Inhibition of CXCR7 extends survival following irradiation of brain tumours in mice and rats.

BACKGROUND: In experimental models of glioblastoma multiforme (GBM), irradiation (IR) induces local expression of the chemokine CXCL12/SDF-1, which promotes tumour recurrence. The role of CXCR7, the high-affinity receptor for CXCL12, in the tumour's response to IR has not been addressed. METHODS: We tested CXCR7 inhibitors for their effects on tumour growth and/or animal survival post IR in three rodent GBM models. We used immunohistochemistry to determine where CXCR7 protein is expressed in the tumours and in human GBM samples. We used neurosphere formation assays with human GBM xenografts to determine whether CXCR7 is required for cancer stem cell (CSC) activity in vitro. RESULTS: CXCR7 was detected on tumour cells and/or tumour-associated vasculature in the rodent models and in human GBM. In human GBM, CXCR7 expression increased with glioma grade and was spatially associated with CXCL12 and CXCL11/I-TAC. In the rodent GBM models, pharmacological inhibition of CXCR7 post IR caused tumour regression, blocked tumour recurrence, and/or substantially prolonged survival. CXCR7 expression levels on human GBM xenograft cells correlated with neurosphere-forming activity, and a CXCR7 inhibitor blocked sphere formation by sorted CSCs. CONCLUSIONS: These results indicate that CXCR7 inhibitors could block GBM tumour recurrence after IR, perhaps by interfering with CSCs.

Pharmacokinetic and pharmacodynamic evaluation of the novel CCR1 antagonist CCX354 in healthy human subjects: implications for selection of clinical dose.

The safety and pharmacokinetic (PK)/pharmacodynamic (PD) profile of the novel CCR1 antagonist CCX354 was evaluated in double-blind, placebo-controlled, single- and multiple-dose phase I studies (1-300 mg/day oral doses). CCX354 was well tolerated and displayed a linear dose-exposure profile, with half-life approaching 7 h at the 300-mg dose. The extent of CCR1 receptor blockade on blood monocytes, which correlated well with plasma concentrations of the drug, was assessed using fluorescently labeled CCL3 binding in whole blood from phase I subjects. High levels of receptor coverage at the 12-h time point were achieved after a single dose of 100 mg CCX354. Preclinical studies indicate that effective blockade of inflammatory cell infiltration into tissues requires ≥90% CCR1 inhibition on blood leukocytes at all times. The comparison of the properties of CCX354 with those published for other CCR1 antagonists has informed the dose selection for ongoing clinical development of CCX354 in rheumatoid arthritis (RA).

Hydrophilic, pro-drug analogues of T138067 are efficacious in controlling tumor growth in vivo and show a decreased ability to cross the blood brain barrier.

The novel anticancer compound T138067 is an irreversible inhibitor of tubulin polymerization. Amides 3-6 were synthesized using standard methodologies and determined to be significantly less lipophilic than T138067 based on logP calculations. Tubulin polymerization and [(3)H]-T138067 competition assays revealed that these amides are pro-drugs for parent aniline 2. Amides 3-5 showed no detectable signs of crossing the blood brain barrier, while amide 6 was found in extremely small amounts (12 ng/g of brain tissue). Aniline 2, which was formed in vivo from these amides, was found in significantly smaller amounts (approximately 20 to >5000 times) in the brain than when 2 was administered directly. The in vivo efficacy of amide 6 approached that of T138067 and was better tolerated when administered to athymic nude mice bearing MX-1 human mammary tumor xenografts.

CI-1017, a functionally M1-selective muscarinic agonist: design, synthesis, and preclinical pharmacology.

The five muscarinic receptor subtypes (M1-M5) are characterized by seven helices that define a transmembrane cavity which serves as the binding pocket for agonists and antagonists. The five cavities appear to be topographically different enough to permit subtype selectivity among antagonists but not among classical agonists which tend to be smaller in size than antagonists. It was reasoned that synthesis of muscarinic agonists longer/larger than their classical counterparts might result in subtype selectivity. M1 subtype selectivity was found in a class of 1-azabicyclo[2.2.1]heptan-3-one, O-(3-aryl-2-propynyl) oximes. One of these, CI-1017, improved spatial memory of hippocampally deficient mice and nbM-lesioned rats at doses of 1.0-3.2 and 0.1-0.3 mg/kg, respectively, while producing parasympathetic side effects only at very high doses (100-178 mg/kg). Additionally, CI-1017 inhibited production of amyloidogenic A beta and increased secretion of soluble APP. Thus, CI-1017, besides treating AD symptomatically, may also retard its progression. CI-1017 has recently completed phase I clinical trials.

