Elevated Electron Temperature Coincident with Observed Fusion Reactions in a Sheared-Flow-Stabilized Z Pinch.

The sheared-flow-stabilized Z pinch concept has been studied extensively and is able to produce fusion-relevant plasma parameters along with neutron production over several microseconds. We present here elevated electron temperature results spatially and temporally coincident with the plasma neutron source. An optical Thomson scattering apparatus designed for the FuZE device measures temperatures in the range of 1-3 keV on the axis of the device, 20 cm downstream of the nose cone. The 17-fiber system measures the radial profiles of the electron temperature. Scanning the laser time with respect to the neutron pulse time over a series of discharges allows the reconstruction of the T_{e} temporal response, confirming that the electron temperature peaks simultaneously with the neutron output, as well as the pinch current and inductive voltage generated within the plasma. Comparison to spectroscopic ion temperature measurements suggests a plasma in thermal equilibrium. The elevated T_{e} confirms the presence of a plasma assembled on axis, and indicates limited radiative losses, demonstrating a basis for scaling this device toward net gain fusion conditions.

Osteoporosis identification among previously undiagnosed individuals with vertebral fractures.

Because osteoporosis is under-recognized in patients with vertebral fractures, we evaluated characteristics associated with osteoporosis identification. Most patients with vertebral fractures did not receive evaluation or treatment for osteoporosis. Black, younger, and male participants were particularly unlikely to have had recognized osteoporosis, which could increase their risk of negative outcomes. INTRODUCTION: Vertebral fractures may be identified on imaging but fail to prompt evaluation for osteoporosis. Our objective was to evaluate characteristics associated with clinical osteoporosis recognition in patients who had vertebral fractures detected on their thoracolumbar spine imaging reports. METHODS: We prospectively identified individuals who received imaging of the lower spine at primary care clinics in 4 large healthcare systems who were eligible for osteoporosis screening and lacked indications of osteoporosis diagnoses or treatments in the prior year. We evaluated characteristics of participants with identified vertebral fractures that were associated with recognition of osteoporosis (diagnosis code in the health record; receipt of bone mineral density scans; and/or prescriptions for anti-osteoporotic medications). We used mixed models to estimate adjusted odds ratios (ORs) and 95% confidence intervals (95% CIs). RESULTS: A total of 114,005 participants (47% female; mean age 65 (interquartile range: 57-72) years) were evaluated. Of the 8579 (7%) participants with vertebral fractures identified, 3784 (44%) had recognition of osteoporosis within the subsequent year. In adjusted regressions, Black participants (OR (95% CI): 0.74 (0.57, 0.97)), younger participants (age 50-60: 0.48 (0.42, 0.54); age 61-64: 0.70 (0.60, 0.81)), and males (0.39 (0.35, 0.43)) were less likely to have recognized osteoporosis compared to white participants, adults aged 65 + years, or females. CONCLUSION: Individuals with identified vertebral fractures commonly did not have recognition of osteoporosis within a year, particularly those who were younger, Black, or male. Providers and healthcare systems should consider efforts to improve evaluation of osteoporosis in patients with vertebral fractures.

Investigating silent pauses in connected speech: integrating linguistic, neuropsychological, and neuroanatomical perspectives across narrative tasks in post-stroke aphasia.

