Critical Assessment of Pharmacokinetic Drug-Drug Interaction Potential of Tofacitinib, Baricitinib and Upadacitinib, the Three Approved Janus Kinase Inhibitors for Rheumatoid Arthritis Treatment.

The introduction of novel, small-molecule Janus kinase inhibitors namely tofacitinib, baricitinib and upadacitinib has provided an alternative treatment option for patients with rheumatoid arthritis outside of traditional drugs and expensive biologics. This review aimed to critically assess the drug-drug interaction potential of tofacitinib, baricitinib and upadacitinib and provide a balanced perspective for choosing the most appropriate Janus kinase inhibitor based on the needs of patients with rheumatoid arthritis including co-medications and renal/hepatic impairment status. Based on the critical assessment, all three approved Janus kinase inhibitors generally provide a favourable opportunity for co-prescription with a plethora of drugs. While cytochrome P450 3A4-related inhibition or induction altered the exposures (area under the curve) of tofacitinib and upadacitinib, it did not impact the exposure of baricitinib. Transporter drug-drug interaction studies revealed that the disposition of baricitinib was altered with certain transporter inhibitors as compared with either tofacitinib or upadacitinib. Adjustment of tofacitinib or baricitinib dosages but not that of upadacitinib is required with the progression of renal impairment from a mild to a severe condition. While the dosage of tofacitinib needs to be adjusted for patients with moderate hepatic impairment status, it is not the case for either baricitinib or upadacitinib. Assessment of the drug-drug interaction potential suggests that tofacitinib, baricitinib and upadacitinib generally show a favourable disposition with no perpetrator activity; however, as victim drugs, they show subtle pharmacokinetic differences that may be considered during polypharmacy. Moreover, careful choice of the three drugs could be made in patients with rheumatoid arthritis with varying degrees of renal/hepatic impairments.

Discovery of a chiral fluorinated azetidin-2-one as a tubulin polymerisation inhibitor with potent antitumour efficacy.

Inhibition of tubulin polymerisation with small molecules has been clinically validated as a promising therapy for multiple solid tumours. Herein, a series of chiral azetidin-2-ones were asymmetrically synthesised and biologically evaluated for antitumour activities. Among them, a chiral fluorinated azetidin-2-one, 18, was found to exhibit the most potent activities against five cancer cell lines, including a drug-resistant cell line, with IC50 values ranging from 1.0 to 3.6 nM. Further mechanistic studies revealed that the compound 18 worked by disrupting tubulin polymerisation, blocking the cell cycle in the G2/M phase, inducing cellular apoptosis, and suppressing angiogenesis. Additionally, 18 exhibited higher human-microsomal metabolic stability and aqueous solubility compared to those of combretastatin A-4. Finally, 18 was also found to effectively inhibit tumour growth in a xenograft mice model with low toxicity and thus might be a promising lead for further clinical development.

GPR30-Expressing Gastric Chief Cells Do Not Dedifferentiate But Are Eliminated via PDK-Dependent Cell Competition During Development of Metaplasia.

