Collaborative study for the establishment of erythropoietin BRP batch 3

The European Pharmacopoeia (Ph. Eur.) monograph on Erythropoietin concentrated solution (1316) specifies that identification and assay are performed using pharmacopoeial methods requiring the use of a reference preparation. To replace the current erythropoietin Biological Reference Preparation (BRP) of Ph. Eur., in 2006, the European Directorate for the Quality of Medicines undertook a collaborative study designed to establish a replacement batch. In order to guarantee continuity, the formulation of the candidate batch was similar to that of previous batches (1 and 2). The methods chosen to qualify the new standard were those included in the current monograph. The study was defined to allow calibration of the candidate by in vivo bioassay in terms of the current World Health Organization (WHO) International Standard (IS) and to assign a unitage. The suitability of the candidate preparation to serve as a reference standard for the other pharmacopoeial analytical procedures was also investigated. The collaborative study involved 16 laboratories from Europe, Australia, Canada, China, Japan, South Korea and the United States of America. Participants carried out biological and physicochemical assays on the candidate erythropoietin BRP batch 3 (cBRP3), using batch 2 (BRP2) and where necessary the 2nd World Health Organization International Standard (WHO 2nd IS) for recombinant erythropoietin as the reference standards. It was demonstrated that the replacement batch is appropriate for use as erythropoietin BRP in the context of the control of erythropoietin concentrated solutions according to the Ph. Eur. monograph (1316). However as regards the potency of BRP2 and cBRP3 in the mouse bioassay unexpected observations were made. Direct calibration of BRP2 against the WHO 2nd IS yielded, in all laboratories, results that were systematically higher than the potency of 32,500 IU/vial assigned by direct calibration against the WHO 2nd IS in the former study. It was therefore recommended to assign the potency of cBRP3 against BRP2, using the average of all results that were not considered as outlying obtained in the collaborative study, in order to guarantee continuity of unitage between the successive BRP batches. The outcome of the study enabled the Ph. Eur. Commission to establish the proposed standard as 'erythropoeitin BRP batch 3' (BRP3). BRP3 was established in June 2007 for use as a reference preparation for the polycythaemic and normocythaemic mouse bioassay, with an assigned potency of 35,280 IU/vial, the identification by capillary zone electrophoresis, by polyacrylamide gel electrophoresis, immunoblotting and peptide mapping and as a reference for checking the system suitability of size-exclusion chromatographic procedures used in the test for dimers and related substances of higher molecular mass in the Ph. Eur. monograph (1316).     

我院抗菌药物应用情况调查分析

目的:对我院抗菌药物的使用进行调查分析,为合理用药提供依据,并提出合理使用抗菌药物的建议。方法:随机抽查我院门、急诊处方7382张进行调查,统计分析了抗菌药物的使用率、药物种类及使用频率和联合用药情况,并进行合理性评价。结果:抗菌药物使用率为34.45%,涉及10个亚类38个品种,以单用抗菌药物为主,占83.88%。合理用药占92.25%。结论:我院抗菌药物使用情况基本合理,但仍需进一步提高。     

Pharmacological characterization of chick and frog beta adrenergic receptors in primary cultures of myocardial cells.

In membrane preparations derived from primary cultures of chick myocardial cells, beta adrenergic receptors modeled for a single low-affinity site for both betaxolol (beta-1-selective) and ICI 118551 (beta-2-selective) displacement of [125I]iodocyanopindolol (ICYP), indicating that the chick beta receptor is pharmacologically distinct from both mammalian beta-1 and beta-2 adrenergic receptors with respect to these antagonists. However, the highly beta-1-selective compound CGP 20712A was able to distinguish two binding sites on ICYP competition curves, a high-affinity "beta-1 site" (75%) and a low-affinity "beta-2 site" (25%). Also, in chick heart cell membranes the relative ability of agonists to displace ICYP produced a profile typical of beta-1 adrenergic receptors with a rank order of potency or efficacy of: isoproterenol greater than epinephrine = norephinephrine. When agonist-mediated adenylyl cyclase stimulation was assessed the order of potency was slightly different, isoproterenol greater than epinephrine greater than or equal to norepinephrine. Additionally, antagonism of isoproterenol stimulation of adenylyl cyclase by CGP 20712A yielded a Kb value (1.16 +/- 0.35 x 10(-7) M) intermediate between the high and low-affinity binding sites of CGP 20712A, suggesting that the low-affinity site is coupled to adenylyl cyclase. In membrane preparations of frog myocardial cells, ICYP/antagonist competition curves modeled for a mixed population of receptors, with subtype percentages varying from 50:50 beta-1:beta-2 to 100% beta-2 depending on the specific antagonist used and the individual cell preparation. For ICYP/agonist competition binding experiments the relative ability to displace ICYP was isoproterenol greater than epinephrine = norepinephrine, a profile typical of beta-1 adrenergic receptors.(ABSTRACT TRUNCATED AT 250 WORDS)     