Introduction: Silent pauses are regarded as integral components of the temporal organization of speech. However, it has also been hypothesized that they serve as markers for internal cognitive processes, including word access, monitoring, planning, and memory functions. Although existing evidence across various pathological populations underscores the importance of investigating silent pauses' characteristics, particularly in terms of frequency and duration, there is a scarcity of data within the domain of post-stroke aphasia. Methods: The primary objective of the present study is to scrutinize the frequency and duration of silent pauses in two distinct narrative tasks within a cohort of 32 patients with chronic post-stroke aphasia, in comparison with a control group of healthy speakers. Subsequently, we investigate potential correlation patterns between silent pause measures, i.e., frequency and duration, across the two narrative tasks within the patient group, their performance in neuropsychological assessments, and lesion data. Results: Our findings showed that patients exhibited a higher frequency of longer-duration pauses in both narrative tasks compared to healthy speakers. Furthermore, within-group comparisons revealed that patients tended to pause more frequently and for longer durations in the picture description task, while healthy participants exhibited the opposite trend. With regard to our second research question, a marginally significant interaction emerged between performance in semantic verbal fluency and the narrative task, in relation to the location of silent pauses-whether between or within clauses-predicting the duration of silent pauses in the patient group. However, no significant results were observed for the frequency of silent pauses. Lastly, our study identified that the duration of silent pauses could be predicted by distinct Regions of Interest (ROIs) in spared tissue within the left hemisphere, as a function of the narrative task. Discussion: Overall, this study follows an integrative approach of linguistic, neuropsychological and neuroanatomical data to define silent pauses in connected speech, and illustrates interrelations between cognitive components, temporal aspects of speech, and anatomical indices, while it further highlights the importance of studying connected speech indices using different narrative tasks.

Hyperbaric oxygen therapy for local late radiation toxicity in breast cancer patients: A systematic review.

PURPOSE: This systematic review aims to provide an overview of the literature on the effect of hyperbaric oxygen therapy (HBOT) on symptoms of local late radiation toxicity (LRT) in patients treated for breast cancer. METHODS: A systematic search was performed in September 2021. All studies with a sample size of ≥10 patients reporting the effect of HBOT for symptoms of LRT after radiotherapy of the breast and/or chest wall were included. The ROBINS-I tool was used for critical appraisal of methodological quality. The toxicity outcomes pain, fibrosis, lymphedema, necrosis/skin problems, arm and shoulder mobility, and breast and arm symptoms were evaluated. RESULTS: Nine studies concerning a total of 1308 patients were included in this review. Except for one study, sample sizes were small. Most studies had inadequate methodology with a substantial risk of bias. Post-HBOT, a significant reduction of pain was observed in 4/5 studies, of fibrosis in 1/2 studies, and of lymphedema of the breast and/or arm in 4/7 studies. Skin problems of the breast were significantly reduced in 1/2 studies, arm- and shoulder mobility significantly improved in 2/2 studies, and breast- and arm symptoms were significantly reduced in one study. CONCLUSION: This systematic review indicates that HBOT might be useful for reducing symptoms of LRT in breast cancer patients, however evidence is limited. A randomized controlled trial in a larger cohort of patients including a combination of patient- and clinician-reported outcome measures would be valuable to assess the effect of HBOT on symptoms of LRT.

Nuclear pore complex assembly studied with a biochemical assay for annulate lamellae formation.

Formation of the nuclear pore is an intricate process involving membrane fusion and the ordered assembly of up to 1,000 pore proteins. As such, the study of pore assembly is not a simple one. Interestingly, annulate lamellae, a cytoplasmic organelle consisting of stacks of flattened membrane cisternae perforated by numerous pore complexes, have been found to form spontaneously in a reconstitution system derived from Xenopus egg extracts, as determined by electron microscopy (Dabauvalle et al., 1991). In this work, a biochemical assay for annulate lamellae (AL) formation was developed and used to study the mechanism of AL assembly in general and the assembly of individual nucleoporins into pore complexes in particular. Upon incubation of Xenopus egg cytosol and membrane vesicles, the nucleoporins nup58, nup60, nup97, nup153, and nup200 initially present in a disassembled form in the cytosol became associated with membranes and were pelletable. The association was time and temperature dependent and could be measured by immunoblotting. Thin-section electron microscopy as well as negative staining confirmed that annulate lamellae were forming coincident with the incorporation of pore proteins into membranes. Homogenization and subsequent flotation of the membrane fraction allowed us to separate a population of dense membranes, containing the integral membrane pore protein gp210 and all other nucleoporins tested, from the bulk of cellular membranes. Electron microscopy indicated that annulate lamellae were enriched in this dense, pore protein-containing fraction. GTP gamma S prevented incorporation of the soluble pore proteins into membranes. To address whether AL form in the absence of N-acetylglucosaminylated pore proteins, AL assembly was carried out in WGA-sepharose-depleted cytosol. Under these conditions, annulate lamellae formed but were altered in appearance. When the membrane fraction containing this altered AL was homogenized and subjected to flotation, the pore protein-containing membranes still sedimented in a distinct peak but were less dense than control annulate lamellae.

