Quantification and pharmacokinetic property of verubecestat an BACE1 inhibitor in rat plasma.

In the present study, an ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) approach was designed to measure the rat plasma levels of verubecestat with diazepam as the internal standard. Acetonitrile-based protein precipitation was applied for sample preparation, then the analyte verubecestat was subjected to gradient elution chromatography with a mobile phase composed of acetonitrile (A) and 0.1% formic acid in water (B). Verubecestat was monitored by m/z 410.1 → 124.0 transition for quantification by multiple reaction monitoring (MRM) in positive ion electrospray ionization (ESI) source. When the concentration of verubecestat ranged from 1 to 2500 ng/mL, the method exhibited good linearity. For verubecestat, the intra- and inter-day precision were determined with the values of 2.9-9.0% and 0.4-6.5%, respectively; and the accuracy ranged from -2.2% to 10.4%. Matrix effect, extraction recovery, and stability data were in line with the standard FDA guidelines for validating a bioanalytical method. The validity of the developed method was confirmed through the pharmacokinetic study.

Safety, Tolerability, and Pharmacokinetics of the ß-Site Amyloid Precursor Protein-Cleaving Enzyme 1 Inhibitor Verubecestat (MK-8931) in Healthy Elderly Male and Female Subjects.

ß-site amyloid precursor protein-cleaving enzyme 1 (BACE1) is required for the production of ß-amyloid peptides, which are implicated in the etiology of Alzheimer's disease. The safety and pharmacokinetics of the BACE1 inhibitor verubecestat have previously been studied in young adults aged 19-45 years. In this randomized, placebo-controlled, phase I study (protocol MK-8931-006), we investigated the safety, tolerability, and pharmacokinetics of a single dose (100 mg) or multiple doses (30, 80, and 120 mg) once daily for 28 days of verubecestat in healthy elderly subjects. Safety end points were assessed at baseline and during the duration of the study period and indicated that verubecestat was generally well tolerated. Verubecestat pharmacokinetics were similar between healthy elderly male and female subjects and similar to those reported in healthy young males in previous studies. These data supported subsequent studies to assess the potential efficacy of verubecestat in subjects with Alzheimer's disease.

Chronic Verubecestat Treatment Suppresses Amyloid Accumulation in Advanced Aged Tg2576-AßPPswe Mice Without Inducing Microhemorrhage.

Verubecestat is a potent BACE1 enzyme inhibitor currently being investigated in Phase III trials for the treatment of mild-to-moderate and prodromal Alzheimer's disease. Multiple anti-amyloid immunotherapies have been dose-limited by adverse amyloid related imaging abnormalities such as vasogenic edema (ARIA-E) and microhemorrhage (ARIA-H) observed in human trials and mice. Verubecestat was tested in a 12-week nonclinical study for the potential to exacerbate microhemorrhage (ARIA-H) profiles in 18-22-month-old post-plaque Tg2576-AßPPswe mice. Animals were treated with verubecestat or controls including the anti-Aß antibody analog of bapineuzumab (3D6) as a positive control for ARIA induction. ARIA-H was measured using in-life longitudinal T2*-MRI and Prussian blue histochemistry at study end. Verubecestat reduced plasma and cerebrospinal fluid Aß40 and Aß42 by >90% and 62% to 68%, respectively. The ARIA-H profile of verubecestat-treated mice was not significantly different than controls. Anti-Aß treatment significantly increased ARIA-H detected by Prussian blue staining; however, anti-Aß antibody treatment did not impact plaque status. Verubecestat treatment significantly suppressed the accumulation of total levels of brain Aß40 and Aß42 and Thioflavin S positive plaque load. Stereological analysis of cortex and hippocampus plaque load similarly revealed significantly reduced area of Aß immunoreactivity and reduced plaque number in verubecestat-treated animals compared to controls. The absence of elevated ARIA events in verubecestat-treated mice was associated with a significant reduction in the level of accumulated CNS amyloid pathology and brain Aß peptides; effects consistent with the desired therapeutic mechanism of verubecestat in AD patients. These data will be compared with longitudinal MRI profiles from ongoing clinical trials.

Cogrinding enhances the oral bioavailability of EMD 57033, a poorly water soluble drug, in dogs.

