Simultaneous determination of Z-SU5416 and its interconvertible geometric E-isomer in rat plasma by LC/MS/MS

SU5416 is a selective inhibitor of vascular endothelial growth factor (VEGF) receptor, which plays a major role in vascular angiogenesis. SU5416 exists as the thermodynamically stable and pharmacologically active cis isomer (Z-isomer) in the solid state. In light-exposed solutions the unstable trans isomer (E-isomer) is formed. The E-isomer is unstable for synthesis and isolation and the analytical standard of the E-isomer is unavailable. A new, simple, fast and reliable LC/MS/MS method was developed to quantify both isomers simultaneously in rat plasma samples in order to support the study of disposition kinetics of Z- and E-SU5416. This method is sensitive (LOQ = 0.5 ng/ml), reproducible, and has a wide linear range (0.5-2500 ng/ml).     

Simultaneous determination of Z-3-[(2,4-dimethylpyrrol-5-yl)methylidenyl]-2-indolinone (SU5416) and its interconvertible geometric isomer (SU5886) in rat plasma by LC/MS/MS

Z-3-[(2,4-Dimethylpyrrol-5-yl)methylidenyl]-2-indolinone (SU5416) is a cytostatic substance in development as an anti-angiogenic agent. SU5416 exists as the thermodynamically stable cis or Z-isomer as a solid. Studies have shown that in light exposed solutions of SU5416, the unstable trans or E-isomer, namely SU5886, is formed. The E-isomer converts back to the Z-isomer when protected from light. The E-isomer is unstable for synthesis and isolation; therefore, the analytical standard of the E-isomer is not available. In this study, a simple, fast and reliable LC/MS/MS method has been developed to determinate both isomers simultaneously in rat plasma samples to support the study of disposition kinetics of SU5416. This method is sensitive (limit of quantitation (LOQ=0.5 ng/mL)), reproducible and has a wide linear range (0.5-2500 ng/mL). There was no conversion between E- and Z-isomer during sample preparation procedure and sample determination with LC/MS/MS. Experimental results proved that SU5416 and SU5886 have identical detection response. Therefore, SU5416 (Z-isomer) was used successfully as analytical standard for SU5886 (E-isomer). This method has been applied to rat plasma samples obtained from a pharmacokinetic study. This study underscores the use of LC/MS/MS technique for bioanalytical methods where analytical standards are not available and analytes are interconvertible.     

A girl with fragile X premutation from sperm donation

We present a girl with the fragile X premutation who obtained the premutation allele from donated sperm. Our patient has clinical characteristics of fragile X syndrome including emotional problems and neuropsychological difficulties presenting as learning disabilities. She is also at high risk for premature ovarian failure and low risk for the fragile X-associated tremor ataxia (FXTAS). We suggest fragile X DNA screening in gamete donor candidates to decrease the chance of fragile X involvement in their offspring.     

Localized delivery of curcumin from injectable gelatin/Thai silk fibroin microspheres for anti-inflammatory treatment of osteoarthritis in a rat model

The previously developed gelatin/silk fibroin microspheres were loaded with curcumin and applied for anti-inflammatory treatment in monosodium iodoacetate (MIA)-induced osteoarthritis (OA) in a rat model. The MIA-induced OA rats received a single intra-articular injection with gelatin or gelatin/silk fibroin (30/70) microspheres encapsulating curcumin. The therapeutic effects of treatment groups [concentration of interleukin-6 (IL-6) in blood serum, radiographic and the histological grading on articular joint] were compared with those of normal saline treated OA and normal rats. The result showed that both microsphere groups reduced the level of IL-6 in serum after 1 week of treatment. The gelatin/silk fibroin (30/70) microspheres encapsulating curcumin delayed the cellular destruction in articular joint and synovial tissue after 8 weeks. The radiographic and histological gradings on articular cartilage lesion and synovial tissue change of rats treated with gelatin/silk fibroin (30/70) microspheres encapsulating curcumin were close to those of the normal rats. It was explained that the slow-degrading gelatin/silk fibroin (30/70) microspheres released curcumin for extended period and showed a prolonged anti-inflammatory effect, compared to the fast-degrading gelatin microspheres. This delivery system of curcumin was suggested to be applied for localized treatment of anti-inflammatory in OA with minimal invasion.     

Cochinin B, a novel ribosome-inactivating protein from the seeds of Momordica cochinchinensis

Cochinin B, a novel ribosome-inactivating protein (RIP) with a molecular weight of 28 kDa, was purified from the seeds of Momordica cochinchinensis (Cucurbitaceae). The isolation procedure entailed ammonium sulfate precipitation, cation-exchange chromatography on SP Sepharose column and size-exclusion chromatography on Superdex 75 column with a fast protein liquid chromatography (FPLC) system. The first twenty N-terminal amino acid residues of Cochinin B showed homology to type I RIPs from other Momordica species. The purified Cochinin B displayed a strong inhibitory activity on protein synthesis in the cell-free rabbit reticulocyte lysate system with IC50 of 0.36 nM. Furthermore, it exhibited N-glycosidase activity and cytotoxicity against Vero cell line with IC50 higher than 1540 nM. Interestingly, Cochinin B manifested strong anti-tumor activities on human cervical epithelial carcinoma (HeLa), human embryonic kidney (HEK293) and human small cell lung cancer (NCI-H187) cell lines with IC50 of 16.9, 114 and 574 nM, respectively.     