A complex of nuclear pore proteins required for pore function.

A family of proteins bearing novel N-acetylglucosamine residues has previously been found to be required to form functional nuclear pores. To begin to determine which of the proteins in this family are essential for pore function, antisera were raised to each of three members of the family, p62, p58, and p54. With these antisera, it was possible to deplete nuclear reconstitution extracts of the proteins and to test the depleted nuclei for nuclear transport. In the course of the experiments, it was found that the three proteins exist as a complex; antisera to any one, while specific on a protein blot, coimmunoprecipitated all three proteins. This complex of pore proteins is stable to 2 M salt, 2 M urea, and the detergent Mega 10, indicating the presence of specific and tight protein-protein interactions. By gel filtration, the complex has a molecular mass of 550-600 kD. Nuclei containing pores depleted of the complex are found to be defective for nuclear transport; moreover, we observe a strict linear correlation between the amount of complex present in nuclei and the amount of nuclear transport of which those nuclei are capable. Thus, the p62-p58-p54 complex defines a group of proteins with strong protein-protein interactions that form a unit of pore structure essential for pore function.

Influenza virus-susceptible mice carry Mx genes with a large deletion or a nonsense mutation.

The interferon-regulated mouse Mx gene encodes the 72-kilodalton nuclear Mx protein that selectively inhibits influenza virus replication. Mice carrying Mx+ alleles synthesize Mx protein and resist influenza virus infection, whereas mice homozygous for Mx- alleles fail to synthesize Mx protein and, as a consequence, are influenza virus susceptible. Southern blot analysis allowed us to define the following three distinct Mx restriction fragment length polymorphism (RFLP) types among classical inbred strains: RFLP type 1 in the Mx+ strains A2G and SL/NiA, RFLP type 2 in BALB/c and 33 other Mx- strains, and RFLP type 3 in CBA/J and 2 other Mx- strains. cDNA clones of Mx mRNAs from BALB/c and CBA/J cells were isolated, and their sequences were compared with that of the wild-type Mx mRNA of strain A2G. Mx mRNA of BALB/c mice has 424 nucleotides absent from the coding region, resulting in a frame shift and premature termination of Mx protein. The missing sequences correspond exactly to Mx exons 9 through 11. These three exons, together with some flanking intron sequences, are deleted from the genomes of all Mx RFLP type 2 strains. The Mx- phenotype of the Mx RFLP type 3 strain CBA/J is due to a point mutation that converts the lysine codon in position 389 to a termination codon. Mx RFLP type 3 strains have an extra HindIII site which maps to an intron and thus probably does not affect the coding capacity of Mx mRNA. We further show that the Mx mRNA levels in interferon-treated BALB/c and CBA/J cells are about 15-fold lower than in similarly treated Mx+ cells. This is probably due to decreased metabolic stabilities of the mutant mRNAs.

TATA-box DNA binding activity and subunit composition for RNA polymerase III transcription factor IIIB from Xenopus laevis.

The RNA polymerase III transcription initiation factor TFIIIB contains the TATA-box-binding protein (TBP) and polymerase III-specific TBP-associated factors (TAFs). Previous studies have shown that DNA oligonucleotides containing the consensus TATA-box sequence inhibit polymerase III transcription, implying that the DNA binding domain of TBP is exposed in TFIIIB. We have investigated the TATA-box DNA binding activity of Xenopus TFIIIB, using transcription inhibition assays and a gel mobility shift assay. Gel shift competition assays with mutant and nonspecific DNAs demonstrate the specificity of the TFIIIB-TATA box DNA complex. The apparent dissociation constant for this protein-DNA interaction is approximately 0.4 nM, similar to the affinity of yeast TBP for the same sequence. TFIIIB transcriptional activity and TATA-box binding activity cofractionate during a series of four ion-exchange chromatographic steps, and reconstituted transcription reactions demonstrate that the TATA-box DNA-protein complex contains TFIIIB TAF activity. Polypeptides with apparent molecular masses of 75 and 92 kDa are associated with TBP in this complex. These polypeptides were renatured after elution from sodium dodecyl sulfate-gels and tested individually and in combination for TFIIIB TAF activity. Recombinant TBP along with protein fractions containing the 75- and 92-kDa polypeptides were sufficient to reconstitute TFIIIB transcriptional activity and DNA binding activity, suggesting that Xenopus TFIIIB is composed of TBP along with these polypeptides.

