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The effect of aging on the repeated-dose liver micronucleus assay using N-nitrosodipropylamine, quinoline, and carbendazim

The repeated dose liver micronucleus (RDLMN) assay has been sufficiently validated in terms of the numbers and types of chemicals studied. However, it remains unclear whether aging affects assay results. The OECD Test Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents) indicates that dosing should begin as soon as feasible after weaning and in any event before 9 weeks of age. Therefore, it is particularly important to determine whether there are age-related differences between 6 and 8 weeks of age at the start of dosing when considering the possibility of integrating this assay into a 4-week repeated dose general toxicity study. We evaluated the impact of the rats’ age on the RDLMN assay with three chemicals: N-nitrosodipropylamine, quinoline, and carbendazim. There were no significant age-related differences for the first two chemicals, whereas a markedly higher frequency of micronucleated hepatocytes (MNHEPs) was observed in younger rats for carbendazim. However, regardless of the age of animals, micronucleus induction was detected in all three chemicals. Combined with the previous reports on clofibrate and diethylnitrosamine, we concluded that animals of any age from 6 to 8 weeks could be used in the RDLMN assay.

Safety and efficacy of the epithelial sodium channel blocker idrevloride in people with primary ciliary dyskinesia (CLEAN-PCD): a multinational, phase 2, randomised, double-blind, placebo-controlled crossover trial

Mucociliary clearance is dysfunctional in people with primary ciliary dyskinesia, resulting in the accumulation of dehydrated mucus in the airways that is difficult to clear. We undertook a study to assess the benefit on lung function of treatment with a nebulised epithelial sodium channel (ENaC) blocker, idrevloride, with or without hypertonic saline, in people with primary ciliary dyskinesia. The CLEAN-PCD trial was a phase 2, randomised, double-blind, placebo-controlled crossover trial conducted at 32 tertiary adult and paediatric care centres and university hospitals in Canada, Denmark, Germany, Italy, the Netherlands, Poland, the UK, and the USA. People with a confirmed diagnosis of primary ciliary dyskinesia, aged 12 years or older, with a percentage of predicted FEV1 (ppFEV1) in the range of 40% to <90%, were randomly assigned in a 2:2:1:1 ratio (block size=6), stratified by ppFEV1 at screening, to one of four sequences: (1) idrevloride in hypertonic saline in treatment period 1 then hypertonic saline in treatment period 2; (2) hypertonic saline in treatment period 1 then idrevloride in hypertonic saline in treatment period 2; (3) idrevloride in treatment period 1 then placebo in treatment period 2; and (4) placebo in treatment period 1 then idrevloride in treatment period 2. The idrevloride dose was 85 μg and hypertonic saline was 4·2% NaCl. 3 mL of each study treatment was nebulised twice daily for 28 days in treatment periods 1 and 2; the two 28-day treatment periods were separated by a 28-day washout period. The primary endpoint was absolute change from baseline in ppFEV1 after 28 days. Safety assessments and reports of adverse events were made at clinic visits during each treatment period and by a follow-up telephone call 28 days after the last dose of study drug. Additionally, adverse events could be reported at a follow-up telephone call 3 days after the start of dosing and as they arose. Participants who received at least one dose of study drug were included in the safety analyses (safety set), and those who also had spirometry data were included in the efficacy analyses (full analysis set). The completed study is registered (EudraCT 2015-004917-26; ClinicalTrials.govNCT02871778). Between Sep 14, 2016, and May 31, 2018, 216 patients were screened and 123 were randomly assigned to one of four crossover sequences. Across the two treatment periods, treatment with idrevloride in hypertonic saline was initiated in 80 patients and completed in 78 patients (all 78 had data available and were included in the analysis); hypertonic saline initiated in 81 patients and completed in 76 patients (75 had data available and were included in the analysis); idrevloride initiated in 37 patients and completed in 35 patients (34 had data available and were included in the analysis); and placebo initiated in 36 patients and completed in 34 patients (all 34 had data available and were included in the analysis). Greater absolute increases in ppFEV1 from baseline to 28 days of treatment were seen with idrevloride in hypertonic saline (least-squares mean absolute change from baseline 1·0 percentage points, 95% CI -0·4 to 2·4) than with hypertonic saline alone (least-squares mean absolute change from baseline of -0·5 percentage points, -2·0 to 0·9; difference 1·5 percentage points, 95% CI <0·1 to 3·0; p=0·044). There was no significant difference in ppFEV1 for the parallel comparison of idrevloride in hypertonic saline compared with placebo or the crossover comparison of idrevloride with placebo. Adverse events were similar across treatments (57 to 65% of patients). Cough occurred in a greater proportion of participants during treatments that contained idrevloride or hypertonic saline compared with placebo, and oropharyngeal pain occurred in a greater proportion of participants during idrevloride treatments than during treatment with hypertonic saline alone or placebo, whereas chest discomfort was more common during treatments that included hypertonic saline. In this phase 2 crossover study, idrevloride in hypertonic saline was safe and associated with improved lung function over a 28-day period in people with primary ciliary dyskinesia compared with hypertonic saline alone. Larger, longer clinical studies are warranted to explore the potential benefits of idrevloride in combination with hypertonic saline in people with primary ciliary dyskinesia. Parion Sciences, under agreement with Vertex Pharmaceuticals.

Comparative Investigation of Raw and Processed Radix Polygoni Multiflori on the Treatment of Vascular Dementia by Liquid Chromatograph-Mass Spectrometry Based Metabolomic Approach.