BACKGROUND & AIMS: Gastric chief cells, a mature cell type that secretes digestive enzymes, have been proposed to be the origin of metaplasia and cancer through dedifferentiation or transdifferentiation. However, studies supporting this claim have had technical limitations, including issues with the specificity of chief cell markers and the toxicity of drugs used. We therefore sought to identify genes expressed specifically in chief cells and establish a model to trace these cells. METHODS: We performed transcriptome analysis of Mist1-CreERT-traced cells, with or without chief cell depletion. Gpr30-rtTA mice were generated and crossed to TetO-Cre mice, and lineage tracing was performed after crosses to R26-TdTomato mice. Additional lineage tracing experiments were performed using Mist1-CreERT, Kitl-CreERT, Tff1-Cre, and Tff2-Cre mice crossed to reporter mice. Mice were given high-dose tamoxifen or DMP-777 or were infected with Helicobacter pylori to induce gastric metaplasia. We studied mice that expressed mutant forms of Ras in gastric cells, using TetO-KrasG12D, LSL-KrasG12D, and LSL-HrasG12V mice. We analyzed stomach tissues from GPR30-knockout mice. Mice were given dichloroacetate to inhibit pyruvate dehydrogenase kinase (PDK)-dependent cell competition. RESULTS: We identified GPR30, the G-protein-coupled form of the estrogen receptor, as a cell-specific marker of chief cells in gastric epithelium of mice. Gpr30-rtTA mice crossed to TetO-Cre;R26-TdTomato mice had specific expression of GPR30 in chief cells, with no expression noted in isthmus stem cells or lineage tracing of glands. Expression of mutant Kras in GPR30+ chief cells did not lead to the development of metaplasia or dysplasia but, instead, led to a reduction in labeled numbers of chief cells and a compensatory expansion of neck lineage, which was derived from upper Kitl+ clones. Administration of high-dose tamoxifen, DMP-777, or H pylori decreased the number of labeled chief cells. Chief cells were eliminated from epithelia via GPR30- and PDK-dependent cell competition after metaplastic stimuli, whereas loss of GRP30 or inhibition of PDK activity preserved chief cell numbers and attenuated neck lineage cell expansion. CONCLUSIONS: In tracing studies of mice, we found that most chief cells are lost during metaplasia and therefore are unlikely to contribute to gastric carcinogenesis. Expansion of cells that coexpress neck and chief lineage markers, known as spasmolytic polypeptide-expressing metaplasia, does not occur via dedifferentiation from chief cells but, rather, through a compensatory response from neck progenitors to replace the eliminated chief cells.

HLA-DR7 and HLA-DQ2: Transgenic mouse strains tested as a model system for ximelagatran hepatotoxicity.

The oral thrombin inhibitor ximelagatran was withdrawn in the late clinical trial phase because it adversely affected the liver. In approximately 8% of treated patients, drug-induced liver injury (DILI) was expressed as transient alanine transaminase (ALT) elevations. No evidence of DILI had been revealed in the pre-clinical in vivo studies. A whole genome scan study performed on the clinical study material identified a strong genetic association between the major histocompatibility complex alleles for human leucocyte antigens (HLA) (HLA-DR7 and HLA-DQ2) and elevated ALT levels in treated patients. An immune-mediated pathogenesis was suggested. Here, we evaluated whether HLA transgenic mice models could be used to investigate whether the expression of relevant HLA molecules was enough to reproduce the DILI effects in humans. In silico modelling performed in this study revealed association of both ximelagatran (pro-drug) and melagatran (active drug) to the antigen-presenting groove of the homology modelled HLA-DR7 molecule suggesting "altered repertoire" as a key initiating event driving development of DILI in humans. Transgenic mouse strains (tgms) expressing HLA of serotype HLA-DR7 (HLA-DRB1*0701, -DRA*0102), and HLA-DQ2 (HLA-DQB1*0202,-DQA1*0201) were created. These two lines were crossed with a human (h)CD4 transgenic line, generating the two tgms DR7xhCD4 and DQ2xhCD4. To investigate whether the DILI effects observed in humans could be reproduced in tgms, the mice were treated for 28 days with ximelagatran. Results revealed no signs of DILI when biomarkers for liver toxicity were measured and histopathology was evaluated. In the ximelagatran case, presence of relevant HLA-expression in a pre-clinical model did not fulfil the prerequisite for reproducing DILI observed in patients. Nonetheless, for the first time an HLA-transgenic mouse model has been investigated for use in HLA-associated DILI induced by a low molecular weight compound. This study shows that mimicking of genetic susceptibility, expressed as DILI-associated HLA-types in mice, is not sufficient for reproducing the complex pathogenesis leading to DILI in man.