First clinical study of a novel complete metal-free ceramic total knee replacement system.

BACKGROUND: The aim of the study was to evaluate the safety and efficacy of a novel metal-free ceramic total knee replacement system. METHODS: Thirty-eight primary total knee arthroplasties (TKAs) were performed on 34 patients using the metal-free BPK-S ceramic total knee replacement system with both the femoral and tibial components of an alumina/zirconia ceramic composite. The clinical outcome was evaluated pre- and postoperatively at 3 (n = 32 TKA) and 12 months (n = 32 TKA) using the Knee Society Score (KSS), the Oxford Knee Score and the EQ-5D. Safety analysis was performed by radiological examination and assessment of adverse events. RESULTS: Postoperatively, the KSS, Oxford Knee Score and EQ-5D improved significantly at 3 and 12 months (p < 0.001). Non-progressive partial radiolucent lines were observed in six cases, but there was no osteolysis and no implant loosening. Induction or exacerbation of allergies did not occur during the follow-up. CONCLUSIONS: The metal-free BPK-S ceramic total knee replacement system proved to be a safe and clinically efficient alternative to metal implants in this short-term follow-up study.

Proteins from disassembled microtubules characterized by oligospecific antisera.

The immunochemical properties of in vitro reassembled microtubules were investigated by immunoelectrophoretic techniques. The tubulin dimer gave no measurable immunochemical response, but the tubulin oligomer, the tau-factor and an antigen of about 135 000 daltons all gave precipitating antibodies. Those four proteins were investigated in reassembled microtubules, in DEAE-cellulose purified tubulin, and after molecular sieve chromatography of disassembled and NaCl-dissociated microtubules. Reconstitution of tubulin oligomer from tubulin dimer and tau-factor was also performed. The presence of a unique antigenic structure on tubulin oligomer which was not found in the dissociated components and the role of this aggregate as a nucleation center or intermediate in the assembly of microtubules is discussed.

3alpha-, 7alpha- and 12alpha-hydroxysteroid dehydrogenase activities from Clostridium perfringens.

25 strains of Clostridium perfringens were screened for hydroxysteroid dehydrogenase activity; 19 contained NADP-dependent 3alpha-hydroxysteroid dehydrogenase and eight contained NAD-dependent 12alpha-hydroxysteroid dehydrogenase active against conjugated and unconjugated bile salts. All strains containing 12alpha-hydroxysteroid dehydrogenase also contained 3alpha-hydroxysteroid dehydrogenase although 12alpha-hydroxysteroid dehydrogenase was invariably in lesser quantity than the 3alpha-hydroxysteroid dehydrogenase. In addition, 7alpha-hydroxysteroid dehydrogenase activity was evident only when 3alpha, 7alpha, 12alpha-trihydroxy-5beta-cholanoate was substrate but notably absent when 3alpha, 7alpha-dihydroxy-5beta-cholanoate was substrate. The oxidation product 12alpha-hydroxy-3, 7-diketo-5beta-cholanoate is rapidly further degraded to an unknown compound devoid of either 3alpha- or 7alpha-OH groups. Group specificity of these enzymes was confirmed by thin-layer chromatography studies of the oxidation products. These enzyme systems appear to be constitutive rather than inducible. In contrast to C. perfringens. Clostridium paraputrificum (five strains tested) contained no measurable hydroxysteroid dehydrogenase activity. pH studies of the C. perfringens enzymes revealed a sharp pH optimum at pH 11.3 and 10.5 for the 3alpha-OH- and 12alpha-OH-oriented activities, respectively. Kinetic studies gave Km estimates of approx. 5 X 10(-5) and 8 X 10(-4) M with 3alpha, 7a-dihydroxy-5beta-cholanoate and 3alpha, 12alpha-dihydroxy-5beta-cholanoate as substrates for two respective enzymes. 3alpha-hydroxysteroid dehydrogenase was active against 3alpha-OH-containing steroids such as androsterone regardless of the sterochemistry of the 5H (Both A/B cis and A/B trans steroides were substrates). There was no activity against 3beta-OH-containing steroids. The 3alpha- and 12alpha-hydroxysteroid dehydrogenase activities, although differing in cofactor requirements cannot be distinguished by their appearance in the growth curve, their mobility on disc gel electrophoresis, elution volume on passage through Sephadex G-200 or heat inactivation studies.