Radix Polygoni Multiflori (PM) is a well-known nootropic used in traditional Chinese medicine (TCM). Considering the efficacy and application discrepancy between raw (RPM) and processed PM (PPM), the similarities and differences between them in the treatment of vascular dementia (VaD) is intriguing. In this study, a VaD rat model was constructed by 2-vessel occlusion (2-VO). During 28 days of treatment, plasma was collected on days 7, 14, 21, and 28 after the start of dosing and the metabolic profile was analyzed by HPLC-MS/MS-based metabolomics. The Morris Water Maze Test, hematoxylin-eosin and Nissl staining, and biochemical analysis were used to assess cognitive function, pathogenic alterations and oxidative stress, respectively. RPM and PPM effectivelyreducedthe 2VO-induced cognitive impairment and mitigated histological alterations in hippocampus tissue. The 2-VO model significantly elevated MDA level and decreased SOD activity and GSH level, indicating severe oxidative stress, which could also be attenuated by RPM and PPM treatment. RPM outperformed PPM in decreasing MDA levels while PPM outperformed RPM in increasing GSH levels. Differential metabolites were subjected to Metabolite Set Enrichment Analysis (MSEA) and genes corresponding to proteins having interactions with metabolites were further annotated with Gene Ontology (GO). Both RPM and PPM ameliorated VaD-relevant vitamin B6 metabolism, pentose phosphate pathways, and taurine and hypotaurine metabolism. In addition, the metabolism of cysteine and methionine was regulated only by RPM, and riboflavin metabolism was modulated only by PPM. The results suggested that raw and processed PM had comparable efficacy in the treatment of VaD but also with some mechanistic differenece.

Safety and efficacy of remimazolam besylate in patients undergoing colonoscopy: A multicentre, single-blind, randomized, controlled, phase Ⅲ trial.

Study objective: The objective of the study was to evaluate the safety and efficacy of remimazolam besylate versus propofol injection in patients undergoing colonoscopy. Design: A multicenter, randomized, non-inferiority, single-blind, parallel-controlled clinical trial. Setting: Operating room. Patients: Patients aged 18–65 years (American Society of Anesthesiologists [ASA] classification I-III) undergoing a diagnostic or therapeutic colonoscopy. Interventions: Patients were administered intravenous injection of remimazolam besylate or propofol (active comparator) for sedation. Measurements: Modified Observer’s Assessment of Alertness/Sedation [MOAA/S] scores of the included patients were assessed before dosing, 1, 1.5, 2, 2.5, and 3 min after the start of dosing, and then every 1 min until the MOAA/S score reached 5 on three consecutive occasions. Main Results: A total of 360 patients received remimazolam and 120 patients received propofol. The incidence of adverse events (67.8% vs. 84.2%, p = 0.001) was significantly lower in patients administered remimazolam compared to propofol. There was no significant difference in sedation success rates (full analysis set [FAS]: 98.9% vs. 99.2%; remimazolam vs. propofol). Remimazolam had a significantly longer onset of action, but the difference was not considered clinically significant (1.45 min vs. 1.24 min, remimazolam vs. propofol). Propofol achieved a deeper level of sedation (mean MOAA/S score 0.5 vs. 0.2; remimazolam vs. propofol). Mean time to discharge after the end of the last administration of study drug (20.3 vs. 21.8 min, p = 0.020) and incidence of injection pain was significantly lower in patients administered remimazolam (2.3% vs. 35.3%, p < 0.0001). Incidence of oxygen desaturation was significantly higher in patients administered propofol compared to patients administered remimazolam (6.7% vs. 1.1%, p = 0.001). Similarly, incidence of hypotension was more frequent in patients administered propofol compared to patients administered remimazolam (29.2% vs. 10.6%, p < 0.0001). Conclusion: Remimazolam besylate had a better safety and tolerability profile and similar sedative efficacy to propofol in patients undergoing a diagnostic or therapeutic colonoscopy in China, suggesting that remimazolam besylate has potential as a sedative agent for colonoscopy.

The effect of aging on the repeated-dose liver micronucleus assay using diethylnitrosamine

BackgroundThe repeated-dose liver micronucleus (RDLMN) assay has been well-developed and applied because of its simplicity and the ease of integration into general toxicity studies which is the preferred method from the 3R’s point of view. In this assay, we observed micronucleated hepatocytes which accumulated during a rather long-term dosing period. When considering integration into general toxicity studies, the effects of age of the animals used in the micronucleus assay becomes a major issue. The effect of age on the micronucleus induction rate has been reported in bone marrow micronucleus assays, and it is considered that the decrease in cell proliferation rate due to aging is the cause of the decrease in sensitivity. A decrease in sensitivity due to aging was also reported in a liver micronucleus assay using clofibrate and the cause is considered to be a decrease in hepatocyte proliferation activity due to aging. However, no actual decrease in hepatocyte proliferation rate due to aging has been reported. In addition, there are no reports, so far, on whether similar effects of aging appear when other substances were administered. To investigate the effects of aging in the RDLMN assay, this study focused on the effects of 14-day repeated administration of DEN, a well-known genotoxic hepatocarcinogen with the hepatocyte toxicity which should cause an elevation of cell proliferation rate as a reflective regeneration.ResultsThe liver micronuclei induced by DEN were equivalent between the two age groups (i.e., six and eight weeks of age at the start of dosing). In the histopathological examination for the liver, single cell necrosis, karyomegaly, and increased mitosis were observed in the hepatocytes, and the frequency and severity were increased dose-dependently. Ki-67 immunohistochemical analysis which can detect all cells in the cell cycle other than those in the G0 phase revealed dose-dependent increase of cell proliferation activity, and the difference between ages was not observed.ConclusionThe effect of aging on the RDLMN assay could not be recognized when DEN was administered for 14 days in rats. Meanwhile, it was supported by the histopathological examination and Ki-67 immunohistochemical analysis that such an effect of aging was masked by the compensatory hepatocyte proliferation which was induced by the hepatocyte toxicity of DEN.

