Fluorothiazinon, a small-molecular inhibitor of T3SS, suppresses salmonella oral infection in mice.

Therapeutic strategies that target bacterial virulence have received considerable attention. The type III secretion system (T3SS) is important for bacterial virulence and represents an attractive therapeutic target. Recently, we developed a new small-molecule inhibitor belonging to a class 2,4-disubstituted-4H-[1,3,4]-thiadiazine-5-ones, Fluorothiazinon (FT-previously called CL-55). FT effectively suppressed T3SS of Chlamydia spp., Pseudomonas aeruginosa, and Salmonella without affecting bacterial growth in vitro. FT was previously characterized by low toxicity, stability, and therapeutic efficacy in animal models. Salmonella T3SS inhibition by FT was studied using in vitro assays for effector proteins detection and estimation of salmonella replication in peritoneal macrophages. The antibacterial effect of FT in vivo was investigated in murine models of salmonella chronic systemic and acute infection. Oral administration of the virulent strain of Salmonella enterica serovar Typhimurium to mice-induced chronic systemic infection with the pathogen persistence in different lymphoid organs such as spleens, Peyer's plaques, and mesenteric lymph nodes. We found that FT suppressed orally induced salmonella infection both with therapeutic and prophylactic administration. Treatment by FT at a dose of 50 mg/kg for 4 days starting from day 7 post-infection (therapy) as well as for 4 days before infection (prevention) led to practically complete eradication of salmonella in mice. FT shows a strong potential for antibacterial therapy and could be used as a substance in the design of antibacterial drugs for pharmaceutical intervention including therapy of antibiotic-resistant infections.

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.

Buprofezin dissipation and safety assessment in open field cabbage and cauliflower using GC/ITMS employing an analyte protectant.

An analytical method for the determination of buprofezin residues in cabbage and cauliflower was developed and validated using gas chromatography with ion trap mass spectrometry. The analyte protectant d-sorbitol was used at a concentration level of 0.5 mg mL-1 in acetonitrile instead of in matrix for constructing the calibration curves of the buprofezin standard. The average recoveries ranged from 91.3 to 96.8%, with an RSD of ≤2.7%. The limits of detection and quantitation of the method in cabbage and cauliflower were 1.3, 1.7 and 4.3, 6.2 µg kg-1 , respectively. The residual levels and dissipation kinetics of buprofezin 25% wettabe powder in cabbage and cauliflower cultivated under open field conditions was investigated at the single (T1) and double (T2) recommended rates of application. Half-life periods were found to be 1.73 and 2.1 days in cabbage, whereas in cauliflower, these values were 1.85 and 2.36 days at T1 and T2, respectively. Based on the dissipation study, and the maximum residue limit value of 0.05 mg kg-1 , the proposed pre-harvest interval of buprofezin in cabbage was 3-6 days and that in cauliflower was 4-10 days. The results showed that buprofezin is safe for application at both recommended application rates.

Pharmacokinetics and Pharmacodynamics of the BACE1 Inhibitor Verubecestat (MK-8931) in Healthy Japanese Adults: A Randomized, Placebo-Controlled Study.

ß-site amyloid precursor protein cleaving enzyme 1 (BACE1) is required for the production of ß-amyloid (Aß) peptides and is considered a potential treatment target for Alzheimer's disease (AD). To support Japan's participation in the global clinical development program, we characterized the safety, pharmacokinetics (PKs), and pharmacodynamics of the BACE1 inhibitor verubecestat (MK-8931) in 24 healthy Japanese adults in a two-part, single-center, randomized, placebo-controlled phase I trial (protocol MK-8931-007) and compared the results with historical data from non-Japanese subjects. Both single (20, 100, and 450 mg) and multiple (80 and 150 mg once daily for 14 days) doses of verubecestat were well tolerated. Verubecestat's PK profile was similar in Japanese and non-Japanese subjects. Verubecestat also reduced mean cerebrospinal fluid concentrations of the Aß proteins Aß40, Aß42, and soluble ß fragment of amyloid precursor protein; the level of reduction was comparable between Japanese and non-Japanese subjects. These results support the continued global development of verubecestat as a potential disease-modifying agent for Japanese and non-Japanese subjects who are at risk for developing AD.