Modification of the catalytic function of the mitochondrial cytochrome b-c1 complex by dicyclohexylcarbodiimide.

N,N'-Dicyclohexylcarbodiimide (DCCD) induces a complex set of effects on the succinate-cytochrome c span of the mitochondrial respiratory chain. At concentrations below 1000 mol per mol of cytochrome c1, DCCD is able to block the proton-translocating activity associated to succinate or ubiquinol oxidation without inhibiting the steady-state redox activity of the b-c1 complex either in intact mitochondrial particles or in the isolated ubiquinol-cytochrome c reductase reconstituted in phospholipid vesicles. In parallel to this, DCCD modifies the redox responses of the endogenous cytochrome b, which becomes more rapidly reduced by succinate, and more slowly oxidized when previously reduced by substrates. At similar concentrations the inhibitor apparently stimulates the redox activity of the succinate-ubiquinone reductase. Moreover, DCCD, at concentrations about one order of magnitude higher than those blocking proton translocation, produces inactivation of the redox function of the b-c1 complex. The binding of [14C]DCCD to the isolated b-c1 complex has shown that under conditions leading to the inhibition of the proton-translocating activity of the enzyme, a subunit of about 9500 Da, namely Band VIII, is the most heavily labelled polypeptide of the complex. The possible correlations between the various effects of DCCD and its modification of the b-c1 complex are discussed.

Peak ages of risk for starting nonmedical use of prescription stimulants.

BACKGROUND: To produce population-level, year- and age-specific risk estimates of first time nonmedical use of prescription stimulants among young people in the United States. METHODS: Data are from the National Surveys on Drug Use and Health 2004-2012; a nationally representative probability sample survey administered each year. Subpopulations included youths aged 12 to 21 years (n=240,160) who had not used prescription stimulants nonmedically prior to their year of survey assessment. A meta-analytic approach was used to produce population-level age-, year-, and cohort-specific risk estimates of first time nonmedical use of prescription stimulants. RESULTS: Peak risk of starting nonmedical use of prescription stimulants was concentrated between ages 16 and 19 years, when an estimated 0.7% to 0.8% of young people reported nonmedical use of these medicines for the first time in the past twelve months. Smaller risk estimates ranging from 0.1% to 0.6% were observed at ages 12 to 15 years and 20 to 21 years. Compared with males, females were more likely to have started nonmedical use of prescription stimulants (odds ratio=1.35; 95% CI, 1.13-1.62), particularly between the ages of 14 and 19. Females showed a peak annual incidence rate of 1% at age 18, while males the same age showed an incidence rate of 0.5%. CONCLUSIONS: Peak annual incidence rates for nonmedical use of prescription stimulants were observed between the ages of 16 and 19 years. There is reason to initiate interventions during the earlier adolescent years to prevent youths from starting nonmedical use of prescription stimulants.

Neurovisual rehabilitation in cerebral blindness.