Pharmacokinetics of bisphenol A in humans following dermal administration

BackgroundHuman exposures to bisphenol A (BPA) are widespread. The current study addresses uncertainties regarding human pharmacokinetics of BPA following dermal exposure. ObjectiveTo examine the absorption, distribution, metabolism and excretion of BPA in humans following dermal administration. MethodsWe dermally administered deuterated BPA (d6-BPA) to 10 subjects (6 men and 4 women) at a dose of 100 µg/kg over a 12-hour period and conducted blood and urine analysis from the beginning of dosing through a three- or six-day period. We present time-course serum and urine concentrations of total and unconjugated (“free”) d6-BPA and used this information to calculate terminal half-life and area under the curve. Results and ConclusionsDetectable serum levels of total d6-BPA were observed at 1.4 h after the start of dosing, and a maximum serum concentration (Cmax) of 3.26 nM was observed. Free d6-BPA was detectable in serum 2.8 h after start of dermal administration, with Cmax of 0.272 nM. Beginning at approximately seven hours and continuing to 12 h (which corresponds to cessation of exposure), the concentration of free and total serum d6-BPA plateaued. The terminal half-lives of total d6-BPA and free d6-BPA in the body were 21.4 ± 9.81 h and 17.6 ± 7.69 h, respectively. Elimination from the body was rate-limited by kinetics in the dermal compartment. Free d6-BPA was a greater percentage of the area under the curve of total serum BPA (8.81%) compared to the 0.56% observed in our previously published oral study. Recovery of total d6-BPA in urine was <2% of the applied dose after six days. Analysis of the area under the curve for dermal and oral administration revealed that 2.2% of the dermal dose became systemically available. These data are in line with prior studies indicating how pharmacokinetics of BPA differ following oral and dermal exposures. Dermal exposure resulted in a longer apparent half-life and higher free:total d6-BPA ratio compared to oral.

A phase Ib/III randomized, double-blind, placebo-controlled study of tazemetostat plus doxorubicin as frontline therapy for patients with advanced epithelioid sarcoma.

TPS11573 Background: Epithelioid sarcoma (ES) is characterized by loss of inhibitor of integrase 1 (INI1), allowing enhancer of zeste homologue 2 (EZH2) to repress cell differentiation and promote tumorigenesis. Tazemetostat (TAZ) is an EZH2 inhibitor approved by the FDA as monotherapy for the treatment of patients (pts) aged ≥16 years with metastatic or locally advanced ES ineligible for complete resection. The most common ( &gt; 20%) adverse events include pain, fatigue, nausea, decreased appetite, vomiting, and constipation. TAZ demonstrated anti-cancer activity in ES pts who previously received doxorubicin (dox), a commonly used front line FDA approved therapy for soft tissue sarcomas (STS), including ES. Preclinical studies have demonstrated synergy between TAZ and dox. This phase 1b/3 study (NCT04204941) is assessing the safety and efficacy of TAZ + dox in pts with advanced ES. Methods: This study is enrolling treatment-naïve adult pts with histologically confirmed STS (phase 1b) or INI1-deficient ES pts (phase 3) with ECOG performance status of 0-2 and life expectancy of ≥3 months. The open-label phase 1b portion is currently enrolling up to 24 patients with STS. The primary objectives are to evaluate the safety and tolerability of TAZ + dox, and to identify the recommended phase 3 dose (RP3D). Using a standard 3 + 3 design, TAZ will be evaluated at 3 dose levels (400 mg, 600 mg, and 800 mg orally BID) with standard fixed dose of dox (75 mg/m2 on day 1 of 21-day cycles for up to 6 cycles). After completion of dox, pts will continue to receive TAZ monotherapy until disease progression or intolerable toxicity. Up to 12 additional pts will be enrolled at the maximum tolerated dose for collection of additional safety and pharmacokinetic (PK) data. The double-blind phase 3 study will randomize (1:1) up to 140 pts to receive either TAZ (RP3D) + dox (75 mg/m2) or placebo + dox. The adaptive study design will allow sample size re-estimation (at interim analysis) to up to 200 pts. Pts may continue TAZ or placebo monotherapy after completing 6 cycles of TAZ + dox. Tumor assessments will be performed at screening and every 6 weeks from the start of dosing. The primary objective of phase 3 will be to evaluate the progression-free survival (PFS) by independent review committee. Secondary objectives will include assessment of PFS by investigators, overall survival, safety, disease control rate, overall response rate, duration of response, time to subsequent anticancer therapy, PFS on next treatment, quality of life, and PK. Clinical trial information: NCT04204941 .

