(50) = 310 nM, HDAC6 IC(50) = 70 nM) that displays 40-fold selectivity over HDAC1 and improved stability in HCT116 cancer cells (t(1/2) = 11 h). Compounds 6h and 2 show inhibition of alpha-tubulin deacetylation in HCT116 cells at 1 microM concentration and antiproliferation effects only at concentrations where inhibition of histone H3 deacetylation is observed.

Turbulent Flow Chromatography (TFC) is a powerful approach for on-line extraction in bioanalytical studies. It improves sensitivity and reduces sample preparation time, two factors that are of primary importance in drug discovery. In this paper the application of the ARIA system to the analytical support of in vivo pharmacokinetics (PK) and in vitro drug metabolism studies is described, with an emphasis in high throughput optimization. For PK studies, a comparison between acetonitrile plasma protein precipitation (APPP) and TFC was carried out. Our optimized TFC methodology gave better S/N ratios and lower limit of quantification (LOQ) than conventional procedures. A robust and high throughput analytical method to support hepatocyte metabolic stability screening of new chemical entities was developed by hyphenation of TFC with mass spectrometry. An in-loop dilution injection procedure was implemented to overcome one of the main issues when using TFC, that is the early elution of hydrophilic compounds that renders low recoveries. A comparison between off-line solid phase extraction (SPE) and TFC was also carried out, and recovery, sensitivity (LOQ), matrix effect and robustness were evaluated. The use of two parallel columns in the configuration of the system provided a further increase of the throughput.

A rapid and sensitive radiometric assay for assessing the potential of drugs to inhibit cytochrome P450 (P450) 2C19 in human liver microsomes is described. The new assay, which does not require high-performance liquid chromatography (HPLC) separation or mass spectrometric detection, is based on the release of tritium as tritiated water that occurs upon CYP2C19-mediated 4′-hydroxylation of ( S )-mephenytoin labeled with tritium in the 4′ position. Because this reaction is subject to an NIH shift, tritium was also introduced into the 3′- and 5′-positions of the tracer to enhance formation of a tritiated water product. Tritiated water was separated from the substrate using 96-well solid-phase extraction plates. The reaction is NADPH-dependent and sensitive to CYP2C19 inhibitors. IC 50 values for 15 diverse drugs differed less than 2.5-fold from those determined by quantification of the unlabeled 4′-hydroxy-( S )-mephenytoin product, using HPLC coupled to mass spectrometric detection. All of the steps of the new assay, namely incubation, product separation, and radioactivity counting, are performed in a 96-well format and can be automated. This assay represents a non-HPLC, high-throughput version of the classic ( S )-mephenytoin 4′-hydroxylation assay, which is the most widely used method to assess the potential for CYP2C19 inhibition of new chemical entities.

A rapid and sensitive radiometric assay for UDP-glucuronosyl-transferase (UGT) is described. UGT substrates are incubated in 96-well plates with microsomes in the presence of [14C]UDP-glucuronic acid, and 14C-labeled glucuronidation products are separated from the unreacted nucleotide sugar by solid-phase extraction using 96-well extraction plates. The assay was validated with 15 structurally diverse UGT substrates containing acidic, phenolic, and hydroxyl reacting groups. Glucuronidation velocities for these compounds were determined using human, rat, and dog liver microsomes, and reaction kinetics were studied with 1-naphthol and 4-methylumbelliferone. Results obtained with the new assay confirmed the previously reported rank order of glucuronidation velocity of several typical UGT substrates and the finding that the glucuronidation of most of these compounds is significantly faster in dog than in human liver microsomes. UGT specificity of five compounds was determined using recombinant human UGTs. The major UGT isoforms identified were UGT1A6, UGT1A7, and UGT1A9 for 4-methylumbelliferone; UGT1A6 and UGT1A8 for 1-naphthol; UGT2B7 for naloxone; UGT1A3 and UGT2B7 for ketoprofen; and UGT1A4 for trifluoperazine. Identical results were obtained with a conventional high-performance liquid chromatography method coupled to mass spectrometric detection. The new assay should prove valuable for rapidly benchmarking recombinant UGTs and microsomal preparations from different species and tissues, identifying high-turnover compounds during drug discovery, and reaction phenotyping studies. Copyright © 2005 by The American Society for Pharmacology and Experimental Therapeutics.