Toxicity, pharmacokinetics and metabolism of a novel inhibitor of IL-6-induced STAT3 activation.

PURPOSE: The oncogenic transcription factor signal transducer and activator of transcription 3 (STAT3) promotes gene transcription involved in cancer, and its activation by IL-6 is found in head and neck squamous cell carcinoma. Four triazolothiadizine STAT3 pathway inhibitors were evaluated to prioritize a single compound for in vivo examination. METHODS: Metabolic stability in mouse liver microsome incubation was used to evaluate four triazolothiadizine analogues, and UPCDC-10205 was administered to mice IV as single or multiple doses to evaluate toxicity. Single-dose pharmacokinetics (PK), bioavailability and metabolism were studied after IV 4 mg/kg, PO 4 mg/kg, or PO 30 mg/kg suspension in 1% carboxymethyl cellulose. Mice were euthanized between 5 min to 24 h after dosing, and plasma and tissues were analyzed by LC-MS. Non-compartmental PK parameters were determined. RESULTS: Of the four triazolothiadizine analogues evaluated, UPCDC-10205 was metabolically most stable. The maximum soluble dose of 4 mg/kg in 10% Solutol™ was not toxic to mice after single and multiple doses. PK analysis showed extensive tissue distribution and rapid plasma clearance. Bioavailability was ~5%. A direct glucuronide conjugate was identified as the major metabolite which was recapitulated in vitro. CONCLUSIONS: Rapid clearance of UPCDC-10205 was thought to be the result of phase II metabolism despite its favorable stability in a phase I in vitro metabolic stability assay. The direct glucuronidation explains why microsomal stability (reflective of phase I metabolism) did not translate to in vivo metabolic stability. UPCDC-10205 did not demonstrate appropriate exposure to support efficacy studies in the current formulation.

Pharmacokinetic study and evaluation of the safety of taurolidine for dogs with osteosarcoma.

BACKGROUND: Osteosarcoma in dogs and humans share many similarities and the dog has been described as an excellent model to study this disease. The median survival in dogs has not improved in the last 25 years. Taurolidine has been shown to be cytotoxic to canine and human osteosarcoma in vitro. The goals of this study were to determine the pharmacokinetics and safety of taurolidine in healthy dogs and the safety of taurolidine in combination with doxorubicin or carboplatin in dogs with osteosarcoma. METHODS: Two percent taurolidine was infused into six healthy dogs (150 mg/kg) over a period of two hours and blood samples were taken periodically. One dog received taurolidine with polyvinylpyrrolidone (PVP) as its carrier and later received PVP-free taurolidine as did all other dogs in this study. Serum taurolidine concentrations were determined using high-performance liquid chromatography (HPLC) online coupled to ESI-MS/MS in the multiple reaction monitoring mode. Subsequently, the same dose of taurolidine was infused to seven dogs with osteosarcoma also treated with doxorubicin or carboplatin. RESULTS: Taurolidine infusion was safe in 6 healthy dogs and there were no significant side effects. Maximum taurolidine serum concentrations ranged between 229 to 646 µM. The dog that received taurolidine with PVP had an immediate allergic reaction but recovered fully after the infusion was stopped. Three additional dogs with osteosarcoma received doxorubicin and taurolidine without PVP. Toxicities included dilated cardiomyopathy, protein-losing nephropathy, renal insufficiency and vasculopathy at the injection site. One dog was switched to carboplatin instead of doxorubicin and an additional 4 dogs with osteosarcoma received taurolidine-carboplatin combination. One incidence of ototoxicity occurred with the taurolidine- carboplatin combination. Bone marrow and gastro-intestinal toxicity did not appear increased with taurolidine over doxorubicin or carboplatin alone. CONCLUSIONS: Taurolidine did not substantially exacerbate bone marrow or gastro-intestinal toxicity however, it is possible that taurolidine increased other toxicities of doxorubicin and carboplatin. Administering taurolidine in combination with 30 mg/m2 doxorubicin in dogs is not recommended but taurolidine in combination with carboplatin (300 mg/m2) appears safe.