471. Safety and Efficacy of Oral and/or Intravenous Tedizolid Phosphate (TZD) in Adolescents with Acute Bacterial Skin and Skin Structure Tissue Infections (ABSSSI)

BackgroundTedizolid phosphate has activity against gram-positive pathogens, including methicillin-resistant Staphylococcus aureus, and was approved for the treatment of ABSSSI in adults in 2014. This study compared the safety and efficacy of TZD with protocol-specified, active comparators for the treatment of ABSSSI in adolescents.MethodsThis was a randomized, assessor-blind, global, multicenter, phase 3 study of TZD vs. active comparator for the treatment of gram-positive ABSSSI in adolescents (aged 12 to < 18 years; NCT02276482). Enrolled patients were stratified by region and randomized 3:1 to TZD 200 mg (IV and/or oral) once daily for 6 days or investigator-selected active comparator per local standard of care (IV vancomycin, linezolid, clindamycin, flucloxacillin, or cefazolin, and/or oral linezolid, clindamycin, flucloxacillin, or cephalexin) for 10 days. The primary endpoint was safety. The percentages of patients with treatment-emergent adverse events (TEAEs) were documented; secondary efficacy endpoints included the blinded investigator’s assessment of clinical success at a test of cure visit (18–25 days after start of dosing) and early clinical response (≥20% reduction from baseline lesion area) at 48–72 h. No hypothesis testing was planned for the treatment groups.ResultsOf the 121 patients enrolled, 120 were treated (TZD, N = 91; comparator, N = 29). Median (range) age was 15 (12–17) years. Most patients were male (62.5%), white (86.7%), and enrolled in Europe (78.3%). Infections included major cutaneous abscess (42.5%), cellulitis/erysipelas (40.0%), and infected wound (17.5%). At baseline, the median (range) lesion surface area was 82.1 (14–978) cm2. Of those with gram-positive cultures (n = 64), S. aureus was most frequently isolated (n = 55 [85.9%]) with 3 isolates (4.7%) being methicillin resistant. TZD was well tolerated, and TEAEs were balanced between treatment arms (TZD, 14.3%; comparator, 10.3%). A total of 3 (3.3%) patients in the TZD group and 1 (3.4%) in the comparator group experienced a single-drug-related TEAE. Clinical success rates were high and similar between treatment groups (table).ConclusionTZD demonstrated comparable safety and efficacy to comparator in the treatment of ABSSSI in adolescents. Disclosures All authors: No reported disclosures.

Toxicological safety evaluation of the human-identical milk oligosaccharide 6'-sialyllactose sodium salt.

Human milk oligosaccharides (HMOs) are abundant in breastmilk, but their presence in infant formula is negligible. Sialylated HMOs, such as 6'-sialyllactose, constitute a significant portion of the HMO fraction of human milk and are linked to important biological functions. To produce infant formula that is more comparable with human milk, biosynthesized sialyllactoses known as human-identical milk oligosaccharides (structurally identical counterparts to their respective naturally occurring HMOs in breastmilk) are proposed for use in infant formula and other functional foods for the general population. To support the safety of 6'-sialyllactose sodium salt (6'-SL), a 90-day oral (gavage) toxicity study and in vitro genotoxicity tests were conducted. The 90-day study is the first to be conducted with 6'-SL using neonatal rats (day 7 of age at the start of dosing), thus addressing safety of 6'-SL for consumption by the most sensitive age group (infants). In the 90-day study, neonatal rats received 6'-SL at doses up to 5000mg/kg body weight (BW)/day and reference controls received 5000mg/kg BW/day of fructooligosaccharide (an ingredient approved for use in infant formula) for comparison with the high-dose 6'-SL group, followed by a 4-week recovery period. There was no evidence of genotoxicity in vitro. No test item-related adverse effects were observed on any parameter in the 90-day study, thus the high dose (5000mg/kg BW/day) was established as the no-observed-adverse-effect level. These results confirm that 6'-SL is safe for use in formula milk for infants and in other functional foods for the general population.

Abstract 627: Antitumor effect of an oligosaccharide API in a genetically engineered mouse-derived allograft (GEDA)

Abstract The use of oligosaccharides as active pharmaceutical ingredients lags behind that of other biological molecules such as proteins and nucleic acids. However, there is now a growing understanding of the complex biological functions of saccharide structures. This has resulted in an increased interest in exploring the pharmaceutical applications of both oligosaccharides and glycoconjugates. Despite recent advances in the treatment of pancreatic adenocarcinoma (PDAC), the median survival remains &amp;lt;12 months. Patients typically present with late stage disease and are often unable to tolerate drug combination regimens due to the associated toxicity. Here we report a novel oligosaccharide drug candidate, RiXOVA (G-blocks). The term “G-blocks” refers to highly defined and specialized short oligomers comprising α-L-guluronate residues derived from alginates extracted from brown seaweed. The G-block oligomers are well tolerated at i.v./i.p. doses &amp;gt;100mg/kg in mice. These oligomers have previously demonstrated the ability to alter matrices of biological macromolecules including mucins, giving rise to the hypothesis that they may have similar effects on the dense desmoplastic tissue within tumours. We here describe the anti-tumour properties of RiXOVA in different PDAC mouse models. Initial efficacy studies were performed in a xenograft model using the CAPAN-2 cells. RiXOVA was found to be non-toxic and non-immunogenic (I.P, 25mg/kg Q3D10 ) in mice. Relative to the start of dosing, the tumour growth in RiXOVA treated mice was 160% +/-30 (n=10) compared to 240% +/- 61 (n=10, p value=0.001) in the vehicle treated mice. Further testing was carried out in a Genetically Engineered Mouse-Derived Allograft (GEDA). The GEDA model recapitulates the dense desmoplastic stroma of the original donor (KPC mouse), unlike cell line-based allografts. KPC (LSL-KrasG12D; LSL-Trp53R172H; Pdx1-cre) mouse tumour fragments (2 donors) were implanted subcutaneously in the flank of recipient LSL-Trp53R172H; Pdx1-cre (PC), immunocompetent mice. Relative to the start of dosing, the tumour growth in the RiXOVA treated group (25 mg/kg TIW) was 260% +/- 160 (n=10), compared to 540% +/- 210 (n=8) for the Vehicle (p = 0.001) and 130 % +/- 57 (n=9) for the combination of RiXOVA + gemcitabine (100 mg/kg BIW p &amp;lt;0.001) compared to the vehicle. Ex vivo analysis of tumour tissue revealed changes in the expression and distribution of ECM proteins. Collectively, these results indicate RiXOVA to be a non-toxic oligosaccharide, that acts at the level of the tumour microenviroment to inhibit tumour growth. Whilst these results are in a preliminary stage they indicate G-block oligomers may represent a novel treatment modality in PDAC. Citation Format: Shalini V. Rao, Tonje S. Steigedal, Aarthi Gopinathan, Synnøve N. Magnussen, Sabina S. Strand, Duncan Jodrell, Fran Richards, Kurt I. Draget, Catherine T. Nordgård. Antitumor effect of an oligosaccharide API in a genetically engineered mouse-derived allograft (GEDA) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 627.