A rapid and sensitive radiometric assay for assessing the potential of drugs to inhibit cytochrome P450 (P450) 3A4/5 in human liver microsomes is described. In contrast to the conventional testosterone 6beta-hydroxylation assay, the new method does not require high-performance liquid chromatography (HPLC) separation and mass spectrometry. The assay is based on the release of tritium as tritiated water that occurs upon CYP3A4/5-mediated 6beta-hydroxylation of testosterone labeled with tritium in the 6beta position. The radiolabeled product is separated from the substrate using 96-well solid-phase extraction plates. Using commercially available [1,2,6,7-(3)H]testosterone as substrate, we demonstrated that the reaction is NADPH-dependent, and sensitive to CYP3A4/5/5 inhibitors and a CYP3A4/5/5-specific inhibitory monoclonal antibody, but not to inhibitors of or antibodies against other P450 enzymes. The method was further improved by synthesis of testosterone specifically tritiated in the 6beta position, which displayed greatly improved conversion rate with an ensuing increase in assay sensitivity. Competition experiments using tritiated and unlabeled testosterone indicated that CYP3A4/5-mediated 6beta-hydroxylation exhibits positive cooperativity and a modest kinetic isotope effect. IC(50) values for more than 40 structurally diverse chemical inhibitors were not significantly different from those determined in the testosterone 6beta-hydroxylation assay, using HPLC-tandem mass spectrometry analysis. All the steps of the new assay, namely, incubation, product separation, and radioactivity counting, are performed in 96-well format and can be automated. This assay thus represents a high-throughput version of the classical testosterone 6beta-hydroxylation assay, which is the most widely used method to assess the potential for CYP3A4/5 inhibition of new chemical entities.

A rapid and sensitive radiometric assay for assessing the potential of drugs to inhibit cytochrome P450 (P450) 2C9 in human liver microsomes is described. In contrast to the conventional diclofenac 4'-hydroxylation assay, the new method does not require high performance liquid chromatography (HPLC) separation and mass spectrometry. The assay is based on the release of tritium as tritiated water that occurs upon CYP2C9-mediated 4'-hydroxylation of diclofenac labeled with tritium in the 4' position. The radiolabeled product is separated from the substrate using 96-well solid-phase extraction plates. The reaction is NADPH-dependent, and sensitive to CYP2C9 inhibitors and inhibitory monoclonal antibodies, but not to inhibitors of or antibodies against other P450 enzymes. Competition experiments using tritiated and unlabeled diclofenac indicated that CYP2C9-mediated diclofenac 4'-hydroxylation exhibits positive cooperativity and no significant kinetic isotope effect or NIH shift. IC(50) values for 18 structurally diverse chemical inhibitors were not significantly different from those determined in the diclofenac 4'-hydroxylation assay, using HPLC-tandem mass spectrometry. All the steps of the new assay, namely, incubation, product separation, and radioactivity counting, are performed in 96-well format and can be automated. This assay thus represents a high-throughput version of the classic diclofenac 4'-hydroxylation assay, which is one of the most widely used methods to assess the potential for CYP2C9 inhibition of new chemical entities.

Endogenous injury response mechanisms likely reduce secondary neuronal loss following CNS trauma by activating growth factor receptors. Therefore, it is important to determine which growth factor receptors are activated in vivo by CNS trauma and which signal transduction pathways are affected in which cell types. We present a model of penetrating brain injury utilizing stereotaxic insertion of a fine needle. This procedure can be used to anatomically characterize injury response mechanisms through immediate, local application of pharmacological agents. We find, through immunohistochemistry, that injury of the rat facial motor nucleus leads to activation of STAT3, a neuronal survival factor, in the dendrites, nuclei and cytoplasm of the motor neurons. A similar response was observed with the trigeminal motor nucleus. Use of the ciliary neurotrophic factor (CNTF) receptor antagonist, AADH-CNTF, indicated that the STAT3 activation resulted largely, and perhaps entirely, from injury-induced activation of CNTF receptors.

SAR on the phenethylamide 1 (Ki 1.2 μM) in the P2- and the P′-position led to potent inhibitors, one of which showed good exposure and low clearance when administered intramuscularly to rat.

Liver microsomal preparations are routinely used to predict drug interactions that can occur in vivo as a result of inhibition of cytochrome P450 (CYP)-mediated metabolism. However, the concentration of free drug (substrate and inhibitor) at its intrahepatic site of action, a variable that cannot be directly measured, may be significantly different from that in microsomal incubation systems. Intact cells more closely reflect the environment to which CYP substrates and inhibitors are exposed in the liver, and it may therefore be desirable to assess the potential of a drug to cause CYP inhibition in isolated hepatocytes. The objective of this study was to compare the inhibitory potencies of a series of CYP2D inhibitors in rat liver microsomes and hepatocytes. For this, we developed an assay suitable for rapid analysis of CYP-mediated drug interactions in both systems, using radiolabelled dextromethorphan, a well-characterized probe substrate for enzymes of the CYP2D family. Dextromethorphan demethylation exhibited saturable kinetics in rat microsomes and hepatocytes, with apparent Km and Vmax values of 2.1 vs. 2.8 microM and 0.74 nM x min(-1) per mg microsomal protein vs. 0.11 nM x min(-1) per mg cellular protein, respectively. Quinine, quinidine, pyrilamine, propafenone, verapamil, ketoconazole and terfenadine inhibited dextromethorphan O-demethylation in rat liver microsomes and hepatocytes with IC50 values in the low micromolar range. Some of these compounds exhibited biphasic inhibition kinetics, indicative of interaction with more than one CYP2D isoform. Even though no important differences in inhibitory potencies were observed between the two systems, most inhibitors, including quinine and quinidine, displayed 2-3-fold lower IC50 in hepatocytes than in microsomes. The cell-associated concentrations of quinine and quinidine were found to be significantly higher than those in the extracellular medium, suggesting that intracellular accumulation may potentiate the effect of these compounds. Studies of CYP inhibition in intact hepatocytes may be warranted for compounds that concentrate in the liver as the result of cellular transport.

