Ziegler-Natta polymerization of propene: Cooperative effects of titanium ligands on the steric control of the first addition of monomer

A ¹³C NMR analysis of the methyl and ethyl end-groups in isotactic polypropylene, prepared in the presence of TiCl₃- and Til₃-based catalysts, was performed and the isotactic regularity of the first propylene unit added to the Ti—alkyl bond was measured. In the presence of the catalyst Til₃/Al(¹³CH₂CH₃)₃ the stereoregulating effects derived from the ethyl and iodine ligands are cooperative, so that the addition of the first monomeric unit is as isotactic as the following propagation steps. A comparison of the chain end-groups of polypropylene obtained in the presence of different catalysts shows that the extent of steric control upon insertion of the first monomer molecule results not only from the presence of alkyl titanium ligands larger than methyl and of titanium halide ligands larger than chlorine, but also from mutual ligand interactions.     

Kinetic Studies of the Copolymerization of Ethylene with Norbornene by ansa‐Zirconocene/Methylaluminoxane Catalysts: Evidence of a Long‐Lasting “Quasi‐Living” Initial Period

Copolymers of ethylene (E) with norbornene (N) were synthesized using the catalysts rac‐Et(Ind)₂ZrCl₂/MAO ( 1 ), 90%rac/10%meso‐Et(4,7‐Me₂Ind)₂ZrCl₂/MAO ( 2 ), and rac‐H₂C(3‐t‐BuInd)₂ZrCl₂/MAO ( 3 ). Catalyst activity, molar mass (MM), and copolymer composition were studied as a function of time. The polymers showed an unusually narrow molar mass distribution (MMD) and a significant increase of their MM with time for up to one hour, suggesting a “quasi‐living” polymerization at 30 °C. The experimental data were fitted to kinetic equations and the propagation and transfer reactions were described in quantitative terms. Norbornene greatly depressed the propagation rate, along with the chain transfer rate. The more sterically hindered catalysts of the series showed lower propagation and chain transfer turnover frequency than 1 and yielded polymers with a low ( 2 ) to very low ( 3 ) norbornene content. The presence of norbornene in solution seemed to be one of the main factors responsible for the observed “quasi‐living” character of the copolymerization, probably due to coordination of norbornene to the active site. Time‐resolved kinetic studies also allowed for the calculation of the fraction of active metal centers, ranging from 56% ( 3 ) and 66–68% ( 1 ) to 94% ( 2 ) of the total zirconium present, depending on catalyst structure.

Left: molar mass (top) and polydispersity (bottom) as a function of the normalized polymer yield. The dashed line is the theoretical curve for ideal living polymerization. Catalysts 1 (□), 2 (?), and 3 (○) at N/E ratio 12.5 and catalyst 1 (?) at N/E ratio 28.4. Right: enlargement of the low yield section.     

On the mechanism of olefin polymerization by titanocene/MAO catalysts: Relationships between metathesis and addition polymerization

Ethylene polymerizations and norbornene oligomerizations catalysed by Cp₂Ti¹³CH₃Cl/MAO (Cp: cyclopentadienyl; MAO: methylaluminoxane) mixtures have been carried out at different temperatures (from -20°C to 20°C), in order to test the validity of carbene mechanisms in α-olefin polymerizations. Depending on the temperature, different ratios of the cationic species [Cp₂Ti¹³CH₃]⁺[Cl · MAO]^? and precursors of the alkylidene Cp₂Ti = ¹³CH₂ exist. The in situ polymerization of ¹³C enriched ethylene was monitored by ¹³C NMR spectroscopy. Moreover, catalytic activity was determined and polyethylene samples were analyzed by ¹³C NMR and gel permeation chromatography (GPC). The following evidence has been provided against the carbene mechanism in the α-olefin polymerization with titanocene based catalysts: (a) in the in situ ethylene polymerization experiments the appearance of polyethylene signals is concurrent with the decrease of cationic [Cp₂Ti¹³CH₃]⁺[Cl · MAO]^? signals and is not related to the intensity of the alkylidene Cp₂Ti = ¹³CH₂ signals; (b) from the ¹³C NMR analysis of polyethylene chain-end groups the ¹³C enrichment of Cp₂Ti¹³CH₃Cl has only been found in the methyl chain-end group and not in the methylene of the propyl chain-end group, as should have been the case if the carbene mechanism had been valid; (c) from norbornene oligomerization (at 0°C) the addition product 2-¹³C enriched methyl-norbornane has been identified. Moreover, the identification of a ¹³C enriched methylidene-norbornane dimer at higher temperatures has revealed the possibility of norbornene addition to titanium carbenes through the formation of titanacyclobutane without the opening of the norbornene ring. However, this process requires higher energies with respect to the Cossee type insertion.     