HDAC inhibitor confers radiosensitivity to prostate stem-like cells

Background:

Radiotherapy can be an effective treatment for prostate cancer, but radiorecurrent tumours do develop. Considering prostate cancer heterogeneity, we hypothesised that primitive stem-like cells may constitute the radiation-resistant fraction.

Methods:

Primary cultures were derived from patients undergoing resection for prostate cancer or benign prostatic hyperplasia. After short-term culture, three populations of cells were sorted, reflecting the prostate epithelial hierarchy, namely stem-like cells (SCs, α₂β₁integrin^hi/CD133⁺), transit-amplifying (TA, α₂β₁integrin^hi/CD133⁻) and committed basal (CB, α₂β₁integrin^lo) cells. Radiosensitivity was measured by colony-forming efficiency (CFE) and DNA damage by comet assay and DNA damage foci quantification. Immunofluorescence and flow cytometry were used to measure heterochromatin. The HDAC (histone deacetylase) inhibitor Trichostatin A was used as a radiosensitiser.

Results:

Stem-like cells had increased CFE post irradiation compared with the more differentiated cells (TA and CB). The SC population sustained fewer lethal double-strand breaks than either TA or CB cells, which correlated with SCs being less proliferative and having increased levels of heterochromatin. Finally, treatment with an HDAC inhibitor sensitised the SCs to radiation.

Interpretation:

Prostate SCs are more radioresistant than more differentiated cell populations. We suggest that the primitive cells survive radiation therapy and that pre-treatment with HDAC inhibitors may sensitise this resistant fraction.     

Surgical technique of en bloc pelvic resection for advanced ovarian cancer

Objective

The aim of this paper was to describe the operative details for en bloc removal of the adnexal tumor, uterus, pelvic peritoneum, and rectosigmoid colon with colorectal anastomosis in advanced epithelial ovarian cancer patients with widespread pelvic involvement.

Methods

The patient presented with good performance status and huge pelvic tumor extensively infiltrating into adjacent pelvic organs and obliterating the cul-de-sac. The patient underwent en bloc pelvic resection as primary cytoreductive surgery. En bloc pelvic resection procedure is initiated by carrying a circumscribing peritoneal incision to include all pan-pelvic disease within this incision. After retroperitoneal pelvic dissection, the round ligaments and infundibulopelvic ligaments are divided. The ureters are dissected and mobilized from the peritoneum. After dissecting off the anterior pelvic peritoneum overlying the bladder with its tumor nodules, the bladder is mobilized caudally and the vesicovaginal space is developed. The uterine vessels are divided at the level of the ureters, and the paracervical tissues (or parametria) are divided. The proximal sigmoid colon is divided above the most proximal extent of gross tumor using a ligating and dividing stapling device. The sigmoid mesentery is ligated and divided including the superior rectal vessels. The pararectal and retrorectal spaces are further developed and dissected down to the level of the pelvic floor. The posterior dissection is progressed and moves to the right and then to the left of the rectum. The rectal pillars including the middle rectal vessels are ligated and divided. Hysterectomy is completed in a retrograde fashion. The distal rectum is divided using a linear stapler. The specimen is removed en bloc with the uterus, adnexa, pelvic peritoneum, rectosigmoid colon, and tumor masses leaving a macroscopically tumor-free pelvis. Colorectal anastomosis was completed using stapling device.

Results

En bloc pelvic resection was performed by total abdominal hysterectomy, bilateral salpingo-oophorectomy, pelvic peritonectomy, and rectosigmoid colectomy with colorectal anastomosis using a stapling device. Complete clearance of pelvic disease leaving no gross residual disease was possible using en bloc pelvic resection.