APL-2, a Complement C3 Inhibitor for the Potential Treatment of Paroxysmal Nocturnal Hemoglobinuria (PNH): Phase I Data from Two Completed Studies in Healthy Volunteers

BackgroundParoxysmal nocturnal hemoglobinuria (PNH) is a rare, acquired, potentially life-threatening disease characterized by complement-mediated hemolytic anemia. PNH arises from a somatic mutation resulting in impairment of an anchor protein responsible for the expression of numerous proteins at the cell surface of the red blood cells, including CD55 and CD59, two complement inhibitory proteins. The subsequent uncontrolled activation of the complement system leads to both intravascular hemolysis triggered by the membrane attack complex (MAC) and extravascular hemolysis, or cell opsonization, mediated by C3b accumulation at the cell surface. Eculizumab, an anti-C5 antibody, is available as a treatment for PNH, however some patients continue to be transfusion-dependent as a result of on-going activation of C3, upstream of C5. Due to the key position of C3 in the complement cascade, APL-2, a cyclic peptide inhibitor of C3, may prevent both intravascular and extravascular hemolysis and could therefore be potential treatment for PNH and beneficial for patients still requiring transfusions despite standard of care treatment.AimsThese initial phase I studies were designed to assess the safety, tolerability, pharmacokinetics (PK) and pharmacodynamics (PD) of single and multiple doses of APL-2 administered by subcutaneous injection (SC) in healthy adult volunteers.MethodsBoth studies were conducted at a single center (Australia) and were double-blind, placebo-controlled designs. Subjects were monitored for up to 43 days following single dosing and 84 days following multiple dosing. Safety was assessed by intensive clinical monitoring. Serial blood sampling was performed for the determination of APL-2 concentrations in serum. Blood samples were also obtained to determine relevant markers of complement activity (C3, CH50 and AP50). Additional relevant PD markers (intact C3, iC3b, C3a, C4a and C5a) were measured in latter Cohorts. Prior to multiple dosing subjects received Neisseria meningitides, Streptococcus pneumoniae, and Haemophilus influenzae Type B (Hib) vaccinations.ResultsIn total 51 subjects were enrolled into the two studies. Twenty-four subjects received APL-2 as a single dose (ranging from 45 to 1,440mg) and 7 subjects received placebo. Sixteen subjects received repeated daily doses of APL-2 for 28 consecutive days (ranging from 30 to 270 mg/day) and 4 subjects received placebo. There were no serious adverse events (SAE's), treatment emergent adverse events (TEAEs) leading to study drug discontinuation, or severe adverse events reported. There were no clinically relevant changes in vital signs, ECG findings or safety laboratory tests. Following single and multiple doses APL-2 exposure increased linearly with increasing dose up to the highest doses tested and APL-2 serum concentrations were close to steady-state after 28 days of daily dosing. A reduction in AP50 was seen following a single dose of 1440mg APL-2 and following multiple daily doses of 180 and 270 mg.ConclusionThe studies concluded that pharmacological doses of APL-2 were safe and well tolerated and that APL-2's PK/PD profile supports daily SC administration. In addition, daily APL-2 doses of 180mg and 270mg significantly reduced hemolytic activity as early as eight days after the start of dosing, and this inhibition was maintained through the dosing period. PK data from these studies has been used to develop a PK/PD model which has been used to aid dose selection in the subsequent studies in PNH patients. DisclosuresGrossi:Apellis Pharmaceuticals Inc: Employment, Equity Ownership. Bedwell:Apellis Pharmaceuticals Inc: Consultancy. Deschatelets:Apellis Pharmaceuticals Inc: Employment, Equity Ownership. Edis:Apellis Pharmaceuticals Inc: Employment, Equity Ownership. Francois:Revon Systems Inc: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Liberate Medical Inc: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Apellis Pharmaceuticals Inc: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Johnson:Apellis Pharmaceuticals Inc: Consultancy. Richardson:Apellis Pharmaceuticals Inc: Consultancy. Tan:Apellis Pharmaceuticals Inc: Consultancy. Vega:Apellis Pharmaceuticals Inc: Employment, Equity Ownership.

Recurrence of dual-strain Clostridium difficile infection in an in vitro human gut model.