Quinoline-azetidinone hybrids: Synthesis and in vitro antiproliferation activity against Hep G2 and Hep 3B human cell lines.

In search of new heterocyclic anticancer agents, a new quinoline-azetidinone hybrid template have been designed, synthesized and screened for their cytotoxic activity against human cancer cell lines such as Hep G2, and Hep 3B by the MTT assay and results were compared with paclitaxel, 5-fluorouracil and doxorubicin. Interestingly, some of the compounds were found significantly active against both cell lines. The compound 6f (IC50=0.04±0.01µM) exhibited potent antiproliferation activity against Hep G2 cell line, and 6j compound (IC50=0.66±0.01µM) demonstrated potent antiproliferation activity against Hep 3B cell line and provide to be more potent as cytotoxic agents than standard drugs. Morphological changes suggest the induction of apoptosis and describe the mechanism of action of these hybrid antitumor agents.

Synthesis and cytotoxic activity of certain trisubstituted azetidin-2-one derivatives as a cis-restricted combretastatin A-4 analogues.

Novel series of 1,3,4-trisubstituted azetidin-2-one derivatives 8a-p were synthesized and proposed as cytotoxic agents acting via inhibition of tubulin at the colchicine binding site. The design of the target compounds was based upon modification in the structure of the vascular targeting agent combretastatin A-4 (CA-4). The cis double bond linker in CA-4 was replaced with the azetidin-2-one ring aiming to prevent the cis/trans isomerization that suppresses the activity of CA-4, thereby enhancing its antiproliferative activity. All new compounds were investigated in vitro against MCF-7 and HCT-116 cell lines. The inhibition of tubulin polymerization by four most potent compounds 8g, 8j, 8n and 8o was also evaluated. The synthesis of the final targets was achieved adopting Staudinger reaction. Molecular modeling studies were performed to rationalize the biological results.

Synthesis, Characterization & Screening for Anti-Inflammatory & Analgesic Activity of Quinoline Derivatives Bearing Azetidinones Scaffolds.

BACKGROUND: Non-steroidal anti-inflammatory drugs (NSAIDS) are clinically used as anti-inflammatory, analgesic and antipyretic agents but they have the drawbacks such as gastric irritation and gastric ulceration. Recently, quinoline derivatives have shown significant anti-inflammatory and less ulcerogenic activity. The present study deals with the synthesis and pharmacological assessment of a series of novel quinoline derivatives bearing azetidinones scaffolds as anti-inflammatory and analgesic agents. METHODS: A series of newer 3-chloro-1-(substituted)-4-(tetrazolo [1,5-a]quinolin-4- yl)azetidin-2-one derivatives (6a-l) was synthesized starting with acetanilide (1). Initially, acetanilide (1) was allowed to react with Vilsmeier-Haack reagent (DMF + POCl3) to form 2- chloro-3-formyl quinoline (2). The 2-chloro-3-formyl quinoline (2) was further treated with p-toluenesulphonic acid and sodium azide which yielded Tetrazolo [1,5-1] quinoline-4- carbaldehyde (3). The reaction of formyl group with various substituted amines (4a-l) formed corresponding Schiff base intermediates (5a-l), which were further allowed to react with chloroacetyl chloride to produce 3-chloro-1-(substituted)-4-(tetrazolo [1,5-a]quinolin-4-yl) azetidin-2-one derivatives (6a-l). The structure of the final analogues (6a-l) has been confirmed on the basis of elemental analysis, IR, 1H NMR, 13C NMR and mass spectra. All the synthesized compounds were evaluated for their anti-inflammatory and analgesic activities by using carrageenan induced rat paw model and Eddy's hot plate method respectively. RESULTS: All the values of elemental analysis, IR, 1H NMR, 13C NMR and mass spectra were found to be prominent. The anti-inflammatory activity test revealed that 3-chloro-1-(4-methoxyphenyl)- 4-(tetrazolo[1,5-a] quinolin-4-yl)azetidin-2-one (6b), 3-chloro-1-(2-methoxyphenyl)- 4-(tetrazolo[1,5-a]quinolin-4-yl)azetidin-2-one (6a) exhibited significant anti-inflammatory and analgesic activity as compared to control group. CONCLUSION: The results of the current study indicate that substitution at quinoline derivatives bearing azetidinones scaffolds showed potent analgesic and anti-inflammatory activities.