Conclusion

En bloc pelvic resection is effective for achieving maximal cytoreduction with the elimination of the pelvic disease in advanced primary ovarian cancer patients with extensive pelvic organ involvement.     

A randomized phase III study of neoadjuvant hormonal therapy in patients with localized prostate cancer

The primary objective of this randomized trial is to evaluate the benefit of the addition of neoadjuvant hormonal therapy to escalated-dose external-beam radiation therapy in the treatment of patients with intermediate-risk carcinoma of the prostate. A secondary objective of this study is to determine prognostic factors for radiation response. All patients will have tissue oxygenation measured and biopsies taken before treatment at the time of fiducial marker insertion for radiation treatment planning and daily monitoring. In addition, patients randomized to the neoadjuvant bicalutamide arm will be asked to consider having these studies repeated before initiation of radiation therapy (after 3 months of hormonal therapy).     

Contextual fear conditioning and baseline startle responses in the rat fear-potentiated startle test: a comparison of benzodiazepine/gamma-aminobutyric acid-A receptor agonists

In the rat, fear-potentiated startle (FPS) test animals are first trained to associate brief light presentations with a mild electric footshock and then tested for startle responses to acoustic stimuli, delivered either in darkness (i.e. baseline startle) or after the conditioning stimulus. Following light presentation the magnitude of the startle response is markedly increased, and the test is commonly used to distinguish anxiolytic drug effects (i.e. a reduction in FPS) from non-specific effects such as sedation/muscle relaxation. However, recent studies suggest that the environment in which the animal is trained may also contribute towards the acquisition of a conditioned fear response (i.e. contextual fear conditioning) and that this may elevate startle responses recorded in the dark. In the present study, therefore, we have compared the benzodiazepine/gamma-aminobutyric acid-A receptor agonist chlordiazepoxide with the partial agonists FG 8205 and bretazenil, which are known to have a reduced propensity to produce sedation/myorelaxation, using two different FPS procedures: (i) conditioning and testing in stabilimeter chambers, and (ii) conditioning and testing in different environments. The results show that FPS can be demonstrated in both procedures and that treatment with chlordiazepoxide, FG 8205 or bretazenil dose-dependently attenuates the response. However, animals conditioned and tested in stabilimeter chambers also showed a significant increase in dark-startle amplitudes compared with non-shocked rats, suggesting that this response was elevated by contextual fear conditioning. Furthermore, despite clear differences in side-effect liabilities, FG 8205 and bretazenil significantly reduced dark-startle responses, suggesting that this measure is also sensitive to the anxiolytic effects of benzodiazepines. In contrast, when animals were conditioned and tested in different environments, dark-startle responses were not significantly different from those recorded in non-shocked rats and treatment with FG 8205 or bretazenil had no effect. Thus, conditioning and testing animals in different environments may provide a more effective means of distinguishing anxiolytic from non-specific drug effects in the rat FPS test.     

Phase 1 Study of Monotherapy with KHK2866, an Anti-Heparin-Binding Epidermal Growth Factor-Like Growth Factor Monoclonal Antibody,in Patients with Advanced Cancer

Background

KHK2866 is a recombinant, humanized, non-fucosylated, monoclonal antibody directed at heparin-binding epidermal growth factor-like growth factor (HB-EGF).

Objective

To determine the safety, tolerability, maximum tolerated dose (MTD), pharmacokinetics, pharmacodynamics, potential immunogenicity, and preliminary clinical efficacy of KHK2866 monotherapy in patients with advanced and refractory cancer in a first-in-human, phase 1 study.

Materials and Methods

Using a standard 3?+?3 dose-escalation design, 20 patients received KHK2866 (0.3, 1, and 3 mg/kg) intravenously once weekly. Two additional patients received 0.1 mg/kg in a cohort which was subsequently added following protocol amendment.