Evaluation of adjunctive perampanel in patients with refractory partial‐onset seizures: Results of randomized global phase III study 305

Purpose: To assess the efficacy and safety of once‐daily doses of perampanel 8 and 12 mg when added to 1–3 concomitantly administered, approved antiepileptic drugs (AEDs) in patients with uncontrolled partial‐onset seizures. Methods: Study 305 was a multicenter, double‐blind, placebo‐controlled trial in patients aged 12 years and older with ongoing seizures despite prior therapy with at least two AEDs, and currently receiving 1–3 AEDs. Equal randomization to once‐daily oral perampanel 8 or 12 mg, or placebo was performed. Patients entered a 19‐week double‐blind treatment phase comprising a 6‐week titration period, with weekly 2‐mg dose increments, followed by a 13‐week maintenance period. Primary efficacy end points were the responder rate (proportion of patients who had a ≥50% reduction in seizure frequency during treatment per 28 days relative to baseline), and the percent change in seizure frequency per 28 days relative to pre‐perampanel baseline. A secondary end point was percent change in the frequency of complex partial plus secondarily generalized seizures. Adverse events (AEs) were monitored throughout the study. Key Findings: Three hundred eighty‐six patients were randomized and treated with study medication. Of these, 321 patients completed the study. The 50% responder rates (intent‐to‐treat analysis) were 14.7%, 33.3%, and 33.9%, respectively, for placebo, perampanel 8 mg, and perampanel 12 mg, with significant improvements over placebo for both perampanel 8 mg (p = 0.002) and 12 mg (p < 0.001). The median percent change from baseline in seizure frequency per 28 days (intent‐to‐treat analysis) was ?9.7%, ?30.5%, and ?17.6% for placebo, 8 mg, and 12 mg, respectively, with significant reductions compared with placebo for both 8 mg (p < 0.001) and 12 mg (p = 0.011). For complex partial seizures plus partial seizures that secondarily generalized, the median percent change in frequency was ?32.7% (8 mg), ?21.9 (12 mg), and ?8.1% (placebo), with significant reductions for both 8 mg (p < 0.001) and 12 mg (p = 0.005). The most frequent (occurring in ≥10% of patients in any treatment group) treatment‐emergent AEs were dizziness, somnolence, fatigue, and headache, with an apparent dose effect suggested for all except headache. Significance: This phase III trial demonstrated that adjunctive treatment with once‐daily perampanel at 8 mg and 12 mg was effective in improving seizure control in patients 12 years and older with refractory partial‐onset seizures. These study results also demonstrated that once‐daily doses of 8 mg and 12 mg were safe and acceptably tolerated in this study. Perampanel demonstrated a favorable risk/benefit ratio in this population.     

Perampanel,a selective,noncompetitive α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid receptor antagonist,as adjunctive therapy for refractory partial‐onset seizures: Interim results from phase III,extension study 307

Purpose: To evaluate safety, tolerability, and seizure outcome data during long‐term treatment with once‐daily adjunctive perampanel (up to 12 mg/day) in patients with refractory partial‐onset seizures. Methods: Study 307 was an extension study for patients completing the double‐blind phase of three pivotal phase III trials (studies 304, 305, and 306). The study consisted of two phases: an open‐label treatment phase (including a 16‐week blinded conversion period and a planned 256‐week maintenance period) and a 4‐week follow‐up phase. Patients were blindly titrated during the conversion period to their individual maximum tolerated dose (maximum 12 mg/day). Adverse events (AEs) were monitored throughout the study and seizure frequency recorded. The interim data cutoff date for analyses was December 1, 2010. Key Findings: In total, 1,218 patients were enrolled in the study. At the interim cutoff date, 1,186 patients were in the safety analysis set; 1,089 (91.8%) patients had >16 weeks of exposure to perampanel, 580 (48.9%) patients had >1 year of exposure, and 19 (1.6%) patients had >2 years of exposure. At the interim analysis, 840 (70.8%) patients remained on perampanel treatment. The large majority of patients (n = 1,084 [91%]) were titrated to 10 mg or 12 mg/day. Median (range) duration of exposure was 51.4 (1.1–128.1) weeks. Treatment‐emergent AEs were reported in 87.4% of patients. The most frequent were dizziness (43.9%), somnolence (20.2%), headache (16.7%), and fatigue (12.1%). Serious AEs were reported in 13.2% of patients. In the intent‐to‐treat analysis set (n = 1,207), the frequency of all seizures decreased over the first 26 weeks of perampanel treatment in patients with at least 26 weeks of exposure to perampanel (n = 1,006 [83.3%]); this reduction was maintained in patients with at least 1 year of exposure (n = 588 [48.7%]). The overall median percent changes in seizure frequency in patients included in each 13‐week interval of perampanel treatment were ?39.2% for weeks 14–26 (n = 1,114), ?46.5% for weeks 40–52 (n = 731), and ?58.1% for weeks 92–104 (n = 59). Overall responder rates in patients included in each 13‐week interval of perampanel treatment were 41.4% for weeks 14–26 (n = 1,114), 46.9% for weeks 40–52 (n = 731), and 62.7% for weeks 92–104 (n = 59). During the blinded conversion period, the reduction in seizure frequency in patients previously randomized to placebo (?42.4%, n = 369) was similar to that in patients previously randomized to perampanel (?41.5%, n = 817). Significance: Consistent with pivotal phase III trials, these interim results demonstrated that perampanel had a favorable tolerability profile in patients with refractory partial‐onset seizures over the longer term. The decrease in seizure frequency was consistent and maintained in those patients over at least 1 year of perampanel exposure.     