Secreted site-1 protease cleaves peptides corresponding to luminal loop of sterol regulatory element-binding proteins.

We describe a permanent line of Chinese hamster ovary cells transfected with a cDNA encoding a truncated form of Site-1 protease (S1P) that is secreted into the culture medium in an enzymatically active form. S1P, a subtilisin-like protease, normally cleaves the luminal loop of sterol regulatory element-binding proteins (SREBPs). This cleavage initiates the two-step proteolytic process by which the NH(2)-terminal domains of SREBPs are released from cell membranes for translocation to the nucleus, where they activate transcription of genes involved in the biosynthesis and uptake of cholesterol and fatty acids. Truncated S1P (amino acids 1-983), produced by the transfected Chinese hamster ovary cells, lacks the COOH-terminal membrane anchor. Like native S1P, this truncated protein undergoes normal autocatalytic processing after residue 137 to release an NH(2)-terminal propeptide, thereby generating an active form, designated S1P-B. Prior to secretion, truncated S1P-B, like native S1P-B, is cleaved further after residue 186 to generate S1P-C, which is the only form that appears in the culture medium. The secreted enzyme, designated S1P(983)-C, cleaves a synthetic peptide that terminates in a 7-amino-4-methyl-coumarin fluorochrome. This peptide, RSLK-MCA, corresponds to the internal propeptide cleavage site that generates S1P-B as described in the accompanying paper (Espenshade, P. J., Cheng, D., Goldstein, J. L., and Brown, M. S. (1999), J. Biol. Chem. 274, 22795-22804). The secreted enzyme does not cleave RSVL-MCA, a peptide corresponding to the physiologic cleavage site in SREBP-2. However, S1P(983)-C does cleave after this leucine when the RSVL sequence is contained within a 16-residue peptide corresponding to the central portion of the SREBP-2 luminal loop. The catalytic activity of S1P(983)-C differs from that of furin/prohormone convertases, two related proteases, in its more alkaline pH optimum (pH 7-8), its relative resistance to calcium chelating agents, and its ability to cleave after lysine or leucine rather than arginine. These data provide direct biochemical evidence that S1P is the protease that cleaves SREBPs and thereby functions to control lipid biosynthesis and uptake in animal cells.

Novel halogenated sulfonamides inhibit the growth of multidrug resistant MCF-7/ADR cancer cells.

In this report, we describe the synthesis of halogenated benzenesulfonamide compounds and their ability to inhibit the growth of HeLa, MCF-7 and MCF-7/ADR tumor cells in vitro. The multidrug resistance (MDR) phenotype of certain cells does not affect their sensitivity to these compounds. These agents belong to a family of compounds previously shown to bind irreversibly to cysteine-239 of beta-tubulin. Consistent with this mechanism of action, the cytotoxicities of these compounds appear to correlate with their ability to undergo nucleophilic aromatic substitution.

Selective, covalent modification of beta-tubulin residue Cys-239 by T138067, an antitumor agent with in vivo efficacy against multidrug-resistant tumors.

Microtubules are linear polymers of alpha- and beta-tubulin heterodimers and are the major constituents of mitotic spindles, which are essential for the separation of chromosomes during mitosis. Here we describe a synthetic compound, 2-fluoro-1-methoxy-4-pentafluorophenylsulfonamidobenzene (T138067), which covalently and selectively modifies the beta1, beta2, and beta4 isotypes of beta-tubulin at a conserved cysteine residue, thereby disrupting microtubule polymerization. Cells exposed to T138067 become altered in shape, indicating a collapse of the cytoskeleton, and show an increase in chromosomal ploidy. Subsequently, these cells undergo apoptosis. Furthermore, T138067 exhibits cytotoxicity against tumor cell lines that exhibit substantial resistance to vinblastine, paclitaxel, doxorubicin, and actinomycin D. T138067 is also equally efficacious in inhibiting the growth of sensitive and multidrug-resistant human tumor xenografts in athymic nude mice. These observations suggest that T138067 may be clinically useful for the treatment of multidrug-resistant tumors.

Benzenesulfonamide derivatives of 2-substituted 4H-3,1-benzoxazin-4-ones and benzthiazin-4-ones as inhibitors of complement C1r protease.

A series of 2-sulfonyl-4H-3,1-benzoxazinones was prepared that inhibit C1r protease in vitro. Several compounds were found to be selective for C1r verses the related serine protease trypsin. Selected compounds demonstrated functional activity in a hemolysis assay.