Target-derived neurotrophic factors are assumed to regulate motoneuron cell death during development but remain unspecified. Motoneuron cell death in the spinal nucleus of the bulbocavernosus (SNB) of rats extends postnatally and is controlled by androgens. We exploited these features of the SNB system to identify endogenously produced trophic factors regulating motoneuron survival. Newborn female rat pups were treated with the androgen, testosterone propionate, or the oil vehicle alone. In addition, females received trophic factor antagonists delivered either into the perineum (the site of SNB target muscles) or systemically. Fusion molecules that bind and sequester the neurotrophins (trkA-IgG, trkB-IgG, and trkC-IgG) were used to block activation of neurotrophin receptors, and AADH-CNTF was used to antagonize signaling through the ciliary neurotrophic factor receptor-α (CNTFRα). An acute blockade of trkB, trkC, or CNTFRα prevented the androgenic sparing of SNB motoneurons when antagonists were delivered to the perineum. Trophic factor antagonists did not significantly reduce SNB motoneuron number when higher doses were injected systemically. These findings demonstrate a requirement for specific, endogenously produced trophic factors in the androgenic rescue of SNB motoneurons and further suggest that trophic factor interactions at the perineum play a crucial role in masculinization of this neural system.

Since ciliary neurotrophic factor (CNTF) inhibits the production of TNF and activates the hypothalamus-pituitary-adrenal axis (HPAA), we investigated whether CNTF can produce antiinflammatory actions and whether it may act through a central mechanism, using the murine air pouch model of inflammation. In this model, inflammation is evaluated by measuring the induction of TNF and IL-6 as well as cell recruitment in the pouch fluid 24 h after carrageenan. Intracerebroventricular injection, but not intravenous or local injection of CNTF markedly inhibited inflammation. This was associated with high serum corticosterone levels, and antiinflammatory action was not observed in adrenalectomized mice, indicating that an intact HPAA is required. A CNTF receptor antagonist increased carrageenan inflammation, suggesting that endogenous CNTF might have a centrally mediated antiinflammatory role.

Receptor subunits for the neurocytokine ciliary neurotrophic factor (CNTF) share sequence similarity with the receptor for leptin, an adipocyte-derived cytokine involved in body weight homeostasis. We report here that CNTF and leptin activate a similar pattern of STAT factors in neuronal cells, and that mRNAs for CNTF receptor subunits, similarly to the mRNA of leptin receptor, are localized in mouse hypothalamic nuclei involved in the regulation of energy balance. Systemic administration of CNTF or leptin led to rapid induction of the tis-11 primary response gene in the arcuate nucleus, suggesting that both cytokines can signal to hypothalamic satiety centers. Consistent with this idea, CNTF treatment of ob / ob mice, which lack functional leptin, was found to reduce the adiposity, hyperphagia, and hyperinsulinemia associated with leptin deficiency. Unlike leptin, CNTF also reduced obesity-related phenotypes in db / db mice, which lack functional leptin receptor, and in mice with diet-induced obesity, which are partially resistant to the actions of leptin. The identification of a cytokine-mediated anti-obesity mechanism that acts independently of the leptin system may help to develop strategies for the treatment of obesity associated with leptin resistance.

Ciliary neurotrophic factor (CNTF) drives the sequential assembly of a receptor complex containing the ligand-specific alpha-receptor subunit (CNTFR) and the signal-transducing beta-subunits gp130 and leukemia inhibitory factor receptor-beta (LIFR). CNTFR can function in either membrane-bound or soluble forms. The membrane-bound form mediates the neuronal actions of CNTF, whereas the soluble form serves to confer cytokine responsiveness to non-neuronal cells expressing gp130 and LIFR. The objective of this work was to analyze whether the two receptor isoforms differ in their ability to interact functionally with CNTF and related proteins. Two new types of CNTF variants, characterized by weakened interactions with either CNTFR or both LIFR and gp130, were developed, and the biological activities of these and other mutants were determined in non-neuronal versus neuronal cells, as well as in non-neuronal cells transfected with an expression vector for CNTFR. Membrane anchoring of CNTFR was found to render the CNTF receptor complex relatively insensitive to changes in agonist affinity for either alpha- or beta-receptor subunits and to promote a more efficient interaction with a gp130-depleting antagonistic variant of CNTF. As a result of this phenomenon, which can be rationalized in terms of the multivalent nature of CNTF receptor interaction, CNTF variants display striking changes in receptor selectivity.