OBJECTIVE: The efficacy of a systematic training of saccadic eye movements was evaluated in hemianopic patients with three main objectives: (1) to determine the role of visual field recovery, (2) to assess the transfer of treatment gains to functional outcome measures, and (3) to evaluate the patients' subjective experience throughout therapy. DESIGN: Within-subject repeated measures design. The mean follow-up interval was 3 months (range, 1 to 10 months). SETTING: Outpatients of a day clinic for the treatment of neuropsychological disorders that is associated with a city hospital. PATIENTS: A consecutive sample of 22 hemianopic patients without neglect after unilateral stroke. Follow-up was possible in all cases. INTERVENTIONS: Saccadic eye movement strategies were treated regularly (30-minute daily sessions 5 days per week; 25 to 27 total treatment sessions). MAIN OUTCOME MEASURES: Visual perimetry results, visual search field within the scotoma, visual search on projected slides with wide eccentricity, search times for identifying objects visually on a table (table test), and standardized rating of the degree of subjective visual impairment due to the field defect. All outcome measures were planned before initiation of the study. RESULTS: (1) Increase in visual search field size (mean, 30 degrees). (2) Training-related visual field increases in 12 (54%) of 22 patients (mean increase, 6.7 degrees; range, 2 degrees to 24 degrees). (3) Transfer of treatment gains to functional measures (table test) and improvement after training in patients' subjective rating of their visual impairments. (4) Stability of improvements at the 3-month follow-up visit. (5) Return to part-time work in 20 (91%) of 22 patients. All mentioned results were significant (nonparametric tests; alpha level, .05; two-sided; adjusted for the number of tests). CONCLUSIONS: Training of compensatory eye movement strategies restores oculomotor functions, improves performance in functional visual activities, and reintegrates hemianopic patients into vocational life.

Pretherapeutic flow cytometric DNA investigations in radiotherapy patients with maxillo-facial carcinomas.

In order to analyze the possible meaning of cellular DNA content and cell cycle phases for the radiosensitivity and the prognosis of human malignant tumors, flow cytometric measurements have been performed in biopsies of 131 patients with histologically proven squamous cell carcinomas of the maxillo-facial region. In two-thirds of the patients (88/131; 67%), aneuploid tumor cell lines have been found, only 33% (43/131) had a diploid DNA distribution pattern. The average DNA index (DI) of the aneuploid carcinomas was 3.4 +/- 0.6 (normal nonmalignant tissue DI = 2.0). The frequency of S-phase cells, which represents the "proliferative activity", was between 4.8 and 63.2%, regardless of the ploidy stages. The aneuploid carcinomas had about twice as many S-phase cells (mean 23.7 +/- 11.8%) than diploid tumors (mean 12.7 +/- 4.8%). Mean survival for patients with diploid carcinoma and aneuploid carcinoma was 12 and 9.5 months, respectively. Concerning the relationship of S-phase frequency and survival times in our material there was a high negative statistical correlation (Spearman-Rank test) in patients with diploid carcinomas. A high S-phase fraction resulted in short survival times. No correlation was found in the aneuploid carcinomas: patients with tumors in high S-phase values in their biopsies showed no difference in prognosis in comparison to tumors with lower S-phase fractions.

Synthesis and pharmacological evaluation of triflate-substituted analogues of clozapine: identification of a novel atypical neuroleptic.

The trifluoromethanesulfonyloxy (TfO) analogues 3 and 4 of 8-chloro-11-(4-methyl-1-piperazinyl)-5H-dibenzo[b,e][1,4]diazepine (clozapine, 1) and its 2-chloro isomer (iso-clozapine, 2), respectively, were synthesized via their OMe and OH analogues with the conventional synthetic method of the tricyclic dibenzodiazepines and evaluated pharmacologically along with their parent drugs. The binding profile of the 2-OTf analogue (4) is comparable to the binding profile of 1, although the affinity for the dopamine (DA) D2 receptors is higher (IC50 values are 31 nM and 330 nM for compounds 4 and 1, respectively). Interestingly, no notable affinity for muscarinic receptors could be detected in compound 4. On the contrary, the 8-OTf analogue 3 only displayed affinity for muscarinic M1 receptors (IC50 value 35 nM) and no affinity (IC50 value > 500 nM) for the other receptors tested. The 10 mumol/kg sc dose, but not the 10 mumol/kg po dose, of compound 4 stimulated the output of DA. Increases of 80% and 35% in DOPAC output from the dorsal striatum were seen after sc and po administrations of 10 mumol/kg of compound 4, respectively. Doses up to 100 mumol/kg of compound 3 had no effect on either parameter. Doses up to 100 mumol/kg of compound 4 were not cataleptogenic, but significantly decreased apomorphine-induced locomotor activity. In conclusion, compound 4 (GMC1-169) is a new clozapine-like neuroleptic candidate, which is lacking anticholinergic properties and displays a higher potency, as compared to clozapine (1) itself.