Stimulation of adenylate cyclase by water-soluble analogues of forskolin

Analogues of forskolin that are more soluble in water than forskolin have been synthesized and tested for their ability to interact with adenylate cyclase. These analogues are esterified with various heterocyclic amino acids at the 6 beta-hydroxyl position of forskolin or at the 6 beta-hydroxyl or 7 beta-hydroxyl position of 7-desacetyl forskolin. Analogues were tested for their ability to activate rat brain adenylate cyclase, activate detergent-solubilized rat brain adenylate cyclase, increase cyclic AMP in intact S49 wild-type cells, and inhibit the binding of 3H-forskolin to rat brain membranes. Forskolin activated rat brain adenylate cyclase with an EC50 of 4 microM and increased cyclic AMP in intact S49 cells with an EC50 of 5 microM. Analogues esterified at the 7 beta-hydroxyl position had EC50 values that ranged from 4 microM to 15 microM for activating adenylate cyclase in membranes and solubilized preparations, and for increasing cyclic AMP in S49 cells. Analogues esterified at the 6 beta-hydroxyl position with no acyl group at the 7 beta-hydroxyl position were generally less potent than the corresponding 7-acyl analogues with EC50 values that ranged from 30 microM to 100 microM. Interestingly, the diacyl analogues of forskolin containing an acetate group at the 7 beta-hydroxyl position and esterified with heterocyclic amino acids at the 6 beta-hydroxyl position were very potent at stimulating adenylate cyclase, with EC50 values that ranged from 1 microM to 25 microM. The 7-acyl analogues and the 6,7-diacyl analogues inhibited the binding of 3H-forskolin to rat brain membranes with IC50 values that ranged from 20 microM to 70 microM, while the 6-acyl analogues had much higher IC50 values that ranged from 100 nM to 375 nM. Aqueous solutions of forskolin were also produced by dissolving forskolin in solutions of hydroxypropyl-gamma-cyclodextrin. These aqueous solutions of forskolin were equipotent with alcoholic solutions of forskolin in stimulating adenylate cyclase. In conclusion, water-soluble derivatives of forskolin may be useful for increasing cyclic AMP in broken cell preparations or in intact cell preparations where the presence of organic solvents, which are necessary to solubilize forskolin, are detrimental. Alternatively, aqueous solutions of forskolin can be produced by dissolving forskolin in solutions of hydroxypropyl-gamma-cyclodextrin.     

Detection by ELISA of laminin released by endothelial cells from a collagen coated layer of Matrigel

Summary We have developed an in vitro model system quantitating the degradation of extracellular matrix by endothelial cells. Collagen is layered over a reconstituted basement membrane matrix (Matrigel) and the release of laminin contained in the Matrigel into the media by endothelial cells is quantitated by ELISA. The cell-specific release of laminin is consistent and reproducible. Incubating an equivalent number of dead endothelial cells in the same manner results in no release of laminin relative to media controls. The cell-specific laminin release is abolished by removing the exogenous growth factor component of the media and plating the cells in media plus 10% serum. Cells plated onto wells coated with collagen alone do not release laminin into the media, indicating that no de novo synthesis and release of laminin occurs during the time frame of the experiment, and that some factor or combination of factors present in the media supplement is essential for the endothelial cells to release laminin degrading enzymes.