Identification and characterization of m1 selective muscarinic receptor antagonists1.

A series of esters of 1,4-disubstituted tetrahydropyridine carboxylic acids (I) has been synthesized and characterized as potential m1 selective muscarinic receptor antagonists. The affinity of these compounds for the five human muscarinic receptor subtypes (Hm1-Hm5) was determined by the displacement of [3H]-NMS binding using membranes from transfected Chinese hamster ovarian cells. One of the most potent and selective compounds of this series is an analogue of I [11, R1 = (CH2)5CH3], which has an IC50 value of 27.3 nM at the m1 receptor and possesses 100-fold (m2), 48-fold (m3), 74-fold (m4), and 19-fold (m5) selectivities at the other receptors. Thus, this analogue appears to be more selective on the basis of binding than the prototypical m1 antagonist, pirenzepine. Functional data, such as the inhibition of carbachol-stimulated phosphatidylinositol hydrolysis, on selected analogues confirmed the muscarinic antagonistic properties of this chemical series.

Design and synthesis of m1-selective muscarinic agonists: (R)-(-)-(Z)-1-Azabicyclo[2.2.1]heptan-3-one, O-(3-(3'-methoxyphenyl)-2-propynyl)oxime maleate (CI-1017), a functionally m1-selective muscarinic agonist.

The synthesis and SAR of a series of (Z)-(+/-)-1-azabicyclo[2.2. 1]heptan-3-one, O-(3-aryl-2-propynyl)oximes are described. The biochemistry and pharmacology of 24Z (PD 142505) and its enantiomers are highlighted. 24Z is functionally an m1-selective muscarinic agonist. Efficacy and m1 selectivity reside in the R enantiomer, (R)-24Z (CI-1017).

2-amino-4H-3,1-benzoxazin-4-ones as inhibitors of C1r serine protease.

A series of 2-amino-4H-3,1-benzoxazin-4-ones have been synthesized and evaluated as inhibitors of the complement enzyme C1r. C1r is a serine protease at the beginning of the complement cascade, and complement activation by beta-amyloid may represent a major contributing pathway to the neuropathology of Alzheimer's disease. Compounds such as 7-chloro-2-[(2-iodophenyl)-amino]benz[d][1,3]oxazin-4-one (32) and 7-methyl-2-[(2-iodophenyl)amino]benz[d][1,3]oxazin-4-one (37) show improved potency compared to the reference compound FUT-175. Many of these active compounds also possess increased selectivity for C1r compared to trypsin and enhanced hydrolytic stability relative to 2-(2-iodophenyl)-4H-3,1-benzoxazin-4-one (1).

Novel antineoplastic agents with efficacy against multidrug resistant tumor cells.

A novel series of pentafluorobenzenesulfonamides has been shown to inhibit the growth of a variety of human tumor cell lines. Among the cell types against which these agents were evaluated were the multidrug resistant (MDR) cell lines MCF-7/ADR and P388/ADR. The cytotoxic activity of members of this series of compounds was not affected by the multidrug resistant pump in MCF-7/ADR or P388/ADR cells.

Identification and characterization of m4 selective muscarinic antagonists.

Our interest in the area of m4 muscarinic antagonists had led us to study a series of benzoxazine isoquinolines. One of the most potent and selective compounds of this series is example 1 with an IC50 value of 90.7 nM at m4 receptors, and 72-fold (m1), 38-fold (m2), 10-fold (m3), and 82-fold (m5) more selective compared to the other receptors. The synthesis and receptor binding affinity of analogs of 1 are reported.

Aryl 1-but-3-ynyl-4-phenyl-1,2,3,6-tetrahydropyridines as potential antipsychotic agents: synthesis and structure-activity relationships.

A novel series of aryl 1-but-3-ynyl-4-phenyl-1,2,3,6-tetrahydropyridines with dopaminergic activity is described. The structure-activity relationships of this series were studied by synthesis of analogs and evaluation of their affinities for the dopamine (DA) D2 receptor and inhibition of locomotor activity (LMA) in rodents. The basic amine, alkyne chain length, and aryl groups were varied. Compounds having a 4-phenyl-1,2,3,6-tetrahydropyridine and an aryl group with hydrogen-bonding substituents separated by a butynyl chain were found to have the most potent dopaminergic activity. Several compounds that were found to have exceptional in vivo activity in LMA inhibition in rodents were evaluated for additional pharmacological activity including binding affinities for other DA receptor subtypes as well as effects on brain DA synthesis, DA neuronal firing, and conditioned avoidance responding in squirrel monkeys.