Sigma ligands with subnanomolar affinity and preference for the sigma 2 binding site. 2. Spiro-joined benzofuran, isobenzofuran, and benzopyran piperidines.

Spiro[isobenzofuran-1(3H),4'-piperidines] and the corresponding benzofuran and benzopyran derivatives have been synthesized and evaluated as sigma ligands. The compounds are related to Lu 28-179 (1'-[4-[1-(4-fluorophenyl)-1H-indol-3-yl]-1- butyl]spiro[isobenzofuran-1(3H),4'-piperidine]) that has been demonstrated to be a selective sigma 2 ligand with affinity in the subnanomolar range. The object of the study was to determine the structural factors governing sigma 1/sigma 2 affinity and selectivity within this class of compounds. The N-substituent in spiro[isobenzofuran-1(3H),4'-piperidines] is highly important, both for affinity and selectivity. Spiropiperidines with no or small N-substituents (H, Me, Et) exert very low affinity for both sigma 1 and sigma 2 binding sites (IC50(sigma 1, sigma 2) > 100 nM), whereas medium-sized substituents (e.g., Pr, Bu, Ph(CH2)2) result in potent, but unselective compounds (IC50(sigma 1, sigma 2) = 2-5 nM). Increasing the chain length and the lipophilicity of the N-substituent result in compounds in which high affinity for sigma 2 binding sites is retained and with selectivity for sigma 2 vs sigma 1 binding sites (e.g., 4-cyclohexyl-1-butyl: IC50-(sigma 1) = 1.5 nM, IC50(sigma 2) = 0.07 nM). Introduction of substituents in the benzene ring of the spiro[isobenzofuran-1(3H),4'-piperidine] ring system of Lu 28-179 mainly affects affinity for sigma 1 binding sites. Compounds with substituents (F, CF3) in the 4- or 7-position of the isobenzofuran display high affinity for sigma 2 binding sites (IC50(sigma 2) = 0.5-2 nM) and very low affinity for sigma 1 binding sites (IC50(sigma 1) > 100 nM). Compounds with substituents (F, CF3, Me) in the 5- or 6-position of the isobenzofuran exert increased affinity for sigma 1 binding sites (IC50(sigma 1) = 5-30 nM, IC50(sigma 2) = 0.3-7 nM), thus rendering unselective compounds. Exchanging the isobenzofuran moiety of Lu 28-179 with thioisobenzofuran, benzofuran, or benzopyran also has a pronounced effect on both affinity and selectivity for sigma binding sites. The position of the oxygen atom and the position of the spiroconnection with the 4-position of the piperidine ring were varied, and only compounds in which both the benzene ring and the heteroatom are attached directly to the piperidine ring retain high affinity and selectivity for sigma 2 binding sites (e.g., 3,4-dihydro-1'-[4-[1-(4-fluorophenyl)-1H-indol-3-yl]-1- butyl]spiro[1H-2-benzopyran-1,4'-piperidine]: IC50(sigma 1) = 53 nM, IC50(sigma 2) = 0.9 nM).(ABSTRACT TRUNCATED AT 400 WORDS)

Sigma ligands with subnanomolar affinity and preference for the sigma 2 binding site. 1. 3-(omega-aminoalkyl)-1H-indoles.

