Systemic and tumor disposition of platinum after administration of cisplatin or STEALTH liposomal-cisplatin formulations (SPI-077 and SPI-077 B103) in a preclinical tumor model of melanoma

Purpose SPI-077 and SPI-077 B103 are formulations of cisplatin encapsulated in pegylated STEALTH liposomes that accumulate in tumors. However, the extent to which active platinum (Pt) is released from the liposome is unknown. Thus, we evaluated the disposition of encapsulated and released Pt in plasma and tumors after administration of STEALTH liposomal and nonliposomal cisplatin.Methods Cisplatin (10 mg/kg), SPI-077 (10 mg/kg), and SPI-077 B103 (5 mg/kg) were administered i.v. to mice bearing B16 murine melanoma tumors. Microdialysis probes were placed into the right and left sides of each tumor, and serial samples were collected from tumor extracellular fluid (ECF) after administration of each agent. After each microdialysis procedure, tumor samples were obtained at each probe site to measure total Pt and Pt-DNA adducts. In a separate study, serial plasma samples (three mice per time point) were obtained. Unbound Pt in tumor ECF and plasma, and total Pt in tumor homogenates were measured by flameless atomic absorption spectrophotometry. Area under the tumor ECF (AUC_ECF) concentration versus time curves of unbound Pt were calculated. Intrastrand GG (Pt-GG) and AG (Pt-AG) Pt-DNA adducts were measured via ³²P-postlabeling.Results Mean±SD peak concentrations of total Pt in tumor homogenates after administration of cisplatin, SPI-077, and SPI-077 B-103 were 3.2±1.9, 11.9±3.0, and 3.5±0.3 g/g, respectively. After cisplatin, mean±SD AUC_ECF of unbound Pt was 0.72±0.46 g/ml·h. There was no detectable unbound Pt in tumor ECF after SPI-077 or SPI-077 B-103 treatment. Mean±SD peak concentration of Pt-GG DNA adducts after administration of cisplatin, SPI-077, and SPI-077 B-103 were 13.1±3.3, 3.5±1.3, and 2.1±0.3 fmol Pt/g DNA, respectively.Conclusion This study suggests that more SPI-077 and SPI-077 B103 distribute into tumors, but release less Pt into tumor ECF, and form fewer Pt-DNA adducts than does cisplatin.     

A phase I study in paediatric patients to evaluate the safety and pharmacokinetics of SPI-77, a liposome encapsulated formulation of cisplatin

Pre-clinical studies indicate that cisplatin encapsulated in STEALTH((R))liposomes (SPI-77) retains anti-tumour activity, but has a much reduced toxicity, compared to native cisplatin. A phase I study was conducted to determine the toxicity and pharmacokinetics of SPI-77 administered to children with advanced cancer not amenable to other treatment. Paediatric patients were treated at doses ranging from 40 to 320 mg m(-2)by intravenous infusion every 4 weeks. Blood samples taken during, and up to 3 weeks after, administration and plasma and ultrafiltrate were prepared immediately. Urine was collected, when possible, for 3 days after administration. SPI-77 administration was well tolerated with the major toxicity being an infusion reaction which responded to modification of the initial infusion rate of SPI-77. Limited haematological toxicity and no nephrotoxicity were observed. No responses to treatment were seen during the course of this phase I study. Measurement of total plasma platinum showed that cisplatin was retained in the circulation with a half life of up to 134 h, with maximum plasma concentrations approximately 100-fold higher than those reported following comparable doses of cisplatin. Comparison of plasma and whole blood indicated that cisplatin was retained in the liposomes and there was no free platinum measurable in the ultrafiltrate. Urine recovery was less than 4% of the dose administered over 72 h. Results from this phase I study indicate that high doses of liposomal cisplatin can safely be given to patients, but further studies are required to address the issue of reformulation of liposomally bound cisplatin.     

Intra- and inter-observer reproducibility of shoulder laxity tests: Comparison of the drawer,modified drawer and load and shift tests

Background

Conventional tests of shoulder laxity have been shown to have poor reliability due to the difficulty in palpating the subtle movements of the shoulder joint beneath the musculature. Modified drawer test that is performed while the soft tissues surrounding the shoulder are loosened has been proposed to facilitate glenohumeral joint movement and improve reliability. We hypothesised that the modified drawer test would have an improved intra- and inter-observer reproducibility in comparison to the drawer and load and shift tests. Correlation of shoulder laxity measured by these tests with generalized joint laxity was also assessed.