Full‐length sequence of genotype 3 hepatitis E virus derived from a pig in Thailand

Hepatitis E virus (HEV) infection in pigs was investigated in two principal swine farming areas in Thailand. Anti‐HEV antibodies and HEV RNA in sera were examined in 258 pigs reared on five commercial farms from age 1 to 6.5 months and sows. Overall, 167 of 258 (64.7%) pigs were positive for anti‐HEV IgG, while 20 of 258 (7.75%) had detectable HEV RNA. Sequence analysis of 20 HEV isolates obtained from viremic pigs revealed that they were 92.3–100% identical to each other and had 82.2–88.2% nucleotide similarity to other reported genotype 3 isolates in 415 nucleotide sequences within ORF2 region. Further characterization by sequencing the complete genome of the Thai swine HEV isolate (named Thai‐swHEV07) and phylogenetic analysis showed that Thai‐swHEV07 segregated into a cluster consisting of swine isolates from Japan, Mongolia, and Kyrgyzstan within the HEV genotype 3. The Thai‐swHEV07 had a genomic length of 7,229 nt excluding the polyadenylated region at 3′ terminus of the genome. Comparison of Thai‐swHEV07 and 27 reported strains of genotype 3 revealed 80.4–85.9% nucleotide identity, with the highest identity of 85.9% to the novel swHEV strain from Mongolia. These findings suggest that genotype 3 HEV isolates are markedly heterogeneous. J. Med. Virol. 81:657–664, 2009 © 2009 Wiley‐Liss, Inc.     

SU14813: a novel multiple receptor tyrosine kinase inhibitor with potent antiangiogenic and antitumor activity

Receptor tyrosine kinases (RTK), such as vascular endothelial growth factor receptor (VEGFR), platelet-derived growth factor receptor (PDGFR), stem cell factor receptor (KIT), and fms-like tyrosine kinase 3 (FLT3), are expressed in malignant tissues and act in concert, playing diverse and major roles in angiogenesis, tumor growth, and metastasis. With the exception of a few malignancies, seemingly driven by a single genetic mutation in a signaling protein, most tumors are the product of multiple mutations in multiple aberrant signaling pathways. Consequently, simultaneous targeted inhibition of multiple signaling pathways could be more effective than inhibiting a single pathway in cancer therapies. Such a multitargeted strategy has recently been validated in a number of preclinical and clinical studies using RTK inhibitors with broad target selectivity. SU14813, a small molecule identified from the same chemical library used to isolate sunitinib, has broad-spectrum RTK inhibitory activity through binding to and inhibition of VEGFR, PDGFR, KIT, and FLT3. In cellular assays, SU14813 inhibited ligand-dependent and ligand-independent proliferation, migration, and survival of endothelial cells and/or tumor cells expressing these targets. SU14813 inhibited VEGFR-2, PDGFR-beta, and FLT3 phosphorylation in xenograft tumors in a dose- and time-dependent fashion. The plasma concentration required for in vivo target inhibition was estimated to be 100 to 200 ng/mL. Used as monotherapy, SU14813 exhibited broad and potent antitumor activity resulting in regression, growth arrest, or substantially reduced growth of various established xenografts derived from human or rat tumor cell lines. Treatment in combination with docetaxel significantly enhanced both the inhibition of primary tumor growth and the survival of the tumor-bearing mice compared with administration of either agent alone. In summary, SU14813 inhibited target RTK activity in vivo in association with reduction in angiogenesis, target RTK-mediated proliferation, and survival of tumor cells, leading to broad and potent antitumor efficacy. These data support the ongoing phase I clinical evaluation of SU14813 in advanced malignancies.     

RNF43 overexpression attenuates the Wnt/β-catenin signalling pathway to suppress tumour progression in cholangiocarcinoma

RING finger protein 43 (RNF43) is a ubiquitin E3 ligase that negatively regulates Wnt/β-catenin signalling. Mutation, inactivation and downregulation of RNF43 in cholangiocarcinoma (CCA) are associated with a less favourable prognosis. Since the functional role of RNF43 in CCA has not yet been demonstrated, the present study aimed to assess the effect of its overexpression in mediating CCA suppression via Wnt/β-catenin signalling pathway inhibition. Accordingly, RNF43 was overexpressed, and various malignant phenotypic changes studied, including cell proliferation, cell migration, chemotherapeutic sensitivity and the expression of several Wnt/β-catenin target genes. Overexpression of RNF43 in the CCA cell-line KKU-213B hindered activation of Wnt/β-catenin signalling, evidenced by: i) Accumulation of β-catenin in the cytoplasmic fraction and downregulation of several known Wnt target genes at the mRNA level [AXIN2, survivin (BIRC5), CCND1, MMP-7, c-MYC and ABCB1 (MDR1)]; ii) a reduction of cell proliferation; iii) a significant decrease in KKU-213B cell migration with RNF43 overexpression via upregulation of E-cadherin (CDH1); and iv) a reduction in N-cadherin (CDH2), MMP-2, MMP-7 and MMP-9. In addition, overexpression of RNF43 increased 5-fluorouracil sensitivity and downregulation of ABC transporter genes [including ABCB1 and ABCC1 (MRP1)]. The current results demonstrate a functional role for RNF43 in CCA by: i) Blocking β-catenin nuclear translocation; and ii) the subsequent downregulation of Wnt/β-catenin target genes (the latter being involved in the progression of CCA and chemotherapeutic drug susceptibility). Therefore, the present findings suggest that RNF43 could serve a tumour suppressive role in CCA.     