A series of 4-(1H-indol-3-yl)-1-butyl-substituted 4-phenylpiperidines, 4-phenyl-1,2,3,6-tetrahydropyridines, and 4-phenylpiperazines was synthesized. The phenyl group was optionally substituted with 4-fluoro or 2-methoxy substituents. High affinity for both sigma 1 and sigma 2 binding sites was achieved with these compounds. Additionally, these compounds had relatively high affinity for serotonin 5-HT1A and 5-HT2A, dopamine D2, and adrenergic alpha 1 receptors. Introduction of a 4-fluorophenyl substituent at the indole nitrogen atom rendered very selective sigma 2 ligands with subnanomolar affinity for the sigma 2 binding site. The prototype of such a compound was 1-(4-fluorophenyl)-3-[4-[4-(4-fluorophenyl)-1-piperidinyl]-1-butyl]-1H- indole, 11a (code no. Lu 29-253). This compound had the following binding affinities: IC50 (sigma 1) = 16 nM, IC50 (sigma 2) = 0.27 nM, IC50 (5-HT1A) = 22,000 nM, IC50 (5-HT2A) = 270 nM, IC50 (D2) = 4200 nM, IC50 (alpha 1) = 220 nM. Spiro-joining of the phenyl and the piperidine rings into a spiro[isobenzofuran-1(3H),4'-piperdine] ring system resulted in even more selective compounds. Variations of the 1-substituent at the indole and of the chain length of the alkylene spacer group were studied. The optimal compound was the spiro analogue of compound 11a. This compound is 1'-[4-[1-(4-fluorophenyl)-1H-indol-3-yl]-1-butyl]spiro[isobenzofuran- 1(3H),4'-piperidine], 14f (code no. Lu 28-179), with the binding affinities: IC50 (sigma 1) = 17 nM, IC50 (sigma 2) = 0.12 nM, IC50 (5-HT1A) = 21,000 nM, IC50 (5-HT2A) = 2000 nM, IC50 (D2) = 800 nM, IC50 (alpha 1) = 330 nM. However, the most selective sigma 2 versus sigma 1 ligand was the tropane derivative 1-(4-fluorophenyl)-3-[4-[3-(4-fluorophenyl)-8-azabicyclo[3.2.1]oct-2- en-8-yl]-1-butyl]-1H-indole, 15a. This compound had the following binding affinities: IC50 (sigma 1) = 1200 nM, IC50 (sigma 2) = 2.5 nM. Potent anxiolytic activity in the black/white box exploration test in rats was found with the two most prominent sigma 2 ligands Lu 29-253 and Lu 28-179. Good penetration into the CNS was documented both after subcutaneous and peroral administration of Lu 28-179 by ex vivo binding studies. Long duration of action was demonstrated both in ex vivo binding (T1/2 approximately 20 h) and in the black/white box exploration test.

Heterocyclic muscarinic agonists. Synthesis and biological activity of some bicyclic sulfonium arecoline bioisosteres.

A number of S-methylsulfonium analogues of the conformationally restricted muscarinic agonists of the 3-alk-oxy-4,5,6,7-tetrahydroisoxazolo[4,5-c]pyridine (O-alkyl-THPO) type have been synthesized. The effects on muscarinic receptors of these 3-alkoxy-5-methyl-6,7-dihydro-4H-thiopyrano[3,4-d]isoxazol-5 -ium (O-alkyl-S-methyl-DHTO) analogues (7a-d) were assessed in receptor-binding experiments with tritiated oxotremorine M, pirenzepine, and quinuclidinyl benzilate as ligands and were supported by studies on the isolated guinea pig ileum. The degree of muscarinic agonist activity of the compounds (M-agonist index) and their selectivity for M-1 or M-2 muscarinic receptor subtypes (M-2/M-1 index) were estimated on the basis of receptor-binding studies. The in vitro pharmacological profiles of the compounds were compared with those of arecoline and its sulfonium and 3-methoxyisoxazole isosteres, sulfoarecoline and O,5-dimethyl-THPO, respectively. While O-methyl-DHTO (5a) and N-methyl-DHTO (6a) were inactive, all of the sulfonium analogues 7a-d were muscarinic agonists with the exception of O-ethyl-S-methyl-DHTO (7b), which showed a muscarinic antagonist profile.