Identification of a 3-hydroxylated tacrine metabolite in rat and man: metabolic profiling implications and pharmacology.

Discrepancies in urinary metabolic profiles in rats administered tacrine (1) suggested the presence of an unidentified metabolite of 1. Chromatographic methods were developed that allowed isolation of a metabolite fraction containing both 1-hydroxytacrine (2) and an unknown metabolite from rat urine. Mass spectral analysis indicated this metabolite to be a monohydroxylated derivative, which upon two dimensional COSY NMR analysis could be assigned as 3-hydroxytacrine (4). This structural assignment was confirmed by independent synthesis of 4. Compound 4 was also identified as a human urinary metabolite of 1. Biologically, 4 was found to have in vitro human red blood cell acetylcholinesterase inhibitory activity similar to that of 2 and 4-hydroxytacrine (5) and approximately 8-fold less than that of 1. These results underscore the need to conduct rigorous structural identification studies, especially in cases where isomeric metabolites are possible, in assessing the accuracy of chromatographic profiling techniques.

Kynurenic acid derivatives inhibit the binding of nerve growth factor (NGF) to the low-affinity p75 NGF receptor.

The ability of a series of substituted kynurenic acids, thienopyridinonecarboxylic acids, and related compounds to inhibit the binding of nerve growth factor (NGF) to the p75 NGF receptor (NGFR) was evaluated in a radioligand binding assay that utilized a biotinylated derivative of the extracellular domain of p75 NGFR (p75ext) fixed to streptavidin-coated plastic wells. Two compounds, 6-aminokynurenic acid (5h) and the 3-methyl ester of 4,7-dihydro-2-methyl-7-oxothieno[3,2-b]pyridine-3,5-dicarboxylic acid (16), were found to inhibit the binding of [125I]NGF to p75ext with IC50 values in the low micromolar range. Other amino-substituted kynurenic acids also possessed activity at slightly higher concentrations. Several structural features seem to be essential, including the carboxylic acid, a polar group on the benzene ring (or thiophene ring, in the case of analogues of 16), and the C-4 carbonyl group in the pyridinone ring. These compounds were also found to inhibit the binding of [125I]NGF to its receptors in membranes from PC12 cells (which express p75 as well as trka receptors for NGF) and DG44-CHO cells (transfected with full length p75 NGFR). The available data for 5h and 16 do not allow the determination of whether the effects of these compounds are mediated by their interaction with NGF or the NGF receptors.

Mutations of aspartate 103 in the Hm2 receptor and alterations in receptor binding properties of muscarinic agonists.

Aspartate 103 (D103) in the third transmembrane domain of the Hm2 receptor was mutated to glutamate (D103E), asparagine (D103N), or alanine (D103A). As measured by [3H]-NMS, no significant binding was observed in D103A, while a 2-fold decrease in ligand affinity was seen in D103E and a 32-fold decrease in affinity was found in the D103N mutant. Examination of reference agonists showed greater loss of affinity in D103N than in D103E with the rank order of change being: L-607,207>carbachol>arecoline>pilocarpine>oxotremorine>McN-A-343. Of the novel 1-azabicyclo[2.2.1]-heptan-3-one oxime agonists examined, arylacetylene oximes showed little alteration in binding in either the D103E or D103N mutants, while the geometric isomers of several bicyclic aryl-ene-yne oximes showed significant changes in affinity, especially in the D103N mutant. Thus, overall size of the agonist and/or spatial orientation of the molecule within the binding pocket contribute to changes measured in binding.

The discovery and structure-activity relationships of 1,2,3,6-tetrahydro-4-phenyl-1-[(arylcyclohexenyl)alkyl]pyridines. Dopamine autoreceptor agonists and potential antipsychotic agents.

A novel dopamine (DA) autoreceptor agonist, 1,2,3,6-tetrahydro-4-phenyl-1- [(3-phenyl-3-cyclohexen-1-yl)methyl]pyridine (14), was identified. The structure-activity relationships surrounding this compound were studied by synthesis of analogues and evaluation of their dopaminergic activity. The cyclohexene substitution pattern was varied along with the length of the chain connecting the 1,2,3,6-tetrahydro-4-phenylpyridine to the cyclohexene. Compound 14, having the 1,3-substitution pattern and a single methylene chain, was the most potent. The 1,2,3,6-tetrahydro-4-phenylpyridine could be replaced by other aryl-cyclic amines with a slight loss in activity. The phenyl group on the cyclohexene ring could be para substituted; electron-donating groups were better tolerated than electron-withdrawing groups. Finally, the enantiomers of 14 were resolved via the 1,1'-binaphthyl-2,2'-diyl hydrogen phosphate salts. Although both isomers were partial DA agonists, the (+)-enantiomer had higher intrinsic activity than the (-)-enantiomer. Syntheses were developed that allowed rapid preparation of analogues. An X-ray crystal structure determination of an intermediate identified the (+)-isomer of 14 as having R configuration. This compound, designated CI-1007 (PD 143188), was found to have antipsychotic-like activity in behavioral tests; in particular, it was orally active in the conditioned avoidance test in squirrel monkeys with an ED50 of 0.6 mg/kg. The overall profile suggests that (R)-(+)-14 may be a clinically useful antipsychotic agent.