Pharmacokinetics and tolerability of single escalating doses of gabapentin enacarbil: A randomized-sequence,double-blind,placebo-controlled crossover study in healthy volunteers

Background: Gabapentin enacarbil is an actively transported prodrug of gabapentin that provides predictable dose-proportional gabapentin exposure with high (≥68%) oral bioavailability.Objectives: The aims of this study were to investigate the pharmacokinetics and tolerability of gabapentin enacarbil up to supratherapeutic doses and the effects of gabapentin enacarbil on cardiac repolarization in healthy volunteers, and to provide a dose reference for a future definitive QT/corrected QT (QTc) study.Methods: This was a randomized-sequence, double-blind, placebo-controlled, single escalating-dose, crossover study of gabapentin enacarbil 600-mg extended-release tablets administered as a single oral dose of 2400, 3600, 4800, or 6000 mg or placebo, with a 1-week washout between administrations. Blood samples were collected over a period of 36 hours after administration and were analyzed using a validated method of liquid chromatography/tandem mass spec-trometry. Blood gabapentin enacarbil and gabapentin concentrations were analyzed using noncompartmental methods. Tolerability was assessed by monitoring adverse events (AEs) (using subject interview/reporting), laboratory parameters, vital sign measurements, and 12-lead electrocardiography (ECG). Holter ECG was also performed.Results: Thirty-two healthy volunteers were included in the study (18 women, 14 men; mean [SD] age, 31.2 [11.4] years; body mass index, 24.9 [3.04] kg/m²). Gabapentin enacarbil was converted rapidly to gaba-pentin after absorption. Gabapentin exposure in blood was proportional to gabapentin enacarbil dose over the range of 2400 to 6000 mg (1250–3125 mg-equivalent gabapentin). Blood concentrations of intact gabapen-tin enacarbil were low and transient (≤0.5% of the released gabapentin concentration at all doses). The most commonly reported AEs were dizziness and nausea (50% and 25% of subjects, respectively). All but 4 AEs were mild to moderate in intensity. Two subjects experienced treatment-emergent AEs rated as severe: psychomotor retardation, vertigo, and sedation (4800-mg dose) and somnolence (6000 mg). All treatment-emergent AEs resolved without medical intervention. No serious AEs were reported, and none of the AEs led to study withdrawal. There were no clinically significant changes in laboratory parameters, vital sign measurements, or ECG values; QTc intervals did not exceed 480 msec or change from baseline >30 msec at any gabapentin enacarbil dose.Conclusions: Gabapentin enacarbil was associated with dose-proportional gabapentin exposure at doses up to 6000 mg and was generally well tolerated in these healthy subjects. These findings support the use of 6000-mg gabapentin enacarbil in a definitive QT/QTc study.     

Proof of target for SU11654: inhibition of KIT phosphorylation in canine mast cell tumors.

PURPOSE: The purpose of this study was to evaluate the effect of the receptor tyrosine kinase inhibitor SU11654 on the activity of its molecular target KIT in canine mast cell tumors (MCT) and correlate target inhibition with mutational status of the c-kit juxtamembrane domain and SU11654 plasma concentration. EXPERIMENTAL DESIGN: Tumor biopsies were obtained from dogs with advanced MCTs before and 8 h after administration of a single oral dose of SU11654, previously shown to be active in dogs with MCTs. Blood samples were taken to determine the plasma concentration of SU11654. Levels of phosphorylated KIT and ERK1/2 were assessed in tumor biopsies by Western blot. Tumors were analyzed by PCR for the presence or absence of an internal tandem duplication (ITD) in the juxtamembrane domain of c-kit. RESULTS: Fourteen dogs with advanced MCTs were enrolled in the study; 11 of these were evaluable for KIT target modulation (the remaining tumor specimens had inevaluable amounts of total KIT protein). Of these, eight MCTs showed reduced levels of phosphorylated KIT relative to total KIT after treatment with SU11654, compared with pretreatment biopsies. All four evaluable MCTs expressing ITD mutant c-kitshowed modulation of KIT phosphorylation, as did four of seven tumors expressing non-ITD c-kit. Phosphorylated ERK1/2 was modulated in seven tumors; this did not correlate with inhibition of KIT phosphorylation CONCLUSION: SU11654 treatment at the efficacious dose results in inhibition of KIT phosphorylation in canine MCTs.