Intravenous alteplase for stroke with unknown time of onset guided by advanced imaging: systematic review and meta-analysis of individual patient data

Background: Patients who have had a stroke with unknown time of onset have been previously excluded from thrombolysis. We aimed to establish whether intravenous alteplase is safe and effective in such patients when salvageable tissue has been identified with imaging biomarkers. Methods: We did a systematic review and meta-analysis of individual patient data for trials published before Sept 21, 2020. Randomised trials of intravenous alteplase versus standard of care or placebo in adults with stroke with unknown time of onset with perfusion-diffusion MRI, perfusion CT, or MRI with diffusion weighted imaging-fluid attenuated inversion recovery (DWI-FLAIR) mismatch were eligible. The primary outcome was favourable functional outcome (score of 0–1 on the modified Rankin Scale [mRS]) at 90 days indicating no disability using an unconditional mixed-effect logistic-regression model fitted to estimate the treatment effect. Secondary outcomes were mRS shift towards a better functional outcome and independent outcome (mRS 0–2) at 90 days. Safety outcomes included death, severe disability or death (mRS score 4–6), and symptomatic intracranial haemorrhage. This study is registered with PROSPERO, CRD42020166903. Findings: Of 249 identified abstracts, four trials met our eligibility criteria for inclusion: WAKE-UP, EXTEND, THAWS, and ECASS-4. The four trials provided individual patient data for 843 individuals, of whom 429 (51%) were assigned to alteplase and 414 (49%) to placebo or standard care. A favourable outcome occurred in 199 (47%) of 420 patients with alteplase and in 160 (39%) of 409 patients among controls (adjusted odds ratio [OR] 1·49 [95% CI 1·10–2·03]; p=0·011), with low heterogeneity across studies (I2=27%). Alteplase was associated with a significant shift towards better functional outcome (adjusted common OR 1·38 [95% CI 1·05–1·80]; p=0·019), and a higher odds of independent outcome (adjusted OR 1·50 [1·06–2·12]; p=0·022). In the alteplase group, 90 (21%) patients were severely disabled or died (mRS score 4–6), compared with 102 (25%) patients in the control group (adjusted OR 0·76 [0·52–1·11]; p=0·15). 27 (6%) patients died in the alteplase group and 14 (3%) patients died among controls (adjusted OR 2·06 [1·03–4·09]; p=0·040). The prevalence of symptomatic intracranial haemorrhage was higher in the alteplase group than among controls (11 [3%] vs two [<1%], adjusted OR 5·58 [1·22–25·50]; p=0·024). Interpretation: In patients who have had a stroke with unknown time of onset with a DWI-FLAIR or perfusion mismatch, intravenous alteplase resulted in better functional outcome at 90 days than placebo or standard care. A net benefit was observed for all functional outcomes despite an increased risk of symptomatic intracranial haemorrhage. Although there were more deaths with alteplase than placebo, there were fewer cases of severe disability or death. Funding: None

Inhibition of cellular ATP-hydrolyzing activity by tricyclic antidepressants and phenothiazine tranquilizers

In a variety of cellular and subcellular preparations, total ATP-hydrolyzing activity was potently inhibited by tricyclic antidepressants (TA) and phenothiazine tranquilizers (PT). In leukocytes, preincubation of the cells with 5 x 10-4 M TA and 1 x 10-4 M PT resulted in up to 87% and 94% inhibition of the ATPase activity. Among TA, dibenzocycloheptadienes were somewhat more potent inhibitors than were derivatives of dibenzazepine. Substitution, at the position 2 of the phenothiazine nucleus, by a halogen and particularly by the CF3 group, increased the inhibitory strength of PT. However, most effective in inhibiting ATPase was thioridazine, structurally differing from mesoridazine, the weakest inhibitor in this study, by a methylmercapto group instead of a methylsulfinyl substituent. The inhibition by the drugs was markedly reduced in the presence of millimolar ATP. The results indicate a possible adverse effect of these drugs on the cellular energy-yielding capacity.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/22073/1/0000493.pd

Mechanism-Based Inactivation of Cytochrome P450 3A4 by 17␣-Ethynylestradiol: Evidence for Heme Destruction and Covalent Binding to Protein

ABSTRACT 17␣-Ethynylestradiol (EE), a major constituent of many oral contraceptives, inactivated the testosterone 6␤-hydroxylation activity of purified P450 3A4 reconstituted with phospholipid and NADPH-cytochrome P450 reductase in a mechanismbased manner. The inactivation of P450 3A4 followed pseudo first order kinetics and was dependent on NADPH. The values for the K I and k inact were 18 M and 0.04 min Ϫ1 , respectively, and the t 1/2 was 16 min. Incubation of 50 M EE with P450 3A4 at 37°C for 30 min resulted in a 67% loss of testosterone 6␤-hydroxylation activity accompanied by a 35% loss of the spectral absorbance of the native protein at 415 nm and a 70% loss of the spectrally detectable P450-CO complex. The inactivation of P450 3A4 by EE was irreversible. Testosterone, an alternate substrate, was able to protect P450 3A4 from EEdependent inactivation. The partition ratio was ϳ50. (HPLC) analysis demonstrated that the inactivation resulting from EE metabolism led to the destruction of approximately half the heme with the concomitant generation of modified heme and EE-labeled heme fragments and produced covalently radiolabeled P450 3A4 apoprotein. Electrospray mass spectrometry demonstrated that the fraction corresponding to the major radiolabeled product of EE metabolism has a mass (M Ϫ H) Ϫ of 479 Da. HPLC and gas chromatography-mass spectometry analyses revealed that EE metabolism by P450 3A4 generated one major metabolite, 2-hydroxyethynylestradiol, and at least three additional metabolites. In conclusion, our results demonstrate that EE is an effective mechanism-based inactivator of P450 3A4 and that the mechanism of inactivation involves not only heme destruction, but also the irreversible modification of the apoprotein at the active site