The oral bioavailability of EMD 57033, a calcium sensitizing agent with poor solubility, was compared in dogs using four solid dosage form formulation approaches: a physical blend of the drug with excipients, micronization of the drug, preparation of coground mixtures and spray-drying of the drug from a nanocrystalline suspension. The formulations contained generally accepted excipients such as lactose, hydroxypropylmethyl cellulose and sodium lauryl sulphate in usual quantities. Drug micronization and cogrinding was realized by a jet-milling technique. Nanoparticles were created by media milling using a bead mill. All formulations were administered orally as dry powders in hard gelatine capsules. While micronization increased the absolute bioavailability of the solid drug significantly compared to crude material (from nondetectable to 20%), cogrinding with specific excipients was able to almost double this improvement (up to 39%). With an absolute bioavailability of 26%, spray-dried nanoparticular EMD 57033 failed to show the superior bioavailability that had been anticipated from in vitro data. The control solution prepared with cyclodextrin was shown to have an absolute bioavailability of 57% (vs. i.v. infusion). It was concluded that cogrinding can be a useful tool to improve the bioavailability of poorly soluble drugs from a solid dosage form format.

Pharmacokinetics of taurolidine following repeated intravenous infusions measured by HPLC-ESI-MS/MS of the derivatives taurultame and taurinamide in glioblastoma patients.

BACKGROUND AND OBJECTIVE: Taurolidine is known to have antimicrobial activity. Furthermore, at lower concentrations, it has been found to exert a selective antineoplastic effect in vitro and in vivo. The aim of this study was to investigate the pharmacokinetics of taurolidine in vivo following repeated intravenous infusion in a schedule used for the treatment of glioblastoma. As a prerequisite, the pharmacokinetics of taurolidine in human blood plasma and whole blood in vitro was investigated. PATIENTS AND METHODS: The pharmacokinetics of taurolidine and its derivatives taurultame and taurinamide were investigated in human blood plasma and in whole blood in vitro using blood from a healthy male volunteer. During repeated intravenous infusion therapy with taurolidine, plasma samples were taken every hour for a period of 13 hours per day in seven patients (three male, four female; mean age 48.4 +/- 12.8 years, range 27-66 years) with a glioblastoma. Following dansyl derivatisation, the concentrations of taurultame and taurinamide were determined using a new method based on high-performance liquid chromatography (HPLC) online coupled to electrospray ionisation tandem mass spectrometry (ESI-MS/MS) in the multiple reaction monitoring mode. Under the experimental conditions used, taurolidine could not be determined directly and was back-calculated from the taurultame and taurinamide values. RESULTS: The new HPLC-ESI-MS/MS method demonstrated high accuracy and reproducibility. In vitro plasma concentrations of taurultame and taurinamide remained constant over the incubation period. In whole blood in vitro, a time-dependent formation of taurinamide was observed. At the start of the incubation, the taurultame-taurinamide ratio (TTR) was 0.95 at an initial taurolidine concentration of 50 microg/mL, and 1.69 at 100 microg/mL. The concentration of taurultame decreased at the same rate as the taurinamide concentration increased, showing logarithmic kinetics. The calculated taurolidine concentration remained largely constant over the 6-hour incubation period. During repeated infusions in patients, calculated plasma concentrations of taurolidine showed a strong increase after the start of each infusion and continued to increase until the end of infusion, followed by a rapid decline. The TTR was found to fluctuate between 0.1 and 0.3, depending on the relation to the previous or next infusion period. The volume of distribution was markedly higher for taurolidine, taurultame and taurinamide than the plasma volume. CONCLUSIONS: Taurolidine displayed a stable pattern of derivatives in plasma in vitro, whereas in whole blood, a time- and concentration-dependent conversion was apparent. In patients, the calculated average taurolidine plasma concentration, achieved with the repeated infusion regimen, was in the antineoplastic-effective concentration range. The tissue concentrations of taurolidine and taurultame are expected to be higher than the plasma concentrations, taking into account the calculated volumes of distribution. Repeated infusion of taurolidine is the therapeutically adequate mode of administration for the indication of glioblastoma.

The pharmacokinetics of taurolidine metabolites in healthy volunteers.