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.

[The estimation of the perspectives of the 1,3,4-thiadiazine derivatives as the protectors means for the health tissues under radiation therapy of malignant tumour patients].

We generalized the results of our own researches of the mechanisms, determined the high (90% BALB-line mice were survived) radioprotection activity by 1,3,4-thiadiazine derivatives. It was determined that this preparat achieves the highest concentrations in the critical for the acute radiation influence tissues. The preparate bind with the cell's membranes, nucleus and mitochondries, blockade the development of the radial reactions on the tissues level. Small quantity passes to the brain marrow, takes part in the regulative processes, which central nervous system is produced, reduces the metabolitical processes in the organism. It doesn't possess the election accumulation in the tumour and it is perspective for the prevention of damage health tissues under irradiation cancroid's therapy.

Taurolidine-Fibrin-Sealant-Matrix using spray application for local treatment of brain tumors.

Malignant gliomas tend to recur in the vast majority of cases. Recurrent gliomas may arise from vital tumor cells present in this zone around the resection margin. It appears promising to combine tumor resection with local chemotherapy using an antineoplastic, but non-toxic agent. Taurolidine exerts a selective antineoplastic effect by induction of programmed cell death and has anti-angiogenic activity. Fibrin sealant is completely degradable and firmly adheres to brain tissue, suggesting that it would provide a suitable matrix for taurolidine delivery--a Taurolidine-Fibrin-Sealant-Matrix (TFM)--in the local treatment of brain tumors. The potential of local delivery of taurolidine out of a fibrin sealant matrix was investigated. Taurolidine could be suspended homogeneously in both the thrombin and the procoagulant protein components of the fibrin sealant. The fibrin sealant matrix was a suitable carrier for the suspension of taurolidine at a concentration that ensured the release of therapeutically effective amounts of the drug over a period of 2 weeks in vitro. The antineoplastic action of taurolidine was not affected by embedding in the fibrin sealant matrix. The described drug delivery system may be suitable for local taurolidine treatment of brain tumors following complete or partial resection or of tumors that are non-resectable because of their location.

Kinetic profile of a heterocyclic HCV replicon RNA synthesis inhibitor.

Recently, a benzo-1,2,4-thiadiazine was shown to be a potent, specific inhibitor of the hepatitis C virus (HCV) RNA polymerase [J. Biol. Chem. 277 (2002) 32327]. Herein, we present several lines of evidence to demonstrate that thiadiazine compound 4 (C(21)H(21)N(3)O(4)S) is highly synergistic with interferon-alpha (IFN-alpha) and disrupts HCV replicon RNA synthesis with a distinct kinetic profile. A time course analysis after a single treatment with 5 microM compound 4 showed a loss of viral RNA consistent with replicon RNA half-life, suggesting inhibition of 90% of ongoing or newly initiated replicative intermediates. This finding is consistent with the mechanism of action recently reported for compound 4, an RNA synthesis initiation inhibitor [J. Biol. Chem. 278 (2003) 16602]. Further, unlike IFN-alpha, an immediate reduction of HCV replicon RNA synthesis was apparent upon addition of compound 4. Treatment with IFN-alpha showed a delay of approximately 4h prior to inhibition of viral RNA replication, consistent with its signaling kinetics.

3-amino-4-sulfonylpyridinone acetamide and related pyridothiadiazine thrombin inhibitors.

We describe a series of highly potent and efficacious thrombin inhibitors based on a 3-amino-4-sulfonylpyridinone acetamide template. The functionally dense sulfonyl group stabilizes the aminopyridinone, conformationally constrains the 4-substituent, and forms a hydrogen bond to the insertion loop tyrosine OH. We also describe a related series of fused bicyclic dihydrothiadiazinedioxide derivatives, of which one had improved pharmacokinetics in dogs after oral dosing.