Discovery of 2-azetidinone and 1H-pyrrole-2,5-dione derivatives containing sulfonamide group at the side chain as potential cholesterol absorption inhibitors.

Cholesterol absorption inhibitor (CAI) targeting Niemann-Pick C1-like1 protein was developed for the treatment of hyperlipidaemia and only ezetimibe was approved so far. For developing novel CAIs, we synthesized sixteen 2-azetidinone derivatives and thirteen 1H-pyrrole-2,5-dione derivatives containing sulfonamide group at the side chain, and their inhibitory activity of cholesterol absorption was evaluated in Caco-2 cell line in vitro. Furthermore, top six compounds were measured by cytotoxicity and partition coefficient, and 2-azetidinone analogue 9e was selected for in vivo study. Finally, 9e considerably reduced total cholesterol, LDL-C, FFA and triglyceride in the serum and increased the rate of HDL-C to total cholesterol, suggesting it could regulate the lipid metabolism and act as a potent CAI.

Novel type of adenylyl cyclase participates in tabtoxinine-ß-lactam-induced cell death and occurrence of wildfire disease in Nicotiana benthamiana.

Tabtoxinine-ß-lactam (TßL), a non-specific bacterial toxin, is produced by Pseudomonas syringae pv. tabaci, the causal agent of tobacco wildfire disease. TßL causes the plant cell death by the inhibiting glutamine synthetase, which leads to an abnormal accumulation of ammonium ions. To better understand the molecular mechanisms involved in TßL-induced cell death and necrotic wildfire lesions, we focused on adenylyl cyclase in Nicotiana benthamiana. We isolated the gene designated as NbAC (Nicotiana benthamiana adenylyl cyclase). Recombinant NbAC protein showed adenylyl cyclase activity in vitro. TßL-induced necrotic lesions were significantly suppressed in NbAC-silenced leaves compared with control plant leaves. However, the amount of ammonium ions was scarcely affected by NbAC-silencing. Furthermore, the silencing of NbAC also suppressed l-methionine sulfoximine-induced cell death without any changes in the amount of ammonium accumulated. When inoculated directly with P. syringae pv tabaci, NbAC-silenced plants showed reduced symptoms. These results suggest that NbAC might play an essential role in intracellular signal transduction during TßL-induced cell death and necrotic wildfire disease development.

Synthesis of azetidines and pyrrolidines via iodocyclisation of homoallyl amines and exploration of activity in a zebrafish embryo assay.

Room temperature iodocyclisation of homoallylamines stereoselectively delivers functionalised 2-(iodomethyl)azetidine derivatives in high yield. Increasing reaction temperature from 20 °C to 50 °C switches the reaction outcome to realise the stereoselective formation of functionalised 3-iodopyrrolidine derivatives. It was shown that these pyrrolidines are formed via thermal isomerisation of the aforementioned azetidines. Primary and secondary amines could be reacted with iodomethyl azetidine derivatives to deliver stable methylamino azetidine derivatives. With subtle changes to the reaction sequences homoallyl amines could be stereoselectively converted to either cis- or trans-substituted 3-amino pyrrolidine derivatives at will. The stereochemical divergent synthesis of cis and trans substituted pyrrolidines supports an ion part, aziridinium, isomerisation pathway for azetidine to pyrrolidine isomerisation. Six azetidine derivatives were probed in a zebrafish embryo developmental assay to detect potential biological effects through the analysis of morphology and motility behaviour phenotypes. The range of effects across the probed molecules demonstrates the suitability of this assay for screening azetidine derivatives. One of the probed molecules, rac-(((cis)-1-benzyl-4-phenylazetidin-2-yl)methyl)piperidine, exhibited particularly interesting effects in the developmental assay presenting with hypopigmentation and reduced circulation amongst others. This shows that the zebrafish embryo provides a fast, sensitive and effective way to screen new compounds and in the future in combination with existing in vivo and in vitro assays it will become an integral part in drug discovery and development.