ESRS和SPI-Ⅱ评分对急性缺血性脑卒中患者复发风险的评估作用

目的 探讨Essen卒中风险分层量表(Essen Stroke Risk Score,ESRS)和卒中预测工具-Ⅱ(Stroke Prognostic Instrument-Ⅱ,SPI-Ⅱ)对急性缺血性脑卒中(Acute Ischemic Stroke,AIS)1 y复发风险的评估作用。方法 前瞻性的连续收集2012年12月~2016年6月北京大学航天临床医学院神经内科住院的AIS患者820例。详细记录入选患者的临床基线资料并完成ESRS及SPI-Ⅱ评分。于发病后1 y进行随访,以复发作为金标准,收集患者的复发情况等资料。通过描绘AIS患者ESRS和SPI-Ⅱ的受试者工作特征曲线(Receive Operating Characteristic curve,ROC),计算曲线下面积(Area Under the Curve,AUC)来比较2个量表预测作用。应用Hosmer-Lemeshow法评价预测模型和实际模型的拟合优度。结果 符合纳入标准的患者共790例,失访30例(3.95%),最终纳入760例。随访1 y时,复发84例(11.05%);死亡67例,死亡率(8.82%)。2个量表ROC曲线下面积AUC值分别为0.576(P0.05,95%CI:0.540~0.611)、0.583(P0.05,95%CI:0.547~0.618),2个量表AUC值差异无统计学意义(P0.05);Hosmer-Lemeshow法检验,χ~2值分别为3.391、5.797(均P0.05)表明预测模型与实际模型拟合良好。结论 ESRS和SPI-Ⅱ评分对AIS患者1 y复发评估作用基本一致。     

Inhibition of cellular Shp2 activity by a methyl ester analog of SPI-112

The protein tyrosine phosphatase (PTP) Shp2 (PTPN11) is an attractive target for anticancer drug discovery because it mediates growth factor signaling and its gain-of-function mutants are causally linked to leukemias. We previously synthesized SPI-112 from a lead compound of Shp2 inhibitor, NSC-117199. In this study, we demonstrated that SPI-112 bound to Shp2 by surface plasmon resonance (SPR) and displayed competitive inhibitor kinetics to Shp2. Like some other compounds in the PTP inhibitor discovery efforts, SPI-112 was not cell permeable, precluding its use in biological studies. To overcome the cell permeation issue, we prepared a methyl ester SPI-112 analog (SPI-112Me) that is predicted to be hydrolyzed to SPI-112 upon entry into cells. Fluorescence uptake assay and confocal imaging suggested that SPI-112Me was taken up by cells. Incubation of cells with SPI-112Me inhibited epidermal growth factor (EGF)-stimulated Shp2 PTP activity and Shp2-mediated paxillin dephosphorylation, Erk1/2 activation, and cell migration. SPI-112Me treatment also inhibited Erk1/2 activation by a Gab1-Shp2 chimera. Treatment of Shp2^E76K mutant-transformed TF-1 myeloid cells with SPI-112Me resulted in inhibition of Shp2^E76K-dependent cell survival, which is associated with inhibition of Shp2^E76K PTP activity, Shp2^E76K-induced Erk1/2 activation, and Bcl-XL expression. Furthermore, SPI-112Me enhanced interferon-γ (IFN-γ)-stimulated STAT1 tyrosine phosphorylation, ISRE-luciferase reporter activity, p21 expression, and the anti-proliferative effect. Thus, the SPI-112 methyl ester analog was able to inhibit cellular Shp2 PTP activity.     