Metabolic Activation of Mifepristone [RU486; 17β-Hydroxy-11β-(4-dimethylaminophenyl)-17α-(1-propynyl)-estra-4,9-dien-3-one] by Mammalian Cytochromes P450 and the Mechanism-Based Inactivation of Human CYP2B6

Mifepristone [RU486; 17β-hydroxy-11β-(4-dimethylaminophenyl)-17α-(1-propynyl)-estra-4,9-dien-3-one] inactivates CYP2B6 in the reconstituted system in a mechanism-based manner. The loss of 7-ethoxy-4-(trifluoromethyl)-coumarin deethylation activity of CYP2B6 is concentration- and time-dependent. The inactivation requires NADPH and is irreversible. The concentration of inactivator required to give the half-maximal rate of inactivation is 2.8 μM, and the maximal rate constant for inactivation at a saturating concentration of the inactivator is 0.07 min-1. Incubation of CYP2B6 with 20 μM RU486 for 15 min resulted in 61% loss of catalytic activity, 60% loss of the reduced cytochrome P450 (P450)-CO complex, and a 40% loss of native heme. The partition ratio is ∼5, and the stoichiometry of binding is ∼0.6 mol RU486/mol P450 inactivated. SDS-polyacrylamide gel electrophoresis and high-pressure liquid chromatography analysis showed that [3H]RU486 was irreversibly bound to CYP2B6 apoprotein. RU486 is metabolized to form three major metabolites and bioactivated to give reactive intermediates by purified P450s in the reconstituted system. After incubation of RU486 with the purified P450s and liver microsomes from rats and humans in the presence of glutathione (GSH) and NADPH, GSH conjugates with MH+ ions at m/z 769, 753, and 751 were detected by liquid chromatography-tandem mass spectrometry. Two GSH conjugates with MH+ ions at m/z 753 are formed from the reaction of GSH with RU486. The adducts are formed after addition of an activated oxygen to the carbon-carbon triple bond of the propynyl moiety. This suggests that oxirene intermediates may be involved in the mechanism of inactivation. It seems that the potential for drug-drug interactions of RU486 may not be limited only to CYP3A4 and should also be evaluated for drugs metabolized primarily by CYP2B6, such as bupropion and efavirenz

Covalent Modification of Thr302 in Cytochrome P450 2B1 by the Mechanism-Based Inactivator 4-tert-Butylphenylacetylene

The mechanism of inactivation of cytochrome P450 2B1 (CYP2B1) by 4-tert-butylphenylacetylene (BPA) has been characterized previously to be caused by the covalent binding of a reactive intermediate to the apoprotein rather than heme destruction (J Pharmacol Exp Ther 331:392–403, 2009). The identification of a BPA-glutathione conjugate and the increase in the mass of the BPA-adducted apoprotein have indicated that the mass of adduct is 174 Da, equivalent to the mass of BPA plus one oxygen atom. To identify the adducted residue, BPA-inactivated CYP2B1 was digested with trypsin, and the digest was then analyzed by using capillary liquid chromatography with a LTQ linear ion trap mass spectrometer as the detector. A mass shift of 174 Da was used for a SEQUEST database search. The tandem mass spectrometry fragmentation of the modified peptide and the identity of modified residue were determined. The results revealed a mass increase of 174 Da for the peptide sequence 296FFAGTSSTTLR308 in the I-helix of CYP2B1 and that the site of adduction formation is Thr302. Homology modeling and ligand docking studies showed that BPA binds in close proximity to both the heme iron and Thr302 with the distances being 2.96 and 3.42 Å, respectively. The identification of Thr302 in the CYP2B1 active site as the site of covalent modification leading to inactivation by BPA supports previous hypotheses that this conserved Thr residue may play a crucial role for various functions in P450s

Mechanism-Based Inactivation of CYP2B1 and Its F-Helix Mutant by Two tert-Butyl Acetylenic Compounds: Covalent Modification of Prosthetic Heme Versus Apoprotein

The mechanism-based inactivation of cytochrome CYP2B1 [wild type (WT)] and its Thr205 to Ala mutant (T205A) by tert-butylphenylacetylene (BPA) and tert-butyl 1-methyl-2-propynyl ether (BMP) in the reconstituted system was investigated. The inactivation of WT by BPA exhibited a kinact/KI value of 1343 min−1mM−1 and a partition ratio of 1. The inactivation of WT by BMP exhibited a kinact/KI value of 33 min−1mM−1 and a partition ratio of 10. Liquid chromatography/tandem mass spectrometry analysis (LC/MS/MS) of the WT revealed 1) inactivation by BPA resulted in the formation of a protein adduct with a mass increase equivalent to the mass of BPA plus one oxygen atom, and 2) inactivation by BMP resulted in the formation of multiple heme adducts that all exhibited a mass increase equivalent to BMP plus one oxygen atom. LC/MS/MS analysis indicated the formation of glutathione (GSH) conjugates by the reaction of GSH with the ethynyl moiety of BMP or BPA with the oxygen being added to the internal or terminal carbon. For the inactivation of T205A by BPA and BMP, the kinact/KI values were suppressed by 100- and 4-fold, respectively, and the partition ratios were increased 9- and 3.5-fold, respectively. Only one major heme adduct was detected following the inactivation of the T205A by BMP. These results show that the Thr205 in the F-helix plays an important role in the efficiency of the mechanism-based inactivation of CYP2B1 by BPA and BMP. Homology modeling and substrate docking studies were presented to facilitate the interpretation of the experimental results