Haizheng Hong

Metabolism and Disposition of [14C]BMS-690514, an ErbB/Vascular Endothelial Growth Factor Receptor Inhibitor, after Oral Administration to Humans

(3R,4R)-4-Amino-1-((4-((3-methoxyphenyl)amino)pyrrolo[2,1-f][1,2,4]triazin-5-yl)methyl)-3-piperidinol (BMS-690514), an oral selective inhibitor of human epidermal growth factor receptors 1 (or epidermal growth factor receptor), 2, and 4, and vascular endothelial growth factor receptors 1, 2, and 3, is being developed as a treatment for patients with non–small-cell lung cancer and metastatic breast cancer. The disposition of [14C]BMS-690514 was investigated in nine healthy male subjects (group 1, n = 6; group 2, n = 3) after oral administration of a 200-mg dose. Urine, feces, and plasma were collected from all subjects for up to 12 days postdose. In group 2 subjects, bile was collected from 3 to 8 h postdose. Across groups, approximately 50 and 34% of administered radioactivity was recovered in the feces and urine, respectively. An additional 16% was recovered in the bile of group 2 subjects. Less than 28% of the dose was recovered as parent drug in the combined excreta, suggesting that BMS-690514 was highly metabolized. BMS-690514 was rapidly absorbed (median time of maximum observed concentration 0.5 h) with the absorbed fraction estimated to be approximately 50 to 68%. BMS-690514 represented ≤7.9% of the area under the concentration-time curve from time 0 extrapolated to infinite time of plasma radioactivity, indicating that the majority of the circulating radioactivity was from metabolites. BMS-690514 was metabolized via multiple oxidation reactions and direct glucuronidation. Circulating metabolites included a hydroxylated rearrangement product (M1), a direct ether glucuronide (M6), and multiple secondary glucuronide conjugates. None of these metabolites is expected to contribute to the pharmacology of BMS-690514. In summary, BMS-690514 was well absorbed and extensively metabolized via multiple metabolic pathways in humans, with excretion of drug-related radioactivity in both bile and urine.

Mechanisms of hexabromocyclododecanes induced developmental toxicity in marine medaka (Oryzias melastigma) embryos

Hexabromocyclododecanes (HBCDs) are widely used as additive brominated flame retardants, and are now ubiquitous contaminants in the environmental media and biota, including the marine environment and marine organisms. However, the impacts of HBCDs on marine fish are not well known. In this study the embryos of marine medaka (Oryzias melastigma) were used to assess the developmental toxicity of HBCDs. Freshly fertilized marine medaka embryos were exposed to various concentrations of technical HBCD (tHBCD, 0, 5, 20 and 50μg/L) until the first fry stage, and hatch success, morphology and cardiac function were examined. In all the exposure groups (5, 20 and 50μg/L) tHBCD significantly increased the embryo heart beats. The measurement of sinus venosus-bulbus arteriosus (SV-BA) distance indicated that tHBCD significantly enlarged the SV-BA distance at exposure concentrations of 20 and 50μg/L. The malformation rate at the first fry stage was also induced by tHBCD in a dose dependent manner, with the formation of pericardial edema and yolk sac edema as the most frequently observed malformation. In addition, the concentrations of total HBCD isomers (ΣHBCDs) in embryos in the current study were comparative with environmental levels and increased with increasing exposure duration. Furthermore, exposure to tHBCD also induced the level of 8-oxodG, a representative oxidative DNA damage. The mechanisms of HBCD-induced developmental toxicity were further explored by TUNEL assay, gel-based quantitative proteomic approach and measurement of the expression of several stress responsive genes, such as p53, TNF-α, IL-1β, CYP1A, COX-1 and COX-2, together with the activities of caspases. The results suggested that HBCDs exposure at environmentally realistic concentrations induced oxidative stress and apoptosis, and suppressed nucleotide and protein synthesis, which all together resulted in developmental toxicity, particularly in the cardiovascular system, in the embryos of O. melastigma.

