Stability of pemetrexed disodium in sodium chloride 0.9% w/v intravenous Viaflo infusion bags.

OBJECTIVES: The aim of this study was to assess the stability of pemetrexed disodium (Alimta), reconstituted in 100 mL sodium chloride 0.9% w/v intravenous infusion bags (Baxter Viaflo) at two target bag concentrations (2.0 and 13.5 mg/mL) during storage at 2-8°C for 28 days (protected from light), followed by 24 hours at 25±2°C with 60±5% relative humidity (RH) (protected from light). This study was commissioned by NHS England and NHS Improvement to generate data to aid shelf life extensions for aseptic products compounded in National Health Service (NHS) hospital aseptic facilities. METHODS: A high performance liquid chromatography (HPLC) assay was developed and validated to monitor pemetrexed concentration and related substance levels in accordance with NHS yellow cover document requirements. This assay and analysis of related substances was used alongside visual inspection, pH monitoring and sub-visible particle count analysis to monitor stability. The stability of three preparations of each concentration of pemetrexed disodium in Viaflo saline bags (0.9% w/v) was assessed at various time points. RESULTS: Pemetrexed assay concentrations remained >97.0% of initial concentration at all points during the study (including the period at elevated temperature). Appearance remained consistent with the Summary of Product Characteristics, particle count data remained within the British Pharmacopoeia limits, and pH remained within 0.43 units of T=0 at all times. The increases in related substance levels during the study were found to be the limiting factor for shelf life assignment. CONCLUSION: The data for appearance, pH, sub-visible particle count analysis and pemetrexed assay would support a shelf life of 28 days stored at 2-8°C (protected from light) followed by 24 hours at 25±2°C with 60±5% RH (protected from light). However, given the increase in related substance levels, a shelf life of 21 days stored at 2-8°C (protected from light) was deemed to be appropriate.

P53 apoptosis mediator PERP: localization, function and caspase activation in uveal melanoma.

p53 apoptosis effector related to PMP-22 (PERP) is a transcriptional target gene of p53 tumour suppressor that is specifically induced during apoptosis and not during cell cycle arrest. In primary uveal melanoma (UM), the most common intraocular malignancy in adults that has a reportedly unaffected signalling pathway upstream of and including p53, PERP expression is down-regulated in the metastatic monosomy 3-type tumours, compared with the less aggressive disomy 3-type tumours. Here, we demonstrate experimentally, by the use of full-length PERP-green fluorescent protein (GFP) fusions and real-time confocal microscopy, the intracellular targeting and plasma membrane localization of PERP in living UM cells and show that expression of PERP induces caspase-mediated apoptosis in UM cells. Induction of PERP expression in GFP-PERP-transfected UM cells leads to increased levels of cleaved caspase-8 forms, as well as to reduction of its full-length substrate Bid, but not to detectable processing of caspase-9. The levels of mature caspase-8, -9 and -3 proteins significantly correlate with PERP expression levels in primary UMs. Transcriptional profiling of PERP and caspase-8 in tumour specimens indicates that the positive association of PERP and caspase-8 proteins is a consequence of post-translational processing, most likely at the level of caspase-8 cleavage, and not of increased transcription of pro-caspase-8. We conclude that PERP expression leads to activation of an extrinsic receptor-mediated apoptotic pathway, with a possible subsequent engagement of the intrinsic apoptotic pathway. The findings underline the apoptotic pathway mediated by PERP as a critical mechanism employed by UM tumours to modulate susceptibility to apoptosis.

An unsuspected ecdysteroid/steroid phosphatase activity in the key T-cell regulator, Sts-1: surprising relationship to insect ecdysteroid phosphate phosphatase.

The insect enzyme ecdysteroid phosphate phosphatase (EPP) mobilizes active ecdysteroids from an inactive phosphorylated pool. Previously assigned to a novel class, it is shown here that it resides in the large histidine phosphatase superfamily related to cofactor-dependent phosphoglycerate mutase, a superfamily housing notably diverse catalytic activities. Molecular modeling reveals a plausible substrate-binding mode for EPP. Analysis of genomic and transcript data for a number of insect species shows that EPP may exist in both the single domain form previously characterized and in a longer, multidomain form. This latter form bears a quite unexpected relationship in sequence and domain architecture to vertebrate proteins, including Sts-1, characterized as a key regulator of T-cell activity. Long form Drosophila melanogaster EPP, human Sts-1, and a related protein from Caenorhabditis elegans have all been cloned, assayed, and shown to catalyse the hydrolysis of ecdysteroid and steroid phosphates. The surprising relationship described and explored here between EPP and Sts-1 has implications for our understanding of the function(s) of both.

Expression and down-regulation of cytochrome P450 genes of the CYP4 family by ecdysteroid agonists in Spodoptera littoralis and Drosophila melanogaster.

The function of CYP4 genes in insects is poorly understood. Some CYP genes are up-regulated by ecdysteroids and a number of Cyp4 genes in Drosophila melanogaster have been shown by microarray to be down-regulated when the ecdysteroid titre is high, suggesting hormonal regulation. Here, we report the utilization of certain cloned CYP4 cDNAs/fragments to probe their developmental/tissue expression in the Lepidopteran, Spodoptera littoralis, including the effects of ecdysteroid receptor agonists (bis-acyl hydrazines). CYP4L8 is expressed essentially throughout the final larval instar of S. littoralis and, together with CYP4M12, is down-regulated by agonist. Furthermore, expression of these genes occurs in midgut, but is undetectable in brain, fat body, and integument. Similarly, in D. melanogaster, Cyp4ac1, Cyp4ac3, Cyp4ad1 and Cyp4d1 gene expression is drastically down-regulated by ecdysteroid agonist. The significance of the results is discussed in relation to the plausible functions of the CYP4 genes in Lepidoptera and mechanisms of down-regulation.

Characterization in relation to development of an ecdysteroid agonist-responsive cytochrome P450, CYP18A1, in Lepidoptera.

Cytochrome P450 enzymes are involved in a number of steps in ecdysteroid (moulting hormone) homeostasis in insects. We report the cloning and characterization of an ecdysteroid agonist-responsive cytochrome P450, CYP18A1, from the cotton leafworm, Spodoptera littoralis. Northern blot analysis showed that the mRNA transcript was expressed at times of increasing ecdysteroid titre in final instar S. littoralis larvae and was induced by the ecdysteroid receptor agonist, RH-5992, in midgut and fat body. In addition, transcript expression was also detected in the prothoracic glands, a major ecdysteroid biosynthetic tissue, in both S. littoralis and the tobacco hornworm, Manduca sexta, at a time of increasing ecdysteroid titre. The exact significance of the temporal and spatial expression of CYP18A1 is unclear. The characterization of a P450 that is ecdysteroid agonist-responsive may provide a future target for exploitation in the development of novel insect control strategies.