Metabolic activation and major protein target of a 1-benzyl-3-carboxyazetidine sphingosine-1-phosphate-1 receptor agonist.

1-{4-[(4-Phenyl-5-trifluoromethyl-2-thienyl)methoxy]benzyl}azetidine-3-carboxylic acid (MRL-A) is a potent sphingosine-1-phosphate-1 receptor agonist, with potential application as an immunosuppressant in organ transplantation or for the treatment of autoimmune diseases. When administered orally to rats, radiolabeled MRL-A was found to undergo metabolism to several reactive intermediates, and in this study, we have investigated its potential for protein modification in vivo and in vitro. MRL-A irreversibly modified liver and kidney proteins in vivo, in a dose- and time-dependent manner. The binding was found to occur selectively to microsomal and mitochondrial subcellular fractions. Following a nonspecific proteolytic digestion of liver and kidney proteins, a single major amino acid adduct was observed. This adduct was characterized with LC/MS/UV and NMR spectroscopy and was found to be the product of an unprecedented metabolic activation of the azetidine moiety leading to the formation of a ring-opened α,ß-unsaturated imine conjugated to the ε-amino group of a lysine residue. The formation of this adduct was not inhibited when rats were pretreated with 1-aminobenzotriazole, indicating that P450 enzymes were not involved in the metabolic activation of MRL-A. Rather, our findings suggested that MRL-A underwent bioactivation via a ß-oxidation pathway. Several other minor adducts were identified from protein hydrolysates and included lysine, serine, and cysteine conjugates of MRL-A. These minor adducts were also detected in microsomal incubations fortified with the cofactors for acyl-CoA synthesis and in hepatocytes. Trypsin digestion of crude liver homogenates from rats treated with radiolabeled MRL-A led to the identification of a single radioactive peptide. Its sequence, determined by LC/MS analysis, revealed that the target of the major reactive species of MRL-A in vivo is Lys676 of long chain acyl-CoA synthetase-1 (ACSL1). This lysine residue has been found to be critical for ACSL1 activity, and its modification has the potential to lead to biological consequences such as cardiac hypertrophy or thermogenesis dysregulation.

Two-year carcinogenicity studies with the oral direct thrombin inhibitor ximelagatran in the rat and the mouse.

The results of 18 months mouse and 24 months rat carcinogenicity studies with the oral direct thrombin inhibitor ximelagatran are presented. In the mouse, gavage doses of ximelagatran up to 180 µmol/kg per d produced no neoplastic changes in any of the tissues examined. In the rat, gavage doses up to 240 µmol/kg per d produced multiple macroscopically detectable nodules in the pancreas, which are seen to be focal/multifocal acinar cell hyperplasia and focal/multifocal acinar cell adenoma upon histological evaluation. There were no other treatment-related effects on tumor incidence or distribution in the rat. The studies show a clear species difference in pancreatic effects between the rat and the mouse to long-term treatment with ximelagatran.

Antiplasmodial and cytotoxicity evaluation of 3-functionalized 2-azetidinone derivatives.

3-Azido-, 3-amino- and 3-(1,2,3-triazol-1-yl)-ß-lactams were synthesized and evaluated for their antiplasmodial activity against four strains of Plasmodium falciparum and KB cells for their cytotoxicity profiles. The presence of a cyclohexyl substituent at N-1 and a phenyl group on the triazole ring markedly improved the activity profiles of triazole-tethered ß-lactam exhibiting IC(50) values of 1.13, 1.21 and 1.00 µM against 3D7, K1 and W2 strains respectively.