The complex effects of ocean acidification on the prominent N2-fixing cyanobacterium Trichodesmium

A future, more acidic ocean could be less productive, despite the fertilizing effects of elevated CO2. Reconciling pH and future productivity The differential effects of reduced seawater pH and increased carbon dioxide on marine phytoplankton productivity have not been resolved. Hong et al. found that previous experimentation did not account for variable metal concentrations or for ammonia contamination. After controlling for these variables, experimentation, protein expression analysis, and field data showed that low pH, coupled with the low ambient iron availability in the open ocean, inhibits nitrogen fixation, whereas elevated CO2 is fertilizing. Overall, the deleterious effects of decreased pH trump the beneficial effects of increased CO2. Thus, it seems that in a future, more acidic ocean, phytoplankton productivity is likely to be suppressed. Science, this issue p. 527 Acidification of seawater caused by anthropogenic carbon dioxide (CO2) is anticipated to influence the growth of dinitrogen (N2)–fixing phytoplankton, which contribute a large fraction of primary production in the tropical and subtropical ocean. We found that growth and N2-fixation of the ubiquitous cyanobacterium Trichodesmium decreased under acidified conditions, notwithstanding a beneficial effect of high CO2. Acidification resulted in low cytosolic pH and reduced N2-fixation rates despite elevated nitrogenase concentrations. Low cytosolic pH required increased proton pumping across the thylakoid membrane and elevated adenosine triphosphate production. These requirements were not satisfied under field or experimental iron-limiting conditions, which greatly amplified the negative effect of acidification.

Developmental toxicity of three hexabromocyclododecane diastereoisomers in embryos of the marine medaka Oryzias melastigma.

The composition of major hexabromocyclododecane (HBCD) diastereoisomers, i.e. α-, β-, and γ-HBCDs, in marine biota is different from that of the commercially available form (technical HBCD), which is used extensively for toxicological studies. To properly evaluate the impact of HBCDs, the embryos of Oryzias melastigma were used to examine the developmental toxicity of the individual diastereoisomers. Results showed that HBCD diastereoisomers at the environmentally realistic concentrations in the embryos induced malformation rate and heartbeat, and caused the appearance of apoptotic heart. In addition, α-, β-, and γ-HBCDs had similar potency to stimulate the generation of reactive oxygen species, consequently leading to apoptosis in O. melastigma embryos. The order of the developmental toxicity of α-, β-, and γ-HBCDs in O. melastigma embryos was different from that in zebrafish embryos studied previously, which highlighted the importance of using species from both fresh and salt water for toxicity assessment.

In vitro characterization of the metabolic pathways and cytochrome P450 inhibition and induction potential of BMS-690514, an ErbB/vascular endothelial growth factor receptor inhibitor.

(3R,4R)-4-Amino-1-((4-((3-methoxyphenyl)amino)pyrrolo[2,1-f][1,2,4]triazin-5-yl)methyl)-3-piperidinol (BMS-690514) is a potent inhibitor of ErbB human epidermal growth factor receptors (HER1, 2, and 4) and vascular endothelial growth factor receptors 1 to 3 that has been under clinical development for solid tumor malignancies. BMS-690514 is primarily cleared by metabolism with the primary metabolic pathways being direct glucuronidation (M6), hydroxylation (M1, M2, and M37), and O-demethylation (M3). In the current investigation, the metabolic drug-drug interaction potential of BMS-690514 was evaluated in a series of in vitro studies. Reaction phenotyping experiments with cDNA-expressed human cytochrome P450 (P450) and UDP-glucuronosyltransferase (UGT) enzymes and human liver microsomes (HLM) in the presence of P450 or UGT inhibitors suggested that CYP3A4, CYP2D6, and CYP2C9 were the major enzymes responsible for the oxidative metabolism of BMS-690514, whereas both UGT2B4 and UGT2B7 were responsible for the formation of M6. BMS-690514 did not cause direct or time-dependent inhibition of P450 enzymes (IC50 values ≥40 μM) in incubations with HLM and probe substrates of CYP1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, or 3A4. The compound also did not substantially induce CYP1A1, CYP1A2, CYP2B6, CYP3A4, or UGT1A1 at concentrations up to 10 μM in cultured human hepatocytes. Considering the submicromolar plasma Cmax concentration at the anticipated clinical dose of 200 mg, BMS-690514 is unlikely to cause clinically relevant drug-drug interactions when coadministered with other medications. In addition, because multiple enzymatic clearance pathways are available for the compound, inhibition of an individual metabolic pathway either via coadministered drugs or gene polymorphisms is not expected to cause pronounced (>2-fold) increases in BMS-690514 exposure.