Clostridium difficile infection (CDI) is still a major challenge to healthcare facilities. The detection of multiple C. difficile strains has been reported in some patient samples during initial and recurrent CDI episodes. However, the behaviour of individual strains and their contribution to symptomatic disease is unclear. An in vitro human gut model was used to investigate the germination and proliferation of two distinct C. difficile strains during initial and recurrent simulated CDI, as well as their response to vancomycin treatment. The gut model was inoculated with a pooled human faecal emulsion and indigenous gut microbiota, C. difficile populations (vegetative and spore forms), cytotoxin levels and antimicrobial activity were monitored throughout the experiment. Both C. difficile strains germinated and proliferated in response to ceftriaxone instillation, with cytotoxin detected during the peak vegetative growth. Vancomycin instillation resulted in a rapid decline in the vegetative forms of both strains, with only spores remaining 2 days after the start of dosing. A recrudescence of both strains occurred following the cessation of vancomycin installation, although this was observed more quickly, and to a greater extent, in one strain than the other. Within a human gut model, multiple C. difficile strains are able to germinate and proliferate concurrently in response to antibiotic challenge (the onset of simulated CDI). Similarly, more than one strain can proliferate during simulated recurrent CDI, although with differences in germination and growth rate and timing. It appears probable that multiple strains can contribute to CDI within an individual patient, with possible implications for management and bacterial transmission.

A urinary metabolomics study of rats after the exposure to acrylamide by ultra performance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry.

Acrylamide (ACR) is known to induce neurotoxicity in humans and occupational exposure to ACR has an effect on human health. Since some animal experiments indicate the metabolic change caused by the ACR based on the metabolomics, increasing concern is the change of metabolite profiles by the low-dose ACR. In the present study, a low-dose of ACR (18 mg kg(-1)) was administered to male Wistar rats for 40 days. Ultra performance liquid chromatography/time of flight mass spectrometry (UPLC-Q-TOF MS) was used to examine urine samples from ACR-dosed and control animals. Multiple statistical analyses with principal component analysis (PCA) were used to investigate metabolite profile changes in urine samples, and to screen for potential neurotoxicity biomarkers. PCA showed differences between the ACR-dosed and control groups 20 days after the start of dosing; a bigger separation between the two groups was seen after dosing for 40 days. Levels of 4-guanidinobutanoic acid and 2-oxoarginine were significantly higher in urine from the ACR-dosed group than in urine from the control group after 10 days (p < 0.05). Receiver operator characteristic (ROC) curve analysis suggested that 4-guanidinobutanoic acid and 2-oxoarginine were the major metabolites. Our results suggest that high levels of 4-guanidinobutanoic acid and 2-oxoarginine may be related to ACR neurotoxicity. These metabolites could, therefore, act as sensitive biomarkers for ACR exposure and be useful for investigating toxic mechanisms. They may also provide a scientific foundation for assessing the effects of chronic low-dose ACR exposure on human health.

Pharmacokinetics of Afatinib, a Selective Irreversible ErbB Family Blocker, in Patients with Advanced Solid Tumours

Afatinib is a potent, irreversible, ErbB family blocker in clinical development for the treatment of a variety of solid tumours. This study evaluated the pharmacokinetics of afatinib (10-100 mg once daily) in cancer patients. Data from 221 patients with advanced solid tumours in four phase I and one phase II trial were analysed using non-compartmental methods. Within each dose group, the shape of the geometric mean plasma concentration-time profiles after single and multiple doses were comparable. Maximum plasma concentration (C(max)) values were achieved 2-5 h after dosing and thereafter declined at least bi-exponentially. Steady-state plasma concentrations were attained within 8 days after the start of dosing. The geometric mean terminal elimination half-life at steady state was about 37 h. Repeated dosing resulted in a 2.77-fold accumulation based on the area under the plasma concentration-time curve (AUC), and 2.11-fold accumulation based on C(max) values. A slightly more than dose-proportional increase in afatinib exposure was observed. There was moderate intra-individual variability in afatinib trough concentration values (the geometric coefficient of variation (gCV) ranged from 22.2 to 67.5 %). The inter-patient variability in plasma concentrations was moderate to high (e.g. at the 40 mg dose, the gCVs ranged from 35.6 to 221 %). The exposure to afatinib (as measured by AUC and C(max)) correlated with the severity of the most common adverse events of afatinib--diarrhoea and rash. The pharmacokinetic profile of afatinib supports a once-daily dosage regimen. As expected for this patient population, the pharmacokinetic parameters of afatinib showed moderate to high inter-patient variability. Afatinib exhibits non-linear pharmacokinetics.

Pharmacokinetic Properties of Single-Dose Lamivudine/Adefovir Dipivoxil Fixed-Dose Combination in Healthy Chinese Male Volunteers