The cowpox virus fusion regulator proteins SPI-3 and hemagglutinin interact in infected and uninfected cells

The serpin SPI-3 and the hemagglutinin (HA) encoded by cowpox virus (CPV) block cell-cell fusion, and colocalize at the cell surface. wtCPV does not fuse cells, but inactivation of either gene leads to fusion. SPI-3 mAb added to wtCPV-infected cells caused fusion, confirming that SPI-3 protein at the cell surface prevents fusion. The SPI-3 mAb epitope mapped to an 85-amino acid region at the C-terminus. Removal of either 44 residues from the SPI-3 C-terminus or 48 residues following the N-terminal signal sequence resulted in fusion. Interaction between SPI-3 and HA proteins in infected cells was shown by coimmunoprecipitation. SPI-3/HA was not associated with the A27L "fusion" protein. SPI-3 and HA were able to associate in uninfected cells in the absence of other viral proteins. The HA-binding domain in SPI-3 resided in the C-terminal 229 residues, and did not include helix D, which mediates cofactor interaction in many other serpins.     

The vaccinia virus fusion inhibitor proteins SPI-3 (K2) and HA (A56) expressed by infected cells reduce the entry of superinfecting virus

The orthopoxvirus SPI-3 (K2) and A56 (hemagglutinin, HA) proteins interact and together prevent cell–cell fusion. SPI-3/A56 has been proposed to prevent the superinfection of previously infected cells by reducing virus–cell fusion. Binding of mature virions of vaccinia virus (VV) to VV-infected cells was unaffected by SPI-3 or A56 on the surface of infected cells. Entry of VV into infected cells was assessed using VV-P_T7-luc carrying the luciferase reporter under T7 control. Cells infected with VV or cowpox virus (CPV) expressing T7 RNA polymerase and lacking SPI-3 and/or A56 were superinfected with VV-P_T7-luc, and luciferase activity was measured. Inactivation of SPI-3 or A56 from the pre-infecting virus resulted in greater luciferase expression from the superinfecting VV-P_T7-luc. Antibody against SPI-3 present during infection with wild-type CPV-T7 increased luciferase expression from superinfecting VV-P_T7-luc. The SPI-3/A56 complex on the infected cell surface therefore appears to reduce the entry of virions into infected cells.     

Time dependent EC′, ECE and EC2E mechanisms at microdisc electrodes: simulations using adaptive finite element methods

In this paper we extend the work in [Electrochem. Commun. 5 (2003) 519] to more complex reaction mechanisms. We use the adaptive finite element algorithm described there to simulate the chronoamperometric current for EC^′ reaction mechanisms (catalytic reaction mechanisms) at inlaid and recessed microdisc electrodes and for ECE and EC₂E reaction mechanisms at microdisc electrodes.     

Immunogenicity and protection efficacy of a Salmonella enterica serovar Typhimurium fnr,arcA and fliC mutant

Salmonella enterica serovar Typhimurium is a major food-borne pathogen that can cause self-limited gastroenteritis or life-threatening invasive diseases in humans. There is no licensed S. Typhimurium vaccine for humans to date. In this study, we attempted to construct a live attenuated vaccine strain of S. Typhimurium based on three genes, namely, the two global regulator genes fnr and arcA and the flagellin subunit gene fliC. The S. Typhimurium three-gene mutant, named SLT39 (ΔfnrΔarcAΔfliC), exhibited a high level of attenuation with a colonization defect in mouse tissues and approximately 10⁴-fold decreased virulence compared with that of the wild-type strain. To evaluate the immunogenicity and protection efficacy of STL39, mice were inoculated twice with a dose of 10⁷ CFU or 10⁸ CFU at a 28-day interval, and the immunized mice were challenged with a lethal dose of the wild-type S. Typhimurium strain one month post second immunization. Compared with mock immunization, SLT39 immunization with either dose elicited significant serum total IgG, IgG1 and IgG2a and faecal IgA responses against inactivated S. Typhimurium antigens at a comparable level post second immunization, whereas the 10⁸ CFU group induced higher levels of duodenal and caecal IgA than the 10⁷ CFU group. Furthermore, the bacterial loads in mouse tissues, including Peyer’s patches, spleen and liver, significantly decreased in the two SLT39 immunization groups compared to those in the control group post challenge. Additionally, all mice in the SLT39 (10⁸ CFU) group and 80% of the mice in the SLT39 (10⁷ CFU) group survived the lethal challenge, suggesting full protection and 80% protection efficacy, respectively. Thus, the S. Typhimurium fnr, arcA and fliC mutant proved to be a potential attenuated live vaccine candidate for prevention of homologous infection.