Metabolism and Disposition of [14C]BMS-690514 after Oral Administration to Rats, Rabbits, and Dogs

(3R,4R)-4-Amino-1-((4-((3-methoxyphenyl)amino)pyrrolo[2,1-f] [1,2,4]triazin-5-yl)methyl)-3-piperidinol (BMS-690514) is a potent inhibitor of human epidermal growth factor receptors 1, 2, and 4 and vascular endothelial growth factor receptors 1 through 3. BMS-690514 is an oral oncologic agent currently being developed for the treatment of patients with advanced non–small cell lung cancer and breast cancer. In this investigation, a series of studies was conducted to determine the biotransformation of [14C]BMS-690514 after oral administration to rats, rabbits, and dogs. After administration of a single oral dose of [14C]BMS-690514 to rats and dogs, the majority of the radioactive dose (61–71%) was recovered in the feces, whereas 18 to 20% was eliminated in urine. In bile duct-cannulated rats, 83 and 17% of the administered radioactivity was recovered in the bile and urine, respectively, suggesting that biliary secretion was a major route for the elimination of BMS-690514-derived radioactivity in rats. The parent compound underwent extensive metabolism in both species, with <12% of the administered radioactivity recovered as BMS-690514 in the excreta samples. Metabolite profiles in plasma were qualitatively similar in rats, rabbits, and dogs. Unchanged BMS-690514 was a prominent drug-related component in the plasma profiles from all the species. However, multiple metabolites contributed significantly to the circulating radioactivity, particularly for rabbit and dog, in which metabolites comprised 73 to 93% of the area under the time curve (0–8 h). Circulating metabolites included M6, a direct O-glucuronide conjugate; M1, a hydroxylated metabolite; and glucuronide conjugates of hydroxylated and O-demethylated metabolites. Overall, the results from these studies suggested that BMS-690514 was well absorbed and highly metabolized through multiple pathways in these preclinical species.

Toxicity and bioaccumulation of three hexabromocyclododecane diastereoisomers in the marine copepod Tigriopus japonicas

The three major hexabromocyclododecane (HBCD) diastereoisomers, i.e. α-, β- and γ-HBCD, have distinct physical and chemical properties that may potentially result in different levels of bioaccumulation and toxicity in aquatic organisms. To assess the impact of diastereomeric variation in HBCDs, the marine copepod Tigriopus japonicus was exposed to α-, β- and γ-HBCD in isolation. Results showed that all the three diastereoisomers had a similar potency to cause growth delay in T. japonicas. Variation was observed in the overall survival rate with exposure to α- and β-HBCD, and this resulted in significantly higher lethal toxicity in T. japonicas than that with exposure to γ-HBCD. Exposure to α-, β- and γ-HBCD led to the generation of ROS in T. japonicas, a possibly toxic mechanism. Both α- and β-HBCD showed a higher potential to induce oxidative stress, which may be a factor in the higher lethal toxicity observed with α- and β-HBCD exposure. It is of note that T. japonicus was found to be more sensitive to all three diastereoisomers in the F1 generation than in the F0 generation. The bioconcentration potential of HBCD diastereoisomers can be ranked in the order α-HBCD>γ-HBCD>β-HBCD and was found to be higher in T. japonicus than has been reported for fish species.

Mechanistic studies on a P450-mediated rearrangement of BMS-690514: conversion of a pyrrolotriazine to a hydroxypyridotriazine.