Evaluation of AR-H067637, the active metabolite of the new direct thrombin inhibitor AZD0837, in models of venous and arterial thrombosis and bleeding in anaesthetised rats.

AZD0837, currently in clinical development, is a once-daily oral anticoagulant that is bioconverted to AR-H067637, a selective, reversible direct thrombin inhibitor (DTI). When developing a new DTI, the antithrombotic effects are commonly investigated in in vivo animal models; this report shows the effect of AR-H067637 in venous and arterial thrombosis and bleeding models in anaesthetised rats. Thrombus formation was induced by topical application of ferric chloride to the carotid artery or to the caval vein with partial stasis. Cutaneous incision bleeding time and muscle transection blood loss were assessed, with or without acetylsalicylic acid (ASA). Activated partial thromboplastin time (APTT), ecarin coagulation time (ECT) and thrombin coagulation time (TCT) were used as plasma biomarkers of anticoagulant effect. Dalteparin was used as a reference compound. AR-H067637, given by continuous infusion, displayed a dose-dependent antithrombotic effect, with 50% inhibition (IC50) of thrombus size in venous and arterial thrombosis models obtained at plasma concentrations of 0.13 µM and 0.55 µM, respectively, without increased bleeding. Dose-dependent increased bleeding and blood loss were seen at plasma concentrations ≥1 µM AR-H067637. At the highest AR-H067637 plasma concentration tested, bleeding time and blood loss increased two and four times the vehicle group. Addition of ASA moderately potentiated bleeding time and blood loss. APTT, ECT and TCT were dose-dependently prolonged. These studies demonstrate that the DTI AR-H067637 inhibits thrombus formation in rat venous and arterial thrombosis models with no or minor increases in bleeding.

Differential effects of TAK-442, a novel orally active direct factor Xa inhibitor, and ximelagatran, a thrombin inhibitor, on factor V-mediated feedback on coagulation cascade and bleeding.

Thrombin amplifies the blood coagulation via factor V (FV)-mediated positive feedback loop. We hypothesised that factor Xa (FXa) inhibitors would interfere more gradually with this feedback activation loop than thrombin inhibitors, thereby achieving a better balance between haemostasis and prevention of thrombosis. In this study, we compared the effects of TAK-442, a novel FXa inhibitor, versus ximelagatran, a thrombin inhibitor, on FV-mediated positive feedback, venous thrombosis and bleeding. In normal plasma, TAK-442 delayed the onset of tissue factor-induced thrombin generation and prolonged prothrombin time (PT) with more gradual concentration-response curve than melagatran, the active form of ximelagatran. The effect of melagatran on the onset of thrombin generation decreased in an FVa-concentration-dependent manner in FV-deficient plasma supplemented with FVa. Furthermore, in FV-deficient plasma, the PT-prolonging potency of melagatran was markedly increased with a change in its concentration-response curve from steep to gradual. In the rat venous thrombosis model, TAK-442 (10 mg/kg, p.o.) prevented thrombus formation by 55% with 1.2 times prolongation of PT; a similar effect was observed in ximelagatran-treated (3 mg/kg, p.o.) rats. TAK-442 at 100 mg/kg prolonged PT by only 2.1 times with no change in bleeding time (BT), whereas ximelagatran at 10 mg/kg prolonged PT by 3.9 times and significantly increased BT. These results suggest that the differential effects of the two agents on FV-mediated amplification of thrombin generation may underlie the observation of a wider therapeutic window for TAK-442 than for ximelagatran.

Synthesis, in-vitro antimicrobial and cytotoxic studies of novel azetidinone derivatives.