Acephate and buprofezin residues in olives and olive oil.

Field trials were carried out to study the persistence of acephate and buprofezin on olives. Two cultivars, pizz'e carroga and pendolino, with very large and small fruits respectively were used. After treatment, no difference was found between the two pesticide deposits on the olives. The disappearance rates, calculated as pseudo first order kinetics, were similar for both pesticides (on average 12 days). Methamidophos, the acephate metabolite, was always present on all olives, and in some pendolino samples it showed higher residues than the maximum residue limit (MRL). During washing, the first step of olive processing, the residue level of both pesticides on the olives did not decrease. After processing of the olives into oil, no residues of acephate or methamidophos were found in the olive oil, while the residues of buprofezin were on average four times higher than on olives.

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.

Tetrahydro-2H-1,3,5-thiadiazine-5-(4-pyridylcarboxamide)-2-thione derivatives as prodrugs for isoniazid; synthesis, investigations and in vitro antituberculous activity.

3-Substituted-5-(4-pyridylcarboxamido)tetrahydro-2H-1,3,5-thiad iazine-2- thione derivatives 5a-e were synthesized as prodrugs for isoniazid (INH) to overcome the resistance developed with its therapeutic use. These prodrugs revealed higher lipophilicity compared with INH. Their degradation kinetics were studied in vitro using aqueous buffer solutions of pH values 1.2 and 7.4 was well as biological media of human plasma and rat liver homogenate at 37 degrees C. They were more stable toward enzymatic degradation in biological than in chemical media. Release of INH from these derivatives was detected as a result of both chemical and enzymatic hydrolysis by HPLC. The antimycobacterial activity of the synthesized compounds and INH was tested in vitro against human type of Mycobacterium tuberculosis. They exhibited a greater antitubercular activity than the parent drug. This result is considered as an indicator for an improved permeation of the synthesized prodrugs through mycobacterial cell membranes relative to INH.

New carriers for representative peptides and peptide drugs.

3,5-Disubstituted tetrahydro-2H-1,3,5-thiadiazine-2-thione (THTT) derivatives; 4a-g were prepared and found to be a promising prodrug approach for peptide drugs. The pH profile for their degradation in aqueous buffer solutions was determined using HPLC technique and accounted for, in terms of specific base-catalyzed reactions. All of the compounds however, showed high acid-stability. Enzymatic (human serum) hydrolysis of the different derivatives offered an advantageous range of t1/2's, the property that permits controlling onset and duration of actions of drugs.

Different inhibition and induction profiles of hepatic drug metabolism in rats and dogs by two structurally related pyridyl diazinone cardiotonic agents.

ICI 153,110 and ICI 170,777, two pyridyl diazinone cardiotonic agents, produced a different profile of effects on hepatic microsomal mixed function oxidase enzymes following multiple oral dosing to rats and dogs; these differences may be related to the molecular dimensions of the two molecules. ICI 153,110 significantly increased levels of total P450, ethoxycoumarin O-deethylase and ethoxyresorufin O-deethylase in rat microsomes, indicating an induction profile (P448) similar to that of beta-naphthoflavone. This was supported by gel electrophoresis (SDS-PAGE) of microsomal proteins; a similar type of induction was observed in dog microsomes. In contrast, ICI 170,777 produced no changes indicating enzyme induction in either rat or dog. Instead, ICI 170,777 appeared to inhibit specifically the activity of aldrin epoxidase in the rat. Inhibitory activity was also indicated in the rat by prolongation of pentobarbitone sleeping time following single oral doses of either ICI 153,110 or ICI 170,777. The time-course of this effect appeared to correlate more closely with the profile of circulating metabolites, although both parent compounds were found to produce type II spectral changes on interaction with control rat microsomes. The molecular dimensions (area/depth2) of the compounds supported the finding that only ICI 153,110 should interact with or induce P448 isozymes.