BackgroundBoth lamivudine and adefovir dipivoxil are approved for the treatment of chronic hepatitis B (CHB) and have established safety profiles. A fixed-dose combination (FDC) formulation of lamivudine/adefovir dipivoxil for the treatment of CHB may provide dosing convenience and improve adherence. ObjectiveThis study compared the pharmacokinetic profiles of an FDC capsule containing lamivudine/adefovir dipivoxil 100/10 mg and conventional lamivudine 100-mg + adefovir dipivoxil 10-mg tablets to determine bioequivalence. MethodsThis randomized, open-label, single-dose, 2-period crossover study was conducted in healthy male Chinese subjects. The study included a screening visit, 2 treatment sessions, and a follow-up visit. Subjects who met the inclusion/exclusion criteria were assigned to receive, in randomized order, 1 FDC capsule or 1 tablet each of lamivudine and adefovir dipivoxil. After a 7- to 10-day washout period, alternate treatment was given to the subjects during the second treatment session. Blood samples were collected immediately before and after dosing for 48 hours for plasma drug concentration measurement. Data on adverse events (AEs) were collected from the start of dosing until the follow-up visit. Tolerability assessments included physical examinations with vital sign measurements and clinical laboratory evaluations throughout the study. ResultsForty subjects were enrolled into the study (mean age, 22.4 years [range, 19–28 years]; weight, 63.8 kg [range, 54–78 kg]). The pharmacokinetic profiles of lamivudine and adefovir were similar between the FDC and reference formulations. The geometric mean ratios (GMRs) for lamivudine Cmax and AUC0–last were 1.02 (90% CI, 0.92–1.12) and 0.99 (90% CI, 0.95–1.04), respectively; adefovir, 0.94 (90% CI, 0.89–0.99) and 0.95 (90% CI, 0.91–1.00). A limited number of mild AEs were reported, with no clinically significant changes in vital signs or laboratory results. ConclusionsThe FDC capsule was bioequivalent to the concurrent administration of lamivudine + adefovir dipivoxil tablets based on the 90% CIs of the GMRs for Cmax, AUC0–∞, AUC0–last, and t12 (all were between 0.80 and 1.25). Both treatments were well-tolerated. Clinicaltrial.gov identifier: NCT01353742.

Fel d 1–derived peptide antigen desensitization shows a persistent treatment effect 1 year after the start of dosing: A randomized, placebo-controlled study

Allergic rhinoconjunctivitis is an increasingly common source of morbidity, with sensitivity to cats accounting for 10% to 15% of disease burden. Allergy to cats is also a major risk factor for the development of asthma. We sought to probe the persistence of the treatment effect of a novel F el d 1-derived peptide antigen desensitization (Cat-PAD) 1 year after the start of treatment in subjects with cat allergy-induced rhinoconjunctivitis after standardized allergen challenge. In a randomized, double-blind, placebo-controlled, parallel-group clinical trial, subjects attended an environmental exposure chamber in which they were exposed to cat allergen before and after treatment with 2 different regimens of Cat-PAD over a 3-month period. Clinical efficacy was assessed as a change in total rhinoconjunctivitis symptom scores 18 to 22 weeks and 50 to 54 weeks after the start of treatment. Treatment with Cat-PAD showed greater efficacy with 4 administrations of a 6-nmol dose 4 weeks apart than with 8 administrations of a 3-nmol dose 2 weeks apart. The treatment effect of 6 nmol persisted 1 year after the start of treatment and was significantly different from that of 3 nmol (P = .0342) and placebo (P = .0104). The treatment effect was apparent on both nasal and ocular symptoms at 1 year. A short course of Cat-PAD improves the ocular and nasal components of rhinoconjunctivitis symptoms in subjects with cat allergy, with the treatment effect persisting 1 year after the start of treatment.

Transient Serotonin Syndrome by Concurrent Use of Electroconvulsive Therapy and Selective Serotonin Reuptake Inhibitor: A Case Report and Review of the Literature

The serotonin syndrome, which is characterized by psychiatric, autonomic nervous and neurological symptoms, is considered to be caused by excessive stimulation of the 5-HT1A and 5-HT2 receptors in the gray matter and spinal cord of the central nervous system, after the start of dosing or increase of the dose of a serotoninergic drug. There have been hardly any reports of induction of serotonin syndrome by electroconvulsive therapy (ECT) in combination with antidepressant. We present the case of a female patient with major depressive disorder (MDD) who developed transient serotonin syndrome soon after the first session of ECT in combination with paroxetine. Paroxetine was discontinued, and her psychiatric, autonomic nervous and neurological symptoms were gradually relieved and disappeared within 2 days. We performed the second ECT session 5 days after the initial session and performed 12 sessions of ECT without any changes in the procedure of ECT and anesthesia, but no symptoms of SS were observed. Finally, her MDD remitted. ECT might cause transiently increased blood-brain barrier (BBB) permeability and enhance the transmissivity of the antidepressant in BBB. Therefore, it is necessary to pay attention to rare side effect of serotonin syndrome by ECT in combination with antidepressant.

Perioperative Antibiotic Regimen in Rats Treated With Pamidronate Plus Dexamethasone and Subjected to Dental Extraction: A Study of the Changes in the Jaws

Osteonecrosis of the jaws is a well-known condition associated with long-term bisphosphonate use. This study analyzed the prophylactic effect of antibiotic treatment in Sprague-Dawley rats treated with pamidronate plus dexamethasone and subjected to oral surgery in the form of dental extraction. One hundred twenty animals were included in a randomized prospective study. Animals in group I (n = 60) were treated with dexamethasone 1 mg/kg and pamidronate 3 mg/kg 3 times per week for 7, 14, and 21 days. All were subjected to right mandibular or maxillary molar extraction 8, 15, and 22 days after the start of dosing. Animals in group II (n = 60) received the same treatment except for the addition of penicillin 0.1 mL/kg per day for 3 days before and 4 days after extraction. Rats in the 2 groups were sacrificed 14 and 28 days after extraction. A clinical and histologic evaluation was performed. In group I, osteonecrosis was documented in 18 cases (34.6%; affecting the upper and lower jaws in 10 and 8 cases, respectively). In group II, osteonecrosis was documented in 5 cases (9.61%; affecting the upper and lower jaws in 3 and 2 cases, respectively). The difference between the 2 groups was statistically significant (P = .002). The adoption of preventive measures (antibiotic prophylaxis) in invasive dental procedures results in a significant decrease in osteonecrosis of the jaws associated with bisphosphonate use.