Developing novel antimicrobial drugs is increasingly important in the modern pharmaceutical industry. A series of novel 3-chloro-4-[4-(2-oxo-2H-chromen-4-ylmethoxy)phenyl]-1-phenylazetidin-2-ones 5a-o have been synthesized from 4-bromomethylcoumarins 1a-e and 4-aryliminomethyl-phenols 3a-c. These compounds were screened for their in-vitro antibacterial activity against two Gram-positive (Staphylococcus aureus and Vancomycin resistant enteroccoccus) and two Gram-negative (Escherichia coli and Shigella dysentery) bacterial strains and antifungal activity against Aspergillus fumigatus, Candida albicans, and Penicillium. Results revealed that compounds 5c, 5f, 5h, 5j, and 5m showed excellent activity against a panel of microorganisms. The brine-shrimp bioassay was also carried out to study their in-vitro cytotoxic properties and two compounds, 5h and 5m, possessing LD(50) = 7.154x10(-4 )M and 5.782x10(-4) M, respectively, displayed potent cytotoxic activity against Artemia salina. The presence of a chlorine group in the coumarin moiety, its effect on their antibacterial, antifungal, and cytotoxic activities is discussed. All newly synthesized compounds were characterized by elemental analysis, IR, (1)H-NMR,( 13)C-NMR, and MS.

An assessment of the carcinogenic potential of ezetimibe using nonclinical data in a weight-of-evidence approach.

Ezetimibe blocks intestinal absorption of sterols via interaction with the Neimann-Pick C1-Like 1 (NPC1L1) transporter and is approved for use in the treatment of primary hyperlipidemia (heterozygous familial and non-familial), homozygous familial hypercholesterolemia, and homozygous sitosterolemia. A recently completed randomized clinical trial [simvastatin and ezetimibe in aortic stenosis (SEAS)] testing the effectiveness of Vytorin (a combination of simvastatin and ezetimibe) in patients with aortic stenosis reported an unexpected safety finding: an increase in overall cancer incidence and cancer-associated mortality (all types) in the treated groups relative to the placebo control. A subsequent meta-analysis utilizing a much larger database from two ongoing clinical trials indicated that the observed findings in the SEAS trial were likely due to chance and not a true drug-induced effect. Nonetheless, it has been suggested by various commentators on the SEAS trial that ezetimibe may be carcinogenic. The extensive nonclinical database for ezetimibe was used to test the hypothesis that ezetimibe may be a direct or indirect carcinogen. Using two different in silico approaches, ezetimibe showed no structural alerts for genetic toxicity or carcinogenicity. Ezetimibe was not genotoxic in two reverse mutation assays, one in vitro clastogenicity assay, and two mouse micronucleus assays. No evidence of proliferative lesions was observed in three species in studies of 1-12 months in duration. Ezetimibe was not carcinogenic in standard 2-year bioassays in mice and rats. Additionally, in these 2-year bioassays, no drug-related non-neoplastic lesions were noted. The absence of drug-induced non-neoplastic or proliferative lesions in these studies indicates that ezetimibe treatment was not associated with findings characteristic of carcinogens (i.e., DNA reactivity or cell proliferation) Administration of pharmacologic doses of ezetimibe to mice did not alter hepatic expression patterns of genes associated with apoptosis, cell proliferation, or epithelial-mesenchymal transition. No evidence of drug-induced tumors was observed in mice in which the molecular target of ezetimibe (NPC 1L1) was knocked out over the life span of the animal. In conclusion, the nonclinical data do not support the proposed hypothesis based on the single observation from the SEAS trial and, rather, support the conclusion that ezetimibe does not represent a carcinogenic hazard to humans using this drug in a therapeutic setting.

Pro-inflammatory response and adverse drug reactions: mechanisms of action of ximelagatran on chemokine and cytokine activation in a monocyte in vitro model.