Taurolidine is an experimental antibacterial and antiendotoxic compound whose clinical utility as an antitumor agent is being investigated in human clinical trials. Taurolidine in aqueous solution exists in equilibrium with taurultam. Taurultam is subsequently transformed to taurinamide. The pharmacokinetic profiles of these metabolites are not well established. In this study, 18 healthy volunteers were administered 5.0 g of taurolidine in 250 mL of 5% polyvinylpyrrolidone in water over 2, 1, or 0.5 hours by intravenous infusion in a parallel-group design. All subjects noted discomfort at the infusion site, although there were no serious adverse events. t(max) generally occurred at the end of infusion for taurinamide, whereas that of taurultam was reached before completion of infusion. The taurolidine metabolite taurultam demonstrated a shorter half-life and lower systemic exposure than taurinamide. Shortening of infusion duration increased the C(max) and AUC of taurultam. Changes in infusion rate did not substantially change the pharmacokinetic parameters of taurinamide.

Cardiovascular profile of the calcium sensitizer EMD 57033 in open-chest anaesthetized pigs with regionally stunned myocardium.

1. Ca(2+) sensitizers enhance systolic function, but may impair relaxation in vitro; these effects may differ in stunned and normal myocardium. We therefore studied the effect of EMD 57033 on systolic and diastolic function of normal and stunned porcine myocardium in vivo. 2. Myocardial stunning by 15 min coronary occlusion and 30 min reperfusion abolished systolic shortening (SS) (baseline 13+/-1%) and decreased end-systolic elastance (E(es)) from 67+/-7 to 47+/-5 mmHg mm(-1) (both P<0.05). Maximum rate of fall of myocardial elastance (dE/dt(min)) decreased from -850+/-100 to -320+/-30 mmHg mm(-1) s(-1), while the time constant tau(e) of the decay of elastance increased from 58+/-3 to 68+/-6 ms (both P<0.05). End-diastolic elastance (E(ed)) was unchanged although the zero pressure intercept (L(0,ed)) had increased. 3. In the stunned region, EMD 57033 (0.2 mg kg(-1) min(-1) for 60 min, i.v., n=7) increased SS to 19+/-2%, E(es) to 287+/-40 mmHg mm(-1), dE/dt(min) to -3630+/-640 mmHg mm(-1) s(-1) and decreased tau(e) to 50+/-3 ms, while E(ed) remained unchanged. In the normal region, 4. EMD 57033 increased SS from 14+/-2 to 18+/-3%, E(es) from 59+/-4 to 263+/-23 mmHg mm(-1), dE/dt(min) from -480+/-70 to -2280+/-700 mmHg mm(-1) s(-1) and decreased tau(e) from 91+/-12 to 61+/-3 ms (all P<0.05), while E(ed) remained unchanged. These responses were minimally affected by adrenoceptor blockade (n=7). Vehicle (n=7) had no effect on either region. EMD 57033 increased cardiac output (up to 27+/-8%) and LVdP/dt(max) (86+/-19%). Mean aortic pressure decreased (19+/-7%) due to systemic vasodilation that was not amenable to blockade of adrenoceptors or NO synthesis. 5. In conclusion, EMD 57033 restored systolic and diastolic function of stunned myocardium, and produced similar improvements in systolic and diastolic function in normal myocardium.

The effect of a thiadiazinone derived Ca2+ sensitizer on the responsiveness of Mg(2+)-ATPase to Ca2+ in myofibrils isolated from stunned and nonstunned porcine and human myocardium.