BMS-690514 ((3R,4R)-4-amino-1-((4-((3-methoxyphenyl)amino)pyrrolo[2,1-f][1,2,4] triazin-5-yl)methyl)-3-piperidinol) is an oral oncologic agent being developed for the treatment of patients with advanced nonsmall cell lung cancer and breast cancer. The compound is metabolized via multiple metabolic pathways, including P450-mediated oxidation at one of the carbons of its pyrrolotriazine group. Oxidation at this site results in the formation of two metabolites, M1 and M37. Mass spectrometric and NMR analysis revealed that M1 underwent an unusual structural change, where the pyrrolotriazine moiety rearranged to yield a hydroxypyridotriazine group. In contrast, the structure of the pyrrolotriazine moiety remained intact in M37. In vitro experiments with liver microsomes and deuterated or tritiated BMS-690514 containing the isotopic label on the carbon that underwent oxidation indicated that during the formation of M1, the isotope label was retained at the site of hydroxylation, while the label was lost during the formation of M37. On the basis of these results, a mechanism for the formation of M1 was proposed as follows: BMS-690514 was first oxidized by P450 enzymes either via epoxidation or an iron-oxo addition pathway to form a zwitterionic intermediate. This was followed by opening of the pyrrolotriazine ring to form an aldehyde intermediate, which could be partially trapped with methoxyamine. The aldehyde intermediate then reacted with the secondary amine of the methoxyaniline group in the molecule to form the pyridotriazine moiety of M1. This mechanism is consistent with the observed retention of the isotope label in M1. Metabolite M37 may be formed either via a common zwitterionic intermediate, shared with M1, or through a direct insertion pathway. In in vitro human liver microsome incubations, the abundance of M1 was higher than M37, suggesting that breaking of the carbon-nitrogen bond to generate the aldehyde intermediate, a process similar to N-dealkylation, was a preferred pathway.

Multigenerational effects of 4-methylbenzylidene camphor (4-MBC) on the survival, development and reproduction of the marine copepod Tigriopus japonicus

One of the most widely used organic UV filters, 4-methylbenzylidene camphor (4-MBC), is present at high concentrations in offshore waters. The marine copepod Tigriopus japonicus was exposed to different concentrations of 4-MBC (i.e., 0, 0.5, 1, 5 and 10μgL-1) for 4 consecutive generations (F0-F3) to evaluate the impact of 4-MBC on marine ecosystems. The results showed that in the F0 generation, 4-MBC caused significant lethal toxicity in T. japonicas at concentrations of 5 and 10μgL-1 and the nauplii were more sensitive to 4-MBC toxicity than the adults. However in the F1-F3 generations, 4-MBC exposure did not affect the survival rate. The hatching rate and the developmental duration from the nauplii to the copepodite (N-C) and from the nauplii to adult (N-A) decreased significantly in the F1-F2 generations and in the F2-F3 generations, respectively, even at the lowest exposure concentration (0.5μgL-1). In the subsequent two generations (i.e., the F4-F5 generations) of recovery exposure in clean seawater, the growth rates of the original 4-MBC exposure groups were still faster than the control in both the N-C and N-A stages, suggesting possible transgenerational genetic and/or epigenetic changes upon chronic 4-MBC exposure. The expression of the ecdysone receptor gene was up-regulated by 4-MBC, which was consistent with the decrease of the N-C/N-A duration. In addition, 4-MBC may induce oxidative stress and trigger apoptosis in T. japonicas, resulting in developmental, reproductive and even lethal toxicity. A preliminary risk assessment suggested that under environmentally realistic concentrations, 4-MBC had significant potential to pose a threat to marine crustaceans and marine ecosystems.

Nitrogen fixation in two coastal upwelling regions of the Taiwan Strait

Recent studies have demonstrated that dinitrogen fixation can be important in nutrient-rich coastal upwelling regions. During a cruise to the Taiwan Strait in summer 2015, we found that the nitrogen fixation rate in surface waters ranged from below detection limits to 7.51 nmol N L−1 d−1. Higher rates accompanied by low N:P ratios (1–10.4:1) associated with low temperatures occurred in the surface water where the Pingtan and the Dongshan upwelling regions met (the NE area). In contrast, insignificant rates were observed in the southwest area of the Dongshan upwelling region (the SW area) with sufficient N and deficient P, and therefore high N:P ratios (e.g., >43 at station C2) due largely to the influence of the Pearl River plume. Diatom-associated symbionts (het-1; 104–106 copies L−1) that are efficient in organic matter export were found to dominate the other diazotrophic groups that were surveyed, which may represent a direct relationship between new nitrogen input and export in the upwelling regions. Our results suggest a hydrographical influence on the diazotroph community and N2 fixation in coastal upwelling regions.