There is a lack of suitable human in vitro systems which can predict drug hepatotoxicity that in many cases involves inflammatory responses mediated by macrophages. In the present investigation we used an in vitro model based on human THP-1 cells to evaluate the inflammatory cytokine/chemokine activation properties of ximelagatran, a drug previously shown to cause elevation of liver transferases in a subset of patients. Treatment of the cells with ximelagatran caused an intracellular accumulation of the metabolites hydroxymelagatran and melagatran. A decreased viability and increased release of the pro-inflammatory cytokines and chemokines IL-8, VEGF and MCP-1 was seen. Ximelagatran exposure caused activation of ERK1/2 and JNK as evident from determination of the phosphorylation status. In accordance, the release of IL-8 was attenuated by inhibitors of the ERK- and JNK-pathways. It is concluded that human monocytes might constitute a valuable additional in vitro model for monitoring the basis for cytotoxic action of drugs.

Prediction of drug-induced liver injury in humans by using in vitro methods: the case of ximelagatran.

OBJECTIVE: To investigate the possible mechanisms underlying the liver enzyme elevations seen during clinical studies of long-term treatment (>35 days) with ximelagatran, and investigate the usefulness of pre-clinical in vitro systems to predict drug-induced liver effects. METHODS: Ximelagatran and its metabolites were tested for effects on cell viability, mitochondrial function, formation of reactive metabolites and reactive oxygen species, protein binding, and induction of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) gene expression or nuclear orphan receptors. Experimental systems included fresh and cryopreserved hepatocytes, human hepatoma cell lines (HepG2 and HuH-7) and subcellular human liver fractions. RESULTS: Loss of cell viability was only seen in HepG2 cells at ximelagatran concentrations 100 microM and in cryopreserved human hepatocytes at 300 microM, while HuH-7 cells were not affected by 24 h exposure at up to 300 microM ximelagatran. Calcium homeostasis was not affected in HepG2 cells exposed to ximelagatran up to 300 microM for 15 min. There was no evidence for the formation of reactive metabolites when cell systems were exposed to ximelagatran. ALT and AST expression in human hepatoma cell lines were also unchanged by ximelagatran. Mitochondrial functions such as respiration, opening of the transition pore, mitochondrial membrane depolarization and beta-oxidation were not affected by ximelagatran or its metabolites. CONCLUSION: Ximelagatran at concentrations considerably higher than that found in plasma following therapeutic dosing had little or no effect on cellular functions studied in vitro. The in vitro studies therefore did not elucidate the mechanism by which ximelagatran induces liver effects in humans, possibly because of limitations in the experimental systems not reflecting characteristics of the human hepatocyte, restricted exposure time, or because the primary mechanism for the observed clinical liver effects is not on the parenchymal liver cell.

Synthesis of new thiazolylthiazolidinylbenzothiazoles and thiazolylazetidinylbenzothiazoles as potential insecticidal, antifungal, and antibacterial agents.

A series of 2-{[2'-(3''-chloro-2''-oxo-4''-substitutedaryl-1''-azetidinyl)-1',3'-thiazol-4'-yl] thio}benzothiazoles (4a-4e) and 2-{[(2'-(2''-substitutedaryl-4''-thiazolidinon-3''-yl)-1',3'-thiazol-4'-yl]thio}benzothiazoles (5a-5e) have been synthesized from 2-[(2'-substitutedarylidenylimino-1',3'-thiazol-4'-yl)thio]benzothiazoles (3a-3e). The structure of these compounds has been elucidated by elemental (C, H, N) and spectral (IR, (1)H-NMR, Mass) analysis. Furthermore, compounds 3a-3e, 4a-4e, and 5a-5e were screened for insecticidal activity against Periplaneta americana and antifungal, antibacterial activities in vitro against different strains of fungi and bacteria. Out of the fifteen compounds tested, compound 5b, 2-{[2'-(2''-p-hydroxy-m-methoxyphenyl)-4''-thiazolidinon-3''-yl)-1',3'-thiazol-4'-yl]thio}benzothiazole, was found to possess most prominent insecticidal activity.