An experimental study of bisphosphonate-induced jaws osteonecrosis in Sprague-Dawley rats

Patients undergoing treatment with bisphosphonates may develop jaw lesions consisting mainly of bone necrosis. The present study describes a model of maxillary osteonecrosis in Sprague-Dawley rats, applying bisphosphonates and examines the changes occurring after tooth extraction. A total 200 animals were included in a randomized prospective study involving the following groups: group I (control, 20 rats without drug treatment), group II (60 animals administered dexamethasone 1 mg/kg/day for 7, 14 and 21 days, in subgroups of 20 animals each), group III (60 animals administered pamidronate daily at a dose of 3 mg/kg) and group IV (60 animals administered pamidronate and dexamethasone). In all groups, molar extraction was carried out on the right upper maxillary or mandibular side 8, 15 and 22 days after the start of dosing. The rats were killed 14 and 28 days after extraction in all groups. A total of 18 cases of osteonecrosis were recorded in the group administered pamidronate and dexamethasone. Osteonecrosis affected the upper maxilla in 10 cases and the mandible in eight cases, and was circumscribed to the extraction zone in all cases. Osteonecrosis was not seen in any of the other groups. The administration of pamidronate and dexamethasone in rats subjected to molar extraction increases the risk of osteonecrosis.

Abstract A42: Antiangiogenic-induced increase in tumor hypoxia in RCC and NSCLC human tumor xenografts and its selective targeting by the hypoxia-activated prodrug TH-302: A model for clinical exploration?

Abstract Several published studies have demonstrated an increase in tumor hypoxia after administration of anti-angiogenesis agents that target VEGF signaling (e.g., sunitinib, sorafenib, bevacizumab). We tested the hypothesis that the hypoxia-activated prodrug (HAP) TH- 302, currently in phase 1/2 trials for the treatment of cancer, would exhibit enhanced efficacy in the context of an antiangiogenic- mediated increase in tumor hypoxia and potentiate the antitumor efficacy of the antiangiogenic. To characterize sunitinib-induced effects on tumor vasculature and tumor hypoxia, 786-O (RCC) and H460 (NSCLC) human ectopic tumor xenografts were treated with sunitinib (20 or 40 mg/kg) daily for 5 days and then 72 hours later animals were injected with pimonidazole to label hypoxic cells and Hoechst 33342 to label vascular perfusion. The NSCLC model (H460) exhibits a baseline hypoxic fraction of 7%. Sunitinib induced a dose-dependent increase in tumor hypoxia volume (24 ± 3.2% with 40 mg/kg vs. 7.3 ± 3.8% with Vehicle, p&amp;lt;0.05) and a corresponding decrease in tumor microvasculature. The 786-O RCC model is a well- vascularized tumor as characterized by CD31 and Hoechst staining with a relatively small baseline hypoxic compartment (&amp;lt;5% volume) in the xenograft tumors. Sunitinib also induced a dose-dependent increase in tumor hypoxia volume (17.2 ± 7.1 % with 40 mg/kg vs. 1.4 ± 0.9% with Vehicle, p&amp;lt;0.05) and a corresponding decrease in functional vasculature in the RCC model. We then tested whether TH-302, a 2-nitroimidazole triggered bromo-isophoramide mustard (Br-IPM) DNA crosslinker, could selectively target the antiangiogenic induced increase in tumor hypoxic fraction. H460 NSCLC tumor- bearing animals (~150 mm3 at start of dosing) were randomized into groups of 10 and treated with sunitinib at 20,40, or 80 mg/kg p.o. daily for 3 weeks (QDx21). After one week of sunitinib monotherapy animals began combination therapy with the addition of TH-302 at 50 mg/kg i.p. daily for 5 days on and 2 days off (QDx5) for 2 weeks. Sunitinib was administered 4 hours before TH-302 on days when both agents were given. Control arms included all 3 doses of sunitinib monotherapy, TH-302 monotherapy, and vehicles-only groups. Sunitinib exhibited dose-dependent antitumor efficacy (by TGI, TGD, and conditional survival), as did TH-302 monotherapy. All three combination therapy treatment groups exhibited superior efficacy compared to the corresponding monotherapy groups. A similarly designed experiment was performed in the RCC (786-O) model. in vitro monolayer cell-based profiling of the two cell lines (H460 and 786-O) with sunitinib and TH-302 in combination, under hypoxic or normoxic conditions, did not show any synergism. Taken together, the combination therapy efficacy observed in the xenograft models are consistent with complementary pharmacological effects at the tumor microenvironmental level, rather than cell autonomous effects. Morphometric analysis of TH-302 effects on hypoxic compartment volume in six different xenograft models (H460 intrapleural and H82 SCLC, H460 and Calu-6 NSCLC, and SU.86.86 and Hs766t CaPanc ectopic models) showed selective targeting of the hypoxic compartment resulting in a decrease in relative hypoxic volumes. Analogous experiments testing TH-302 effects on sunitinib-induced increases in tumor hypoxia volume are underway. The results support the hypothesis that the hypoxia-activated prodrug TH-302 may be an effective addition to antiangiogenic-containing chemotherapy regimens. Citation Information: Clin Cancer Res 2010;16(14 Suppl):A42.