Results

The first three patients enrolled experienced grade 2 hypersensitivity (acute infusion reactions) after the first dose of KHK2866. After prophylactic treatment with an H1-blocker and corticosteroids in subsequently recruited patients, two grade 2 hypersensitivity reactions were observed in the remaining 19 patients. Grade 2/3 neurotoxicity appeared to be dose-limiting at 3 mg/kg in the original dose-escalation cohorts (n?=?2), at 1 mg/kg in the MTD dose expansion cohort (n?=?1), and at 0.1 mg/kg (n?=?1). Neurotoxicity was manifested as complex partial seizure activity, aphasia, and confusion after first-dose administration. Pharmacokinetic exposure to KHK2866 increased proportionally to dose. Mean elimination half-life was 71.9–118 h over the dose range from 0.3 to 3 mg/kg. All KHK2866 doses decreased serum free HB-EGF levels, generally below the lower limit of quantification.

Conclusions

The study was terminated because of neuropsychiatric toxicity. The only predictive factor for neuropsychiatric toxicity was administration of KHK2866. These effects were reversible, but were not predictable. Their etiology is not presently understood. [Study registered at ClinicalTrials.gov #NCT0179291]

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Contextual synthetic lethality of cancer cell kill based on the tumor microenvironment

Acute and chronic hypoxia exists within the three-dimensional microenvironment of solid tumors and drives therapy resistance, genetic instability, and metastasis. Replicating cells exposed to either severe acute hypoxia (16 hours with 0.02% O(2)) followed by reoxygenation or moderate chronic hypoxia (72 hours with 0.2% O(2)) treatments have decreased homologous recombination (HR) protein expression and function. As HR defects are synthetically lethal with poly(ADP-ribose) polymerase 1 (PARP1) inhibition, we evaluated the sensitivity of repair-defective hypoxic cells to PARP inhibition. Although PARP inhibition itself did not affect HR expression or function, we observed increased clonogenic killing in HR-deficient hypoxic cells following chemical inhibition of PARP1. This effect was partially reversible by RAD51 overexpression. PARP1(-/-) murine embryonic fibroblasts (MEF) showed a proliferative disadvantage under hypoxic gassing when compared with PARP1(+/+) MEFs. PARP-inhibited hypoxic cells accumulated γH2AX and 53BP1 foci as a consequence of altered DNA replication firing during S phase-specific cell killing. In support of this proposed mode of action, PARP inhibitor-treated xenografts displayed increased γH2AX and cleaved caspase-3 expression in RAD51-deficient hypoxic subregions in vivo, which was associated with decreased ex vivo clonogenic survival following experimental radiotherapy. This is the first report of selective cell killing of HR-defective hypoxic cells in vivo as a consequence of microenvironment-mediated "contextual synthetic lethality." As all solid tumors contain aggressive hypoxic cells, this may broaden the clinical utility of PARP and DNA repair inhibition, either alone or in combination with radiotherapy and chemotherapy, even in tumor cells lacking synthetically lethal, genetic mutations.     

ATM-dependent phosphorylation of 53BP1 in response to genomic stress in oxic and hypoxic cells

The ATM kinase is activated by chromatin modification following exogenous and endogenous DSBs or cell stress, including acute anoxia. The p53 binding protein 1 (53BP1) contains multiple ATM-consensus phosphorylation sites in its N- and C-termini and may therefore be a distal read-out of ATM function. We have examined the cellular activation of these phosphorylation sites for the first time in situ following anoxic/hypoxic stress and IR-induced exogenous DSBs. We show that multiple residues of 53BP1 are phosphorylated and that these phosphoforms form discrete nuclear foci following IR or during DNA replication as exogenous or endogenous DNA double strand breaks (DSBs), respectively. Novel data pertaining to the phosphorylation of 53BP1^Ser25in situ supports its dependency on the ATM kinase; but this occurs independently of p53 function. We show that 53BP1^Ser25 is activated specifically in S-phase cells during anoxia in an ATM-dependent manner. Exogenous DSBs form discrete IR-induced foci whereas oxygen stress induced non-localized 53BP1^Ser25 activation. Our in vitro data are supported by irradiated xenograft studies in vivo whereby 53BP1^Ser25 phosphorylation does not occur in sub-regions positive for the hypoxia marker EF5. We propose a model whereby DSBs induce chromatin modification at sites of DNA damage which are tracked by the ATM substrates γ H2AX and 53BP1^Ser25 in a mechanism distinct from p53-mediated cell cycle arrest. Together this work indicates 53BP1^Ser25, and possibly other 53BP1 phosphoforms, as a bona fide DSB-biomarkers for surveying ongoing DNA-damage related signaling in oxic and hypoxic cells during clinical radiotherapy.