Evaluation of the effects of the enantiomers of reduced haloperidol, azaperol, and related 4-amino-1-arylbutanols on dopamine and sigma receptors.

The enantiomers of reduced haloperidol (3a), azaperol (3b), and the related compound BMY-14802 (3c) were prepared in high optical purity. The affinity of these compounds for dopamine D2 and D3 receptors, and sigma S1 and S2 sites was determined in vitro. Both enantiomers of 3a display greatly decreased affinity for D2 and D3 receptors compared to haloperidol, although they still possess affinities in the 100-200-nM range. Both enantiomers of 3a possess potent and equal affinity for S1 sites (Ki: 1-2 nM), only slightly weaker than haloperidol (Ki: 0.33 nM). At S2 sites, (R)-(+)-3a displays similar affinity to haloperidol (Ki: 31 and 26 nM, respectively), while (S)-(-)-3a is slight more potent (Ki: 8.2 nM). The stereoselectivity profile of the enantiomers of 3b at D2 and D3 receptors is quite similar to that of 3a, (S)-(-)-3b being about 4 times more potent than its enantiomer at both receptors. (R)-(+)-3b binds preferentially to sigma S1 over S2 sites, while (S)-(-)-3b displays the opposite selectivity profile. Both enantiomers of 3c possess very weak affinity for D2 and D3 receptors. In a manner similar to the enantiomers of 3b, the affinity of (R)-(+)-3c is greater for S1 than S2 sites, while (S)-(-)-3c displays the opposite selectivity profile. Following parenteral administration of both enantiomers of 3a, dopamine synthesis and turnover in rat striatum, cortex, and mesolimbic areas were increased, in a manner similar to the effects produced by haloperidol itself. Additional studies will be required to assess with certainty whether the effects were due to the compounds themselves or simply were a consequence of the in vivo oxidation to haloperidol.

Pharmacological profile of the dopamine partial agonist, (+/-)-PD 128483 and its enantiomers.

(+/-)-PD 128483, ((+/-)-4,5,5a,6,7,8-hexahydro-6-methylthiazolo[4,5-f]-quinolin+ ++-2-amine, maleate (1:1)), is a racemic compound that is a p.o. active dopamine (DA) partial agonist that has DA autoreceptor agonist effects and displays antipsychoticlike activity in preclinical tests. In in vitro receptor binding assays, (+/-)-PD 128483 and its enantiomers bound selectively to DA D-2 receptors vs. DA D-1 receptors and showed no affinity for adrenergic alpha-1 or serotonin1A receptors, but had affinity for adrenergic alpha-2 receptors. In tests of DA agonist effects, including reversal of the tau-butyrolactone-stimulated increase in brain dopa synthesis in striatum and inhibition of DA neuronal firing, the rank order of efficacy was (+)-PD 128483 > (+/-)-PD 128483 > (-)-PD 128483. (+/-)-PD 128483 and (+)-PD 128483 inhibited, whereas (-)-PD 128483 increased, brain DA synthesis in normal rats. (+/-)-PD 128483 and (-)-PD 128483 inhibited spontaneous locomotion in rats and did not produce locomotor stimulation or stereotypies. In contrast, (+)-PD 128483 inhibited locomotor activity at low doses, but at relatively high doses increased locomotion and induced stereotypy in rats. (-)-PD 128483 consistently inhibited Sidman avoidance responding in squirrel monkeys. (+/-)-PD 128483 inhibited Sidman avoidance responding in one group of monkeys, but had minimal effects in another group. (+)-PD 128483 did not inhibit avoidance responding. In squirrel or cebus monkeys sensitized to the acute dystonic effects of haloperidol, only (-)-PD 128483 induced extrapyramidal dysfunction. These results indicate that (+/-)-PD 128483 is a DA partial agonist which produces DA autoreceptor agonist effects and has a preclinical behavioral profile suggestive of antipsychotic activity.