Previously, we showed, in an in situ porcine model, that the thiadiazinone derivative [+]EMD 60263, a putative Ca2+ sensitizer with minimal phosphodiesterase III inhibitory properties, increased contractility more profoundly in stunned than in nonstunned myocardium. The aim of the present investigation was to study the mechanism of action by determining the in vitro effects of [+]EMD 60263 on the Ca2+ responsiveness of the Mg(2+)-dependent ATPases of myofibrils and sarcoplasmic reticulum membrane vesicles, isolated from normal ventricle of swine and hypertrophic septum of cardiomyopathic patients. Contamination of the myofibrils with sarcoplasmic reticulum membranes was excluded by testing the effect of the sarcoplasmic reticulum Ca(2+)-pumping ATPase inhibitor thapsigargin. The plasma concentrations at which [+]EMD 60263 exerted its inotropic effect in the in situ porcine model were found to be submicromolar. [+]EMD 60263 stimulated concentration-dependently (1-10 microM) the submaximally activated Mg(2+)-ATPases (at pCa 6.1) of pig heart myofibrils. [+]EMD 60263 (10 microM) shifted the pCa50 of porcine myofibrillar Ca(2+)-stimulated, Mg(2+)-dependent ATPase from 6.00 +/- 0.05 to 6.67 +/- 0.05, whereas the [-]enantiomer EMD 60264 had no significant effect. Although the effect was much less at 1 and 3 microM, [+]EMD 60263 (10 microM) also stimulated maximal myofibrillar Mg(2+)-ATPase activity. The Hill coefficient, reflecting the steepness of the fitted pCa/Mg(2+)-ATPase curve at half-maximal activation, was not affected by [+]EMD 60263 (10 microM). [+]EMD 60263 (10 microM) had no effect on sarcoplasmic reticulum Ca(2+)-stimulated, Mg(2+)-dependent ATPase from swine heart. The thiadiazinone derivative [+]EMD 57033 (10 microM), but not its [-]enantiomer EMD 57439, had similar, although less potent, effects on pig heart myofibrillar Mg(2+)-ATPase activity as compared to [+]EMD 60263. [+]EMD 60263 (3 microM) produced a significantly larger leftward shift of the pCa2+/Mg(2+)-ATPase activity curve of myofibrils isolated from the stunned compared to the adjacent nonstunned myocardium (Delta pCa50s caused by the presence of [+]EMD 60263 amounted to +0.57 +/- 0.04 and +0.42 +/- 0.05, respectively) in the in situ porcine model. The effects of [+]EMD 60263 on myofibrillar Mg(2+)-ATPase of hypertrophic human heart were identical to those observed with porcine heart myofibrils. The results indicate that the positive inotropic action of [+]EMD 60263 observed in the in situ porcine model of stunned myocardium may be primarily due to myofilament sensitization to Ca2+, and that this compound may have a similar action on diseased human myocardium.

In-vitro antibacterial activity of noxythiolin and taurolidine.

The minimum inhibitory concentrations (MIC) of noxythiolin and taurolidine were determined for strains of Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa. Tests were performed in broth alone and in broth plus 25% v/v serum or 25% v/v urine. Inoculum density was either 10(3), 10(5) or 10(7) colony forming units per mL-1. Slight inoculum-dependent variation in the activity of both agents was observed for some, but not all, strains of P. aeruginosa and S. aureus. A more pronounced medium-dependent increase in activity was observed with both drugs, with up to 8-fold reduction of values for MIC when tested in the presence of serum or urine. These observations may help to clarify the disparity between the observed clinical efficacy of these agents and relatively poor in-vitro activity when tested using conventional methods in synthetic media.

[Pharmacokinetics of Taurolin]. / Uber die Pharmakokinetik von Taurolin.

In order to study the pharmacodynamics of taurolin (4,4'-methylene-bis(tetrahydro-2H-1,2,4-thiadiazine-1,1-dioxide) we labelled the substance in two different ways. 3*T contained 3 atoms of 14C, of which one served as a methylene bridge between the two ring systems. 4*T was labelled with 4 14C-atoms, two for every ring within the taurinamide part. Radioactivity was counted in blood, urine, faeces and expiration air of rats. For this purpose both labelled drugs were administered p.o., i.v. and i.p. After using 3*T radioactivity was excreted mainly by expiration, independently of the way of administration. The blood radioactivity diminished very slowly after a first short period. After administration of 4*T radioactivity was found mainly in the urine and only a small portion in the expiration air. Blood radioactivity diminished here very quickly. These results were confirmed by whole-body autoradiographic studies using 3*T as well as 4*T. No plasma-binding was found using the 4*T compound.

Peritoneal absorption of the antibacterial and antiendotoxin taurolin in peritonitis.

1 Taurolin metabolite plasma concentrations were measured in two groups of patient undergoing abdominal surgery, one group with peritonitis and the other without peritonitis, each group receiving taurolin by intraperitoneal instillation. 2 There was no significant difference in the area under the curves, for the two groups, for one of the metabolites. This would suggest that the absorption of taurolin was not modified in inflammatory conditions such